ES2543890T3 - Activin-ActRII antagonists for use in the treatment of anemia - Google Patents

Abstract

An activin-ActRII antagonist for use in a method of treatment or prevention of anemia in a human patient in need thereof, wherein said antagonist is chosen from: an antibody that binds to the activin and destabilizes the binding to ActRIIa, an antibody which binds to activin and destabilizes binding to ActRIIb, an anti-activin antibody ßA, ßB, ßC or ßE, an anti-ActRIIa antibody, an anti-ActRIIb antibody, folistatin, and inhibin.

Description

Activin-ActRII antagonists for use in the treatment of anemia

Background of the invention

The mature red blood cell, or erythrocyte, is responsible for the transport of oxygen in the vertebrate circulatory system. Red blood cells carry high concentrations of hemoglobin, a protein that binds to oxygen in the lungs at a relatively high partial oxygen pressure (pO2) and supplies oxygen to areas of the body with a relatively low pO2.

Mature red blood cells are produced in pluripotent hematopoietic stem cells in a process called erythropoiesis. In postnatal individuals, erythropoiesis occurs mainly in the bone marrow and red pulp of the spleen. The coordinated action of several signaling pathways controls the balance of proliferation, differentiation, survival and cell death. Under normal conditions, red blood cells are produced at a rate that maintains a constant mass of red blood cells in the body, and production can increase or decrease in response to various stimuli, including increased or decreased oxygen tension or tissue demand. The erythropoiesis process begins with the formation of specific lineage precursor cells and advances through a series of different types of precursor cells. The final stages of erythropoiesis occur as reticulocytes are released into the bloodstream and lose their mitochondria and ribosomes while they assume the morphology of mature red blood cells. An elevated level of reticulocytes, or a high reticulocyte / erythrocyte ratio in the blood, indicates an increase in red blood cell production rates.

Erythropoietin (Epo) is widely recognized as the most important positive regulator of erythropoiesis in postnatal vertebrates. The Epo regulates the compensatory erythropoietic response to decreased tissue oxygen tension (hypoxia) and low levels of red blood cells or low levels of hemoglobin. In humans, elevated levels of Epo promote the formation of red blood cells, stimulating the generation of erythroid progenitors in the bone marrow and spleen. In the mouse, Epo increases erythropoiesis mainly in the spleen.

Doctors use various forms of recombinant Epo to increase red blood cell levels in various clinical settings and especially for the treatment of anemia. Anemia is a widely defined condition, characterized by lower than normal levels of hemoglobin or red blood cells. In some cases, anemia is caused by a primary disorder in the production or survival of red blood cells. More commonly, anemia is a disease secondary to diseases of other systems (Weatherall and Provan (2000) Lancet 355, 1169-1175). Anemia can result from a decrease in the production rate of red blood cells or an increase in the rate of destruction of red blood cells or by loss of red blood cells due to hemorrhage. Anemia can occur due to various disorders that include, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis and bone marrow transplantation.

Epo treatment normally causes an increase in hemoglobin of approximately 1-3 g / dL in healthy humans over a period of weeks. When administered to anemic individuals, this treatment regimen often provides substantial increases in hemoglobin and red blood cell levels, and improves the quality of life and prolongs survival. Epo is not uniformly effective, and many individuals are resistant even at high doses (Horl et al., (2000) Nephrol Dial Trasplant 15, 43-50). More than 50% of cancer patients have an inadequate response to Epo, approximately 10% with end-stage renal disease have a lower than normal response (Glaspy et al (1997) J Clin Oncol 15, 1218-1234; Demetri et al (1998) J Clin Oncol 16, 3412-3425), and less than 10% with myelodysplastic syndrome respond favorably (Estey (2003) Curr Opin Hematol 10, 60-67). Several factors, including inflammation, iron and vitamin deficiency, inadequate dialysis, aluminum toxicity and hyperparathyroidism can predict a poor therapeutic response, the molecular mechanisms of Epo resistance are still unclear.

Therefore, it is an objective of the present disclosure to provide alternative compositions and methods to increase red blood cell levels in patients.

Summary of the Invention

The present invention provides an activin-ActRII antagonist for use in a treatment method.

or prevention of anemia in a human patient who needs it, where said antagonist is chosen from:

an antibody that binds to activin and destabilizes ActRIIa binding,

an antibody that binds to activin and destabilizes ActRIIb binding,

an anti-activin antibody A, B, C or E,

an anti-ActRIIa antibody,

an anti-ActRIIb antibody,

folistatin e

inhibin

The antagonist can be folistatin-288 or folistatin-315. Anemia can be associated with multiple myeloma, chronic kidney disease or insufficiency, acute renal disease or insufficiency, chemotherapy treatment of the patient, a myelodysplastic syndrome or thalassemia.

In part, the disclosure demonstrates that activin antagonists, as well as ActRIIa and ActRIIb antagonists, can be used to increase the levels of red blood cells and hemoglobin. In particular, the disclosure demonstrates that a soluble form of ActRIIa acts as an inhibitor of activin and, when administered in vivo, increases blood levels of red blood cells. A milder effect can be observed with a soluble form of ActRIIb, which binds to activin A with less affinity than soluble ActRIIa. While soluble ActRIIa and ActRIIb can affect red blood cell levels through a different mechanism of activin antagonism, however the disclosure demonstrates that desirable therapeutic agents can be selected, based on activin antagonism or ActRII antagonism or both . Such drugs are collectively called activin-ActRII antagonists. Accordingly, in certain embodiments, the present invention provides certain activin-ActRII antagonists for the treatment or prevention of anemia in a human patient in need thereof. As described in U.S. Patent Application No. 2007-0249022A1, activin-ActRIIa antagonists can be used to promote bone growth and increase bone density. As described herein, the effects of these antagonists on red blood cell levels are faster and occur at lower doses than the effects of such antagonists on the bone. Therefore, in certain embodiments, an activin-ActRIIa antagonist can be used to increase the levels of red blood cells or hemoglobin without causing a significant increase in bone density. For example, a method may cause an increase of less than 3%, 5%, 10% or 15% of bone density. This selective effect can be achieved using, for example, lower doses of Actin-ActRIIa antagonist, less frequent doses or a shorter serum half-life Actin-ActRIIa antagonist in doses and frequencies calculated to provide a lower serum concentration.

This document discloses polypeptides that consist of a soluble Actin-binding polypeptide that binds to activin. The activin-binding polypeptide can be an ActRIIa polypeptide or an ActRIIb polypeptide. ActRII polypeptides can be formulated as a pharmaceutical preparation containing the Actin-binding activin polypeptide and a pharmaceutically acceptable carrier. The ActRII-binding activin polypeptide can bind to the activin with a KD of less than 1 micromolar or less than 100, 10 or 1 nanomolar. Optionally, the activin-binding ActRII polypeptide binds selectively to activin against GDF11 and / or GDF8, and optionally with a KD that is at least 10 times, 20 times or 50 times lower with respect to activin than with respect to GDF11 and / or GDF8. Without wishing to adhere to a particular mechanism of action, it is expected that this degree of selectivity for inhibition of activin with respect to GDF11 / GDF8 inhibition is the reason for the effects on bone

or erythropoiesis without a regularly measurable effect on the muscle. An ActRII polypeptide can be selected to cause 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 components of the polypeptide, evaluated by size exclusion chromatography, and optionally, the composition is at least 98% pure. An ActRIIa-binding activin polypeptide for use in said preparation may be any of those disclosed herein, as a polypeptide having an amino acid sequence selected from SEC. ID NO: 2, 3, 7 or 12 or having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selected from SEC. ID NO: 2, 3, 7, 12 or 13. An ActRIIa-binding activin 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 No.: 1 to 3 or a sequence of SEC. ID Nº: 2, lacking the 10 to 15 amino acids Cterminales (the "tail"). An ActRIIb-binding activin polypeptide for use in said preparation may be any of those disclosed herein, as a polypeptide having an amino acid sequence selected from SEC. ID No.: 16, 17, 20, or 21 or having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selected from SEC. ID Nº: 16, 17, 20 or

21. An activin-binding ActRIIb polypeptide may include a functional fragment of a natural ActRIIb polypeptide, such as one comprising at least 10, 20 or 30 amino acids of SEQ ID NO: 15 to 17 or a sequence lacking 10 to 15 C-terminal amino acids (the "tail") as SEC. ID Nº: 17.

A soluble Actin II binding polypeptide may include one or more alterations in the amino acid sequence (for example in the ligand binding domain) with respect to the naturally occurring ActRII polypeptide. Examples of ActRIIa and ActRIIb polypeptides altered are provided in WO 2006/012627, pp. 59-60 and pp. 55-58, respectively. For example, the alteration in the amino acid sequence can, for example, alter the glycosylation of the polypeptide when it occurs in a mammalian cell, insects or other eukaryotic cell, or alter the proteolytic cleavage of the polypeptide in relation to the ActRII polypeptide of origin natural.

An ActRII-binding activin polypeptide may be a fusion protein having, as a domain, an ActRII polypeptide, (eg, a ligand binding portion of an ActRIIa or ActRIIb) and one or more additional domains that provide a desirable feature, such as better pharmacokinetics, easier purification, which targets particular tissues, etc. For example, a domain of a fusion protein can improve one or more of the following: stability in vivo, half-life in vivo, absorption / administration, tissue localization or distribution, formation of protein complexes, multimerization of fusion protein and / or purification. An activin-binding ActRII fusion protein may include an immunoglobulin Fc domain (wild type or mutant) or a serum albumin or other portion of polypeptide that provides desirable properties such as better pharmacokinetics, greater solubility or greater stability. An ActRII-Fc fusion may comprise a relatively unstructured linker placed between the Fc domain and the extracellular domain of ActRII. This unstructured connector may correspond to the unstructured region of approximately 15 amino acids at the C-terminal end of the ActRII extracellular domain (the "tail"), or it may be an artificial sequence of 1, 2, 3, 4 or 5 amino acids or a length between 5 and 15, 20, 30, 50 or more amino acids that lack relatively secondary structure, or a mixture of both. A connector may be rich in glycine and proline residues and may, for example, contain a single threonine / serine sequence and glycine or repeated threonine / serine and glycine sequences (e.g., TG4, or singlets or repeats of SG4 ). A fusion protein can include a purification sequence, such as a marker epitope, a FLAG marker, a polyhistidine sequence and a GST fusion. Optionally, a soluble ActRII polypeptide includes one or more modified amino acid residues, selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid portion and a conjugated amino acid to an organic derivatizing agent. A pharmaceutical preparation may also include one or more additional compounds as a compound that is used to treat a bone disorder. Preferably, a pharmaceutical preparation is substantially non-pyrogenic. In general, it is preferable that an ActRII protein is expressed in a mammalian cell line that adequately mediates the natural glycosylation of the ActRII protein to decrease the likelihood of an unfavorable immune response in a patient. The human and CHO cell lines have been used successfully, and other common mammalian expression systems are expected to be useful.

As described herein, ActRIIa proteins called ActRIIa-Fc (a form with a minimum connector between the ActRIIa portion and the Fc portion) have desirable properties, including selective binding to activin against GDF8 and / or GDF11, large ligand binding affinity and longer serum half-life than two weeks in animal models. ActRII-Fc polypeptides and pharmaceutical preparations containing said polypeptides and a pharmaceutically acceptable excipient are disclosed herein.

The disclosure also provides nucleic acids encoding a soluble Actin II binding polypeptide, such as an ActRIIa or ActRIIb polypeptide. An isolated polynucleotide may consist of a coding sequence of a soluble Actin-binding activin polypeptide, as described above. For example, an isolated nucleic acid may include a coding sequence of an extracellular domain (e.g., a ligand binding domain) of an ActRII and a sequence that would encode part or all of the transmembrane domain and / or the cytoplasmic domain of a ActRII, but of a stop codon placed within the transmembrane domain or the cytoplasmic domain, or placed between the extracellular domain and the transmembrane or cytoplasmic domain. For example, an isolated polynucleotide may contain a complete ActRIIa polynucleotide sequence as SEC. ID Nº: 4 or 5

or a complete ActRIIb polynucleotide sequence as SEC. ID No. 18, or a partially truncated version of ActRIIa or ActRIIb, wherein said isolated polynucleotide further comprises a transcription termination codon of at least 600 nucleotides before the 3 'end or otherwise positioned so that the translation of the polynucleotide gives place an extracellular domain optionally fused to a truncated portion of the entire ActRII. A nucleic acid sequence for ActRIIa is SEC. ID NO: 14. The nucleic acids disclosed herein are operatively linked to a promoter for expression, and the disclosure provides cells transformed with said recombinant polynucleotides. Preferably the cell is a mammalian cell like a CHO cell.

The disclosure also provides methods for preparing a soluble Actin II binding polypeptide. Said method may include the expression of any of the nucleic acids (eg, SEQ ID NO: 4, 5, 14, 18 or 19) disclosed herein in a suitable cell, such as the ovary cell line of Chinese hamster (CHO). Said method may comprise: a) culturing a cell under conditions suitable for the expression of the soluble ActRII polypeptide, wherein said cell is transformed with a soluble ActRII expression construct; and b) recovering the soluble ActRII polypeptide so expressed. Soluble ActRII polypeptides can be recovered as crude, partially purified or highly purified fractions. Purification can be carried out 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 (eg, Q sepharose), hydrophobic interaction chromatography (eg, phenylsepharose), size exclusion chromatography and cation exchange chromatography.

An activin-ActRII antagonist disclosed herein, such as a soluble Actin-binding polypeptide ActRIIa, or a soluble Actin-activating-binding polypeptide ActRIIb, can be used in a method to promote red blood cell production or increase blood levels. red blood cells in a subject. Methods for treating a disorder associated with low red blood cell counts or low hemoglobin levels (eg anemia), or to promote red blood cell production, are disclosed in patients who

they need One method may comprise administration to a subject in need of an effective amount of an activin-ActRII antagonist. In certain aspects, the disclosure provides uses of activin-ActRII antagonists to prepare a medicament for the treatment of a disorder or condition such as those described herein.

Also, the disclosure provides a method to identify a drug that stimulates the production of red blood cells. The method comprises: a) identifying a test drug that binds to activin or a ligand binding domain of an ActRII polypeptide; and b) evaluate the effect of the drug on the levels of red blood cells, hemoglobin and / or precursors of red blood cells (eg reticulocyte levels).

Brief description of the figures

Figure 1 shows the purification of ActRIIa-hFc expressed in CHO cells. The protein is purified as a single, well-defined peak as visualized by size classification column (left panel) and SDS-PAGE with Coomassie staining (right panel) (left lane: molecular weight standards, right lane: ActRIIa -hFc).

Figure 2 shows the binding of ActRIIa-hFc to activin and GDF-11, measured by the BiaCore ™ assay.

Figure 3 shows the effects of ActRIIa-hFc on the red blood cell count in female nonhuman primates. Female Filipino 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 Figure 3A shows red blood cell counts (RGR). Figure 3B shows hemoglobin levels. Statistical significance is in relation to the baseline (values at the beginning) for each treatment group. On day 57, two monkeys remained in each group.

Figure 4 shows the effects of ActRIIa-hFc on the red blood cell count in male nonhuman primates. Male Filipino 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 Figure 4A shows red blood cell counts (RGR). Figure 4B shows hemoglobin levels. Statistical significance is in relation to the baseline for each treatment group. On day 57, two monkeys remained in each group.

Figure 5 shows the effects of ActRIIa-hFc on the reticulocyte count in female nonhuman primates. Filipino 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. Figure 5A shows the absolute reticulocyte counts.

Figure 5B shows the percentage of reticulocytes in relation to red blood cell counts. The statistical significance is with respect to the baseline for each group. On day 57, two monkeys remained in each group.

Figure 6 shows the effects of ActRIIa-hFc on the reticulocyte count in female nonhuman primates. Filipino 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. Figure 6A shows the absolute reticulocyte counts. Figure 6B shows the percentage of reticulocytes in relation to red blood cell counts. The statistical significance is with respect to the baseline for each group. On day 57, two monkeys remained in each group.

Figure 7 shows the results of the human clinical trial described in Example 5, where the area under the curve (AUC) and the administered dose of ActRIIa-hFc have a linear correlation, regardless of whether ActRIIahFc was administered intravenously (IV ) or subcutaneous (SC).

Figure 8 shows a comparison of serum ActRIIa-hFc levels in patients given IV or SC.

Figure 9 shows the levels of bone alkaline phosphatase (BAP) in response to different dose levels of ActRIIa-hFc. BAP is a marker for anabolic bone growth.

Figure 10 illustrates the mean change from baseline of hematocrit levels of the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dose.

Figure 11 illustrates the mean change from baseline of hemoglobin levels in the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dose.

Figure 12 illustrates the mean change from the baseline of the red blood cell (erythrocyte) count of the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dose.

Figure 13 illustrates the mean change from the baseline of the reticulocyte count of the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dose.

Detailed description of the invention

1. Overview

The transforming growth factor beta superfamily (TGF-beta) contains various growth factors that share common sequence elements and structural motifs. It is known that these proteins exert biological effects on a wide variety of cell types in both vertebrates and invertebrates. Superfamily members perform important functions during embryonic development in pattern formation and tissue specification and can influence various differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis and differentiation of epithelial cells. The family is divided into two general branches: the BMP / GDF and TGF-beta / Activina / BMP10 branches, whose members have diverse, often complementary effects. By manipulating the activity of a member of the TGF-beta family, it is often possible to cause significant physiological changes in an organism. For example, Piedmontese and Belgian blue cattle breeds have a loss of function mutation in the GDF8 gene (also called myostatin) that causes a marked increase in muscle mass. Grobet et al., Nat Genet. 1997, 17 (1): 71-4. In addition, in humans, inactive GDF8 alleles are associated with greater muscle mass and, it is said, an exceptional strength. Schuelke et al., N Engl J Med 2004, 350: 2682-8.

Activins are growth factors, dimeric polypeptides, which belong to the TGF-beta superfamily. There are three main forms of activin (A, B and AB) that are homo / heterodimers of two closely related β subunits (βAβA, βBβB and βAβB, respectively). The human genome also encodes an activin C and an activin E, which are expressed primarily in the liver, and heterodimeric forms containing βC or βE are also known. In the TGF-beta superfamily, activins are unique and multifunctional factors that can stimulate hormonal production in ovarian and placental cells, help the survival of neuronal cells, positively or negatively influence the progress of the cell cycle depending on the type of cell and induce mesodermal differentiation at least in amphibian embryos (DePaolo et al., 1991, Proc Soc Ep Biol Med. 198: 500-512; Dyson et al., 1997, Curr Biol. 7: 81-84; Woodruff, 1998, Biochem Pharmacol. 55: 953-963). In addition, the erythroid differentiation factor (EDF) isolated from human stimulated 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 gland, activin promotes the secretion and synthesis of FSH, while inhibin prevents secretion and synthesis of FSH. Other proteins that can regulate the bioactivity of activin and / or bind to activin include folistatin (FS), folistatin-related protein (FSRP) and α2-macroglobulin.

TGF-β signals are mediated by heteromeric serine / threonine kinase receptor type I and type II complexes, which phosphorylate and activate down Smad proteins sequentially after ligand stimulation (Massagué, 2000, Nat. Rev. Mol Cell Biol. 1: 169-178). These type I and type II receptors are transmembrane proteins, composed of an extracellular domain of ligand binding with a region rich in cysteine, a transmembrane domain and a cytoplasmic domain with predicted serine / threonine specificity. Type I receptors are essential for signaling; and type II receptors are necessary to bind ligands and for the expression of type I receptors. Type I and II activin receptors form a stable complex after ligand binding, which results in phosphorylation of type I receptors. by type II receptors.

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

As demonstrated herein, a soluble ActRIIa polypeptide (sActRIIa), which shows a substantial preference for binding to activin A as opposed to other members of the TGF-beta family, such as GDF8 or GDF11, is effective in increasing levels of red blood cells in vivo. Without wishing to adhere to any particular mechanism, the effect of sActRIIa is expected to be caused primarily by an antagonistic effect of activin, given the very strong binding to activin (picomolar dissociation constant) exhibited by the particular sActRIIa construct used in those studies. . Regardless of the mechanism, it is clear from this disclosure that ActRIIa-activin antagonists increase red blood cell levels in rodents, monkeys and humans. It should be noted that hematopoiesis is a complex process, regulated by various factors, including erythropoietin homeostasis, G-CSF and iron. The terms "increases red blood cell levels" and "promotes red blood cell formation" refer to clinically observable measurements, such as hematocrit, red blood cell count and hemoglobin measurements and are intended to be neutral in terms of the mechanism by which these changes occur.

As also demonstrated herein, a soluble ActRIIb polypeptide (sActRIIb) is effective in increasing the level of reticulocytes in vivo, an effect that is expected to cause an increase in hemotocrit levels over a longer period.

The data reported herein with respect to non-human primates are also reproducible in mice, rats and humans, and therefore, ActRII polypeptides and other antagonists of

Activin-ActRII to promote the production of red blood cells and increase the levels of red blood cells in mammals, from rodents to humans. Activin-ActRII antagonists include, for example, soluble ActRIIa binding polypeptides, soluble ActRIIb polypeptides binding to active activin, antibodies that bind to activin (particularly to activin A or B subunits, also referred to as βA or βB) and destabilize the binding of ActRIIa and / or ActRIIb, antibodies that bind ActRIIa and destabilize the binding of activin, antibodies that bind ActRIIb and destabilize the binding of activin, non-antibody proteins selected for the binding of activin, ActRIIb or ActRIIa (see for example, WO / 2002/088171, WO / 2006/055689 and WO / 2002/032925 for examples of such proteins and methods for designing and selecting them), random peptides selected for the binding of activin, ActRIIb or ActRIIa, often set to an Fc domain. Two different proteins (or other portions) with activin binding activity, ActRIIb or ActRIIa, especially activin binders that block the binding sites of the type I receptor (eg, a soluble type I receptor) and type II (for example, a soluble type II activin receptor), respectively, can be linked together to create a bifunctional binding molecule. Nucleic acid aptamers, small molecules and other agents that inhibit the axis of activin-ActRII signaling are included as activin-ActRII antagonists. Various proteins have activin-ActRII antagonist activity, including inhibin (i.e., the alpha subunit of inhibin), although inhibin does not universally antagonize activin in all tissues, folistatin (e.g., folistatin-288 and folistatin315 ), FSRP, activin C, alpha (2) -macroglobulin and a mutant of activin A M108A (change from methionine to alanine at position 108). In general, alternative forms of activin, particularly those with alterations in the binding domain of the type I receptor can bind to type II receptors and cannot form an active ternary complex, thus acting as antagonists. In addition, nucleic acids, such as antisense molecules, siRNA or ribozymes that inhibit activin A, B, C or E, or, particularly, the expression of ActRIIa or ActRIIb, can be used as antagonists of activin-ActRII. The activin-ActRII antagonist for use may have selectivity for inhibition of activin-mediated signaling against other members of the TGF-beta family, and particularly with respect to GDF8 and GDF11.

The terms used in this specification generally have the current meanings in the area, in the context of this invention and in the specific context in which each term is used. Some terms are discussed below or elsewhere in the specification, to provide additional guidance to those who practice it when describing the compositions and methods of the invention and how to prepare and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used.

"Around" and "approximately" will generally mean an acceptable degree of error for the amount measured given the nature or accuracy of the measurements. Typically, degrees of example error are usually within 20 percent (%), preferably within 10% and more preferably within 5% of a given value or range of values.

Alternatively and, in particular in biological systems, the terms "around" and "approximately" may mean values that are within an order of magnitude, preferably in an environment of 5 times and more preferably in an environment of 2 times a given value. The numerical quantities presented in this document are approximate unless otherwise indicated, which means that the term "around" or "approximately" can be inferred when it has not been expressly indicated.

The sequences can be compared with each other, including the natural sequence and one or more mutants (sequence variants). Such comparisons usually comprise polymer sequence alignments, for example, using sequence alignment programs and / or algorithms that are well known in the area (such as BLAST, FASTA and MEGALIGN, to name a few). The expert can easily appreciate that, in such alignments, when a mutation contains an insertion or deletion of a residue, sequence alignment will introduce a "gap" (normally represented by a hyphen, or "A") into the sequence. of the polymer that does not contain the residue inserted or removed.

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

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

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

2. ActRII polypeptides

ActRII polypeptides are disclosed herein. As used herein, the term "ActRII" refers to the family of type II activin receptors. This family includes the type IIa activin receptor and the type IIb activin receptor.

ActRIIa polypeptides are disclosed herein. As used herein, the term "ActRIIa" is

5 refers to proteins of a family of type IIa (ActRIIa) receptors of any species, and variants derived from said ActRIIa proteins by mutagenesis or other modification. The reference to ActRIIa in this document is understood as a reference to any of the forms currently identified. The members of the ActRIIa family are generally transmembrane proteins, composed of an extracellular ligand binding domain with a cysteine-rich region, a transmembrane domain and a domain.

10 cytoplasmic with predicted serine / threonine kinase activity.

The term "ActRIIa polypeptide" includes polypeptides containing any naturally occurring polypeptide of a member of the ActRIIa family, as well as any of its variants (including mutants, fragments, fusions and peptidomimetic forms) that retain useful activity. See for example WO / 2006/012627. For example, ActRIIa polypeptides include polypeptides derived from the sequence of any known ActRIIa that has a sequence at least 80% identical to the sequence of an ActRIIa polypeptide and optionally at least 85%, 90%, 95%, 97%, 99% or more identity. For example, an ActRIIa polypeptide of the invention can bind to, and can inhibit the function of, an ActRIIa protein and / or activin. An ActRIIa polypeptide can be selected to have red cell formation promotion activity in vivo. Examples of ActRIIa polypeptides include the human ActRIIa precursor polypeptide (SEQ ID NO: 1) and human soluble ActRIIa polypeptides (e.g.,

20 SEQ ID NO: 2, 3, 7 and 12).

The sequence of the human ActRIIa precursor protein is as follows:

The signal peptide is underlined simple; the extracellular domain is in bold and the possible glycosylation sites linked to N are double underlined.

The C-terminal "tail" end of the extracellular domain is underlined. The sequence with the "tail" removed (a ∆15 sequence) is as follows:

The nucleic acid sequence encoding the human ActRIIa precursor protein is as follows (nucleotides 164-1705 of Genbank entry NM_001616):

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

ActRIIb polypeptides are disclosed herein. As used herein, the term "ActRIIb" refers to a family of proteins that are type IIb (ActRIIb) receptors of any species and to variants derived from said ActRIIb proteins by mutagenesis or other modification. The reference to ActRIIb in this document is understood as a reference to any of the forms currently identified. The

5 members of the ActRIIb family are generally transmembrane proteins, composed of an extracellular domain of ligand binding with a region rich in cysteine, a transmembrane domain and a cytoplasmic domain with expected serine / threonine kinase activity.

The term "ActRIIb polypeptide" includes polypeptides containing any naturally occurring polypeptide of a member of the ActRIIb family, as well as its variants (including mutants, fragments, fusions, and peptidomimetic forms) that retain useful activity. See for example WO / 2006/012627. For example, ActRIIa polypeptides are polypeptides derived from the sequence of any known ActRIIa having a sequence at least 80% identical to the sequence of an ActRIIa polypeptide and optionally at least 85%, 90%, 95%, 97%, 99% or more of identity. For example, an ActRIIb polypeptide of the invention can bind to, and can inhibit the function of, an ActRIIb protein and / or activin. An ActRIIb polypeptide can be selected to have

15 activity promoting the formation of red blood cells in vivo. Examples of ActRIIb polypeptides include the human ActRIIb precursor polypeptide (SEQ ID NO: 15) and human soluble ActRIIb polypeptides (eg, SEQ ID NO: 16, 17, 20 and 21).

The sequence of the human ActRIIb precursor protein is as follows:

The signal peptide is simple underlined; the extracellular domain is in bold and the possible glycosylation sites linked to N are in squares.

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

The C-terminal "tail" of the extracellular domain is weakened. The sequence with the "tail" removed (a sequence 25 ∆15) is as follows:

The nucleic acid sequence encoding a human ActRIIb precursor protein is as follows (nucleotides 5-1543 of Genbank entry NM_001106)

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

As described herein, the term "soluble ActRII polypeptide" generally refers to polypeptides that contain an extracellular domain of an ActRIIa or ActRIIb protein. The term "soluble ActRII polypeptide" as used herein, includes any naturally occurring extracellular domain of an ActRIIa or ActRIIb protein, as well as any of its variants (including mutants, fragments and peptidomimetic forms). An Actin-binding activin polypeptide is one that retains the ability to bind to activin, including, for example, AA, AB, BB, or forms that include the C or E subunit. Optionally, an Actin-binding Actin II polypeptide is will bind AA activin with a dissociation constant of 1 nM or less. The extracellular domain of an ActRII protein binds to activin and is generally soluble and therefore can be termed ActRII soluble polypeptide binding to activin. Examples of soluble Actin-binding polypeptides ActRIIa include the soluble polypeptides illustrated in SEQ ID NO: 2, 3, 7, 12 and 13. SEC. ID No.: 7 is known as ActRIIa-hFc and is described more deeply in the examples. Other examples of soluble Actin-binding activin polypeptides comprise a signal sequence in addition to the extracellular domain of an ActRIIa protein, for example, the honey honey melitin leader sequence (SEQ ID NO: 8), the tissue activating leader sequence of the plasminogen (TPA) (SEQ ID NO: 9) or the natural ActRIIa leader sequence (SEQ. ID NO: 10). The ActRIIa-hFc polypeptide illustrated in SEQ. ID No.: 13 uses a TPA leader sequence. Examples of soluble Actin-binding activin polypeptides include the soluble polypeptides illustrated in SEQ ID NO: 16, 17, 20. Actin-binding actin-binding polypeptides may also contain a signal sequence in addition to the extracellular domain of an ActRIIb protein, by example, the honey honey melitin leader sequence (SEQ ID NO: 8) or the tissue plasminogen activator leader sequence (TPA) (SEQ ID NO: 9).

Functionally active fragments of ActRII polypeptides can be obtained by screening for recombinantly produced polypeptides from the corresponding nucleic acid fragment encoding an ActRII polypeptide. In addition, the fragments can be chemically synthesized using techniques known in the area such as Merrifield solid phase technique or conventional 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 act as antagonists (inhibitors) of the ActRII protein or activin-mediated signaling.

Functionally active variants of ActRII polypeptides can be obtained by screening collections of modified polypeptides recombinantly produced from the corresponding mutated nucleic acids encoding all ActRII polypeptides. Variants can be produced and tested to identify those that can act as antagonists (inhibitors) of the ActRII protein or activin-mediated signaling. A functional variant of the ActRIIa polypeptides may contain an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NO: 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 NO: 2 or 3. A functional variant of ActRIIb polypeptides may contain a sequence of amino acids that are at least 75% identical to an amino acid sequence selected from SEQ ID NO: 16 or 17. 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 NO: 17 or 18.

Functional variants can be generated by modifying the structure of an ActRII polypeptide for purposes such as increasing therapeutic efficacy, or stability (eg, ex vivo lifespan and resistance to proteolytic degradation in vivo). Such modified ActRII polypeptides when selected to retain activin binding, are considered functional equivalents of natural ActRII polypeptides. Modified ActRII polypeptides can also be produced, for example, by substitution, deletion or addition of amino acids. For example, it is reasonable to expect an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (for example, conservative mutations) It will not have an important effect on the biological activity of the resulting molecule. Conservative replacements are those that take place in a family of amino acids that are related in their side chains. If a change in the amino acid sequence of an ActRII polypeptide results in a functional homologue, it can be readily determined by evaluating the ability of the variant ActRII polypeptide to produce a response in the cells in a manner similar to the natural ActRII polypeptide.

Specific mutations of ActRII polypeptides can also be prepared so as to alter glycosylation of the polypeptide. Such mutations can be selected in order to introduce or eliminate one or more glycosylation sites, such as glycosylation sites linked to I or N. The glycosylation recognition sites linked to asparagine generally comprise a sequence of three peptides, asparagine-X- Threonine or asparagine-X-serine (where "X" is any amino acid) that is specifically recognized by suitable cellular glycosylation enzymes. The alteration can also be made by adding, or substituting, one or more serine or threonine residues in the sequence of the natural ActRII polypeptide (for O-linked glycosylation sites). Various amino acid substitutions or deletions at one or both positions of the first or third amino acid of a glycosylation recognition site (and / or amino acid deletion in the second position) results in noglycosylation in the modified tripeptide sequence. Another means of increasing the number of carbohydrate portions in an ActRII polypeptide is by chemical or enzymatic coupling of glycosides to the ActRII polypeptide. Depending on the coupling mode used, the sugar (s) may bind to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as cysteine; (d) free hydroxyl groups such as serine, threonine or hydroxyproline; (e) aromatic residues such as phenylalanine, tyrosine or tryptophan; or (f) the glutamine amide group. The removal of one or more carbohydrate portions present in an ActRII polypeptide can be done chemically and / or enzymatically. Chemical deglycosylation may involve, for example, exposure of the ActRII polypeptide to the trifluoromethanesulfonic acid compound, or to an equivalent compound. This treatment results in the cleavage of most or all sugars except the binding sugar (Nacethylglucosamine or N-acetylgalactosamine), leaving the amino acid sequence intact. Enzymatic cleavage of carbohydrate portions in ActRII polypeptides can be achieved through the use of various endo- and exoglucosidases as described by Thotakura et al. (1987) Meth. Enzymol 138: 350 The sequence of an ActRII polypeptide can be adjusted, as appropriate, depending on the type of expression system used, since mammalian, yeast, insect and plant cells can all introduce different glycosylation patterns that may be affected by the sequence. of peptide amino acids. In general, ActRII proteins for use in humans can be expressed in a mammalian cell line that provides adequate glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are also expected to be useful.

This disclosure further contemplates a method for generating mutants, especially sets of combinatorial mutants of an ActRII polypeptide, as well as truncation mutants; Combinatorial mutant mixtures are especially useful for identifying sequences of functional variants. The purpose of screening said combinatorial collections may be, for example, to generate variants of the ActRII polypeptide that bind to activin or other ligands. Various screening tests are provided below, and such tests can be used to evaluate the variants. For example, a variant of the ActRII polypeptide can be screened to determine if it has the ability to bind an ActRII ligand, to prevent binding of an ActRII ligand to an ActRII polypeptide or to interfere with signaling caused by an ActRII ligand.

The activity of an ActRII polypeptide or its variants can also be tested in an in vivo or cell-based assay. For example, the effect of a variant of the ActRII polypeptide on the expression of genes involved in hematopoiesis can be evaluated. This can be performed, as necessary, in the presence of one or more recombinant ActRII ligand proteins (eg, activin), and the cells can be transinfected to produce an ActRII polypeptide and / or its variants, and optionally an ActRII ligand. . Also, an ActRII polypeptide can be administered to a mouse or other animal, and one or more blood determinations, such as red blood cell count, hemoglobin or reticulocyte count can be evaluated.

Combinatorially derived variants can be generated that have a selective or generally greater potency relative to a natural ActRII polypeptide. Likewise, mutagenesis can give rise to variants that have markedly different intracellular half-lives from that corresponding to a natural ActRII polypeptide. For example, the altered protein may become more stable or less stable to proteolytic degradation or other cellular processes that result in the destruction, or otherwise inactivation, of a natural ActRII polypeptide. Such variants and the genes that encode them, can be used to alter levels of ActRII polypeptide by modulating the half-life of ActRII polypeptides. For example, a short half-life may result in more transient biological effects and, when part of an inducible expression system, may allow more stringent control of the levels of recombinant ActRII polypeptide within the cell. In an Fc fusion protein, mutations can be made in the connector (if any) and / or in the Fc portion to alter the protein's half-life.

A combinatorial library can be produced by a degenerate library of genes encoding a collection of polypeptides which include at least a portion of the possible ActRII polypeptide sequences. For example, a mixture of synthetic oligonucleotides can be enzymatically linked in gene sequences such that the degenerate set of possible nucleotide sequences of the ActRII polypeptide can be expressed as individual polypeptides, or alternatively, as a set of larger fusion proteins (by example, for phage expression).

There are many ways in which the collection of potential homologues can be generated from a sequence of degenerate oligonucleotides. The chemical synthesis of a sequence of degenerate genes can be carried out in an automatic DNA synthesizer and the synthetic genes can then be linked to a suitable vector for expression. The synthesis of degenerate oligonucleotides is well known in the area (see

for example, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al., (1983) Nucleic Acid Res. 11: 477). Such techniques were employed in the direct evolution of other proteins (see, for example, Scott et al., (1990) Science 249: 386-390; Roberts et al., (1992) PNAS USA 89: 2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as United States patents No.: 5,223,409, 5,198,346 and 5,096,815).

Alternatively, other forms of mutagenesis can be used to generate a combinatorial collection. For example, variants of the ActRII polypeptide from a collection can be generated and isolated 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. 268: 2888-2892; Lowman et al., (1991) Biochemistry 30: 10832-10838; and Cunningham et al., (1989) Science 244: 1081- 1085), connector scan mutagenesis (Gustin et al., (1993) Virology 193: 653-660; Brown et al., (1992) Mol. Cell Biol. 12: 2644-2652; McKnight et al., (1982 ) Science 232: 316); saturation mutagenesis (Meyers et al., (1986) Science 232: 613); PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol 1: 11-19); random mutagenesis, which includes chemical mutagenesis, etc. (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in Mol Biol 7: 32-34). Connector scan mutagenesis, particularly in a combinatorial framework, is an attractive method to identify truncated (bioactive) forms of ActRII polypeptides.

A wide range of techniques are known in the area for the screening of gene products from combinatorial collections made by point mutations and truncation and, in this regard, to screen cDNA libraries in search of gene products that have a specific property. Such techniques will generally be adaptable for rapid screening of libraries generated by combinatorial mutagenesis of ActRII polypeptides. The most widely used techniques for screening large libraries typically comprise cloning the library into replicable expression vectors, transforming suitable cells with the resulting vector collection and expressing the combinatorial genes under conditions in which the detection of a desired activity makes relatively easy. the isolation of the vector encoding the gene whose product was detected. Preferred assays include activin binding assays and activin mediated cell signaling assays.

ActRII polypeptides may also contain additional post-translational modifications to all those naturally present in ActRII polypeptides. Such modifications include, but not exclusively, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. As a result, the modified ActRII polypeptides may contain non-amino acid elements, such as polyethylene glycols, lipids, poly or mono saccharides and phosphates. The effects of said non-amino acid elements on the functionality of an ActRII polypeptide can be analyzed as described herein for other variants of the ActRII polypeptide. When an ActRII polypeptide is produced in the cells by cleavage of an incipient form of the ActRII polypeptide, post-translational processing may also be important for the correct folding and / or function of the protein. Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have a specific cellular machinery and characteristic mechanisms for such post-translational activities and can be chosen in order to guarantee the correct modification and processing of the polypeptides ActRII.

Functional variants or modified forms of ActRII polypeptides include fusion proteins that have at least a portion of ActRII polypeptides and one or more fusion domains. Well-known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, a constant heavy chain region of immunoglobulin (Fc), binding protein to maltose (MBP) or human serum albumin. A fusion domain can be selected in order to confer a desired property on it. For example, some fusion domains are particularly useful for the isolation of fusion proteins by affinity chromatography. For the purpose of affinity purification, matrices applicable to affinity chromatography are used, such as glutathione, amylase and nickel or cobalt conjugated resins. Many of these matrices are available in kit form, such as the Pharmacia GST purification system and the QIAexpress ™ (Qiagen) system useful with fusion partners (HIS6). As another example, a fusion domain can be selected in order to facilitate the detection of ActRII polypeptides. Examples of such detection domains include various fluorescent proteins (eg, GFP) as well as "marker epitopes" which are generally short peptide sequences for which a specific antibody is available. Well-known marker epitopes for which there are readily available specific monoclonal antibodies include FLAG markers, influenza virus hemagglutinin (HA) and c-myc. In some cases, the fusion domains have a protease cleavage site, as for factor Xa or thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby release the recombinant proteins therefrom. Then, the released proteins can be isolated from the fusion domain by subsequent chromatographic separation. In some preferred embodiments, an ActRII polypeptide is fused to a domain that stabilizes the ActRII polypeptide in vivo (a "stabilizer" domain). By "stabilizing" is meant anything that increases the serum half-life, regardless of whether it is due to less destruction, less clearance by the kidney or other pharmacokinetic effect. It is known that fusions with the Fc portion of an immunoglobulin confer desirable pharmacokinetic properties to a wide range of proteins. Also, human serum albumin fusions can confer desirable properties. Other types of

Fusion domains that can be selected include multimerization domains (eg dimerization, tetramerization) and functional domains (which confer an additional biological function, such as additional stimulation of muscle growth).

As a specific example, a fusion protein containing an extracellular domain of soluble ActRIIa fused to an Fc domain (for example SEQ ID NO: 6) is disclosed herein.

As a further specific example, a fusion protein containing a soluble extracellular domain of ActRIIb fused to an Fc domain (eg, SEQ ID NO: 21) is disclosed herein.

Optionally, the Fc domain has one or more mutations in residues such as Asp-265, lysine 322 and Asn-434. In certain cases, the mutant Fc domain that has one or more of those mutations (for example, Asp-265 mutation) has a lower ability to bind to Fcγ receptors compared to a natural Fc domain. In other cases, the mutant Fc domain that has one or more of these mutations (for example, the Asn-434 mutation) has a greater ability to bind to the Fc receptor related to a MHC class I (FcRN) compared to a domain Natural fc.

It is understood that the different elements of fusion proteins can be organized in any way that is compatible with the desired functionality. For example, an ActRII polypeptide may be placed C-terminal in relation to a heterologous domain, or, alternatively, a heterologous domain may be placed C-terminal in relation to an ActRII polypeptide. The ActRII polypeptide domain and the heterologous domain need not be adjacent to a fusion protein, and there may be other C- or N-terminal amino acid domain or sequences in relation to each domain or between the domains.

ActRII polypeptides may contain one or more modifications capable of stabilizing ActRII polypeptides. For example, such modifications enhance the in vitro half-life of ActRII polypeptides, increase the circulatory half-life of ActRII polypeptides or reduce proteolytic degradation of ActRII polypeptides. Such stabilizing modifications include, but not exclusively, fusion proteins (among others, for example, fusion proteins containing an ActRII polypeptide and a stabilizing domain), modifications of a glycosylation site (including, for example, the addition of a glycosylation site to an ActRII polypeptide) and modifications of the carbohydrate portion (such as, for example, the removal of carbohydrate portions of an ActRII polypeptide). As used herein, the term "stabilizing domain" not only refers to a fusion domain (eg, Fc) as in the case of fusion proteins, but also includes non-protein modifications such as a carbohydrate portion, or non-protein portion, such as polyethylene glycol.

In this document, isolated or purified forms of ActRII polypeptides, which are isolated, or otherwise substantially free, of other proteins are disclosed. ActRII polypeptides will generally be produced by the expression of recombinant nucleic acids.

3. Nucleic acids encoding ActRII polypeptides

Isolated and / or recombinant nucleic acids encoding any of the ActRII polypeptides (eg, soluble ActRIIa polypeptides and soluble ActRIIb polypeptides) which include fragments, functional variants and fusion proteins disclosed herein are disclosed herein. For example, SEC. ID NO: 4 encodes the precursor polypeptide of natural human ActRIIa, while SEC. ID No.: 5 encodes the processed extracellular domain of ActRIIa. For example, SEC. ID NO: 18 encodes the natural human ActRIIb precursor polypeptide, while SEC. ID No.: 19 encodes the processed extracellular domain of ActRIIb. The subject nucleic acids can be single stranded or double stranded. Said nucleic acids may be DNA or RNA molecules.

These nucleic acids can be used, for example, in methods for preparing ActRII polypeptides or as direct therapeutic agents (for example, in genotherapy).

Nucleic acids encoding ActRIIa polypeptides may include nucleic acids that are variants of SEC. ID NO: 4 or 5 and the nucleic acids encoding ActRIIb polypeptides may include nucleic acids that are variants of SEC. ID No.: 18 or 19. Variant nucleotide sequences include sequences that differ in one

or more substitutions, additions or deletions of nucleotides, as allelic variants.

This document discloses isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4, 5 , 18 or 19. An expert will understand that complementary nucleic acid sequences of SEC can be provided. ID No. 4, 5, 18 or 19, and variants of SEC. ID No. 4, 5, 18 or 19. The nucleic acid sequence can be isolated, recombined and / or fused with a heterologous nucleic sequence, or in a library.

Nucleic acids disclosed herein may also include nucleotide sequences that hybridize under very stringent conditions to the nucleotide sequence designated in SEC. ID Nº: 4, 5, 18 or 19, the complementary sequence of SEC. ID Nº: 4, 5, 18 or 19, or its fragments. As discussed above, an expert will readily understand that suitable rigorous conditions that promote DNA hybridization can be varied. An expert will easily understand that suitable stringent conditions that promote DNA hybridization can be varied. For example, hybridization could be performed in 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by a washing of 2.0 x SSC at 50 ° C. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0 x SSC at 50 ° C to a high stringency of about 0.2 x SSC at 50 ° C. In addition, the temperature of the washing step can be increased from low stringency conditions at room temperature, around 22 ° C, to high stringency conditions at around 65 ° C. The temperature and salt concentration can be varied, or the temperature or salt concentration can be kept constant while changing the other variable. In one embodiment, the invention provides nucleic acids that hybridize under conditions of low stringency of 6 x SSC at room temperature followed by a 2 x SSC wash at room temperature.

Isolated nucleic acids that differ from the nucleic acids indicated in SEQ ID No. 4, 5, 18 or 19 can also be provided due to degeneracy in the genetic code. For example, a number of amino acids is designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) can cause "silent" mutations that do not affect the amino acid sequence of the protein. However, it is expected that the DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteins will exist in mammalian cells. An expert will understand that such variations in one or more nucleotides (up to about 3-5% of the nucleotides) of nucleic acids encoding a particular protein may exist between individuals of a given species due to natural allelic variation.

Recombinant nucleic acids can be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequences will generally be suitable for the host cell used for expression. Numerous types of appropriate expression vectors and regulatory sequences suitable for various host cells are known in the area. Typically, said one or more regulatory nucleotide sequences may include, but not exclusively, promoter sequences, leader or signal sequences, ribosomal binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer sequences. or activators. The constitutive or inducible promoters known in the area are considered by the invention. The promoters may be naturally occurring promoters or hybrid promoters that combine elements of more than one promoter. There may be an expression builder present in a cell in an episome, such as a plasmid, or the expression construct can be inserted into a chromosome. The expression vector may contain a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the area and will vary according to the host cell used.

The subject nucleic acid can be provided in an expression vector that contains a nucleotide sequence encoding an ActRII polypeptide and that is operably linked to at least one regulatory sequence. Regulatory sequences are known in the area and are selected to direct the expression of an ActRII polypeptide. Consequently, the expression regulatory sequence includes promoters, enhancers and other expression control elements. Examples of regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any sequence, of a wide variety of expression control sequences, that controls the expression of a DNA sequence when operably linked to it in those vectors, can be used to express DNA sequences encoding an ActRII polypeptide . Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the immediate early promoter of adenovirus or cytomegalovirus, RSV promoters, the lac system, the trp system, the TAC system or TRC, the T7 promoter whose expression is directed by the T7 RNA polymerase, the main promoter and operator regions of the lambda phage, the control regions of the fd envelope protein, the 3-phosphoglycerate kinase promoter or other enzymes glycolytics, promoters of acid phosphatase, for example, Pho5, promoters of the factors of

α mating of yeast, the polyhedron promoter of the baculovirus system and other sequences that are known to control gene expression of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed and / or the type of protein to be expressed. On the other hand, the number of copies of the vector, the ability to control that number of copies and the expression of any other protein encoded by the vector, as antibiotic markers, should also be considered.

A recombinant nucleic acid can be produced by linking the cloned gene, or a portion thereof, in a vector suitable for expression in prokaryotic cells or eukaryotic cells (yeast, birds, insects or mammals) or both. Expression vehicles for the production of a recombinant ActRII polypeptide include plasmids and other vectors. For example, suitable vectors include plasmids of the types: plasmids derived from pBR322, plasmids derived from pEMBL, plasmids derived from pEX, plasmids derived from pBTac and plasmids derived from pUC for expression in prokaryotic cells, such as E. coli.

Some mammalian expression vectors contain 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 vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with bacterial plasmid sequences, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, virus derivatives such as bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, derived from pREP and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral expression systems (including retroviral) can be found later in the description of genotherapy delivery systems. The various methods used in the preparation of plasmids and in the transformation of host organisms are well known in the area. For other expression systems suitable for prokaryotic and eukaryotic cells, as well as for general recombination procedures, see Molecular Cloning A Laboratory Manual, 3rd Ed., Ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some cases, it may be convenient to express recombinant polypeptides by using a baculovirus expression system. Examples of such baculovirus expression systems include vectors derived from pVL (such as pVL1392, pVL1393 and pVL941), vectors derived from pAcUW (such as pAcUW1) and vectors derived from pBlueBac (such as β-gal containing pBlueBac III).

A vector can be designed for the production of ActRII polypeptides of the subject in CHO cells; as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wise). As will be apparent, the gene constructs of the subject can be used to cause the expression of ActRII polypeptides of the subject in cells propagated in culture, for example, to produce proteins, including fusion proteins or variant proteins, for purification.

This disclosure also applies to a host cell transinfected with a recombinant gene that includes a coding sequence (e.g., SEQ ID NO: 4, 5, 18 or 19) for one or more of the subject ActRII polypeptides. The host cell can be any prokaryotic or eukaryotic cell. For example, an ActRII polypeptide can be expressed in: bacterial cells, such as E. coli, insect cells (for example, using a baculovirus expression system), yeast, or mammalian cells. Experts know other suitable host cells.

Consequently, production methods of the subject ActRII polypeptides are also disclosed. For example, a host cell transinfected with an expression vector encoding an ActRIIa polypeptide or an ActRIIb polypeptide can be cultured under suitable conditions to allow expression of the ActRII polypeptide to occur. The ActRII polypeptide can be secreted and isolated from a mixture of cells and medium containing the ActRII polypeptide. Alternatively, the ActRII polypeptide can be retained cytoplasmically or in a fraction of the membrane and harvest and lyse the cells, and isolate the protein. A cell culture includes host cells, media and other byproducts. Suitable cell culture media are known in the area. The subject ActRII polypeptides can be isolated from cell culture medium, host cells or both, using techniques known in the area for protein purification, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis , immunoaffinity purification with antibodies specific for particular epitopes of ActRII polypeptides and affinity purification with an agent that binds to a domain fused to ActRII polypeptide (for example, a protein A column can be used to purify an ActRIIa-Fc fusion or ActRIIb-Fc). The ActRII polypeptide can be a fusion protein that contains a domain that facilitates its purification. Purification can be achieved by a series of column chromatography steps, such as, for example, three or more of the following, in any order: protein A chromatography, sepharose Q chromatography, phenylsepharose chromatography, size exclusion chromatography and cation exchange chromatography. Purification could be completed with viral filtration and exchange of buffer solution. As demonstrated herein, the ActRIIa-hFc protein was purified to a purity> 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 and nonhuman primates.

A fusion gene encoding a purification leader sequence, such as a poly (His) -nterokinase cleavage site sequence at the N-terminal end of the desired portion of the recombinant ActRII polypeptide, may allow purification of the fusion protein expressed by affinity chromatography using a Ni2 + metal resin. The leader purification sequence can then be removed by treatment with enterokinase to provide purified ActRII polypeptide (eg, see Hochuli et al., (1987) J. Chromatography 411: 177; and Janknecht et al., PNAS USA 88: 8972).

The techniques for preparing fusion genes are well known. Essentially, the binding of several DNA fragments encoding different polypeptide sequences is performed according to conventional techniques, employing blunt or irregular ends for ligation, digestion with restriction enzymes to provide suitable ends, filling of cohesive ends as appropriate, treatment with alkaline phosphatase to avoid undesirable binding and enzymatic ligation. The fusion gene can be synthesized by conventional techniques that include automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary protrusions between two consecutive gene fragments that can subsequently be paired to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

4. Alternative antagonists of activin and ActRII

The data presented in this document demonstrate that activin-ActRII signaling antagonists can be used to increase red blood cell or hemoglobin levels. Although soluble ActRIIa and ActRIIb polypeptides, and particularly ActRIIa-Fc and ActRIIb-Fc, can be used as antagonists, and although such antagonists can affect red blood cell levels through a mechanism different from that of activin antagonism (e.g. ., the inhibition of activin 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-ActRII antagonists are expected to be useful, including anti-activin antibodies (e.g., βA, βB, βC and βE activin), anti-ActRIIa antibodies, anti-ActRIIb antibodies, acids antisense, RNAi or ribozyme nuclei that inhibit the production of ActRIIa and / or ActRIIb, and other activin, ActRIIb or ActRIIa inhibitors, particularly those that destabilize the activin-ActRIIa binding and / or activin-ActRIIb.

An antibody that is specifically reactive with an ActRII polypeptide (e.g., a soluble ActRIIa or ActRIIb polypeptide) and that binds competitively to the ActRII polypeptide or otherwise inhibits ActRII-mediated signaling can be used as an antagonist of the ActRII polypeptide activities. Likewise, an antibody that is specifically reactive with a βA, βB, βC or βE activin polypeptide, or any of its heterodimers, and that destabilizes the binding to ActRIIa and / or ActRIIb can be used as an antagonist.

Using immunogens derived from an ActRIIa polypeptide, an ActRIIb polypeptide or an activin polypeptide, anti-protein / anti-peptide antisera or monoclonal antibodies can be prepared following standard protocols (see, for example, Antibodies: A Laboratory Manual ed. By Harlow and Lane (Cold Spring Harbor Press: 1988)). 1988)). A mammal, such as a mouse, a hamster or a rabbit, can be immunized with an immunogenic form of the activin polypeptide, ActRIIa or ActRIIb, an antigenic fragment that is capable of eliciting an antibody response, or a fusion protein. Techniques for conferring immunogeny on a protein or peptide include carrier conjugation or other techniques well known in the area. An immunogenic portion of an ActRII polypeptide or activin can be administered in the presence of adjuvant. The progress of immunization can be monitored by detecting antibody titers in plasma or serum. A standard ELISA or other immunoassays with the immunogen can be used as an antigen to assess antibody levels.

After immunization of an animal with an antigen preparation of an activin polypeptide, ActRIIa or ActRIIb, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be collected from an immunized animal and fused by standard methods of fusion of somatic cells with immortalizing cells such as myeloma cells to produce hybridoma cells. Such techniques are well known in the area and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al. ., (1983) Immunology Today, 4:72) and the hybridoma-EBV technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77- 96). The hybridoma cells can be screened immunochemically in relation to the production of antibodies specifically reactive with an activin, ActRIIa or ActRIIb polypeptide and isolate the monoclonal antibodies from a culture containing said hybridoma cells.

The term "antibody" as used herein is intended to include whole antibodies, eg, of any isotype (IgG, IgA, IgM, IgE, etc.), and includes immunoglobulin fragments or domains that are reactive with a chosen antigen. Antibodies can be fragmented using conventional techniques and screen fragments in relation to utility and / or interaction with a specific epitope of interest. Therefore, the term includes segments of proteolytically cleaved or recombinantly prepared portions of a molecule of

antibody, that are able to react selectively with a certain protein. Non-limiting examples of said 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 linked by a peptide linker. Those of scfv can covalently or non-covalently bind to form antibodies that have two or more binding sites. The term antibody also includes polyclonal, monoclonal antibodies or other purified preparations of antibodies and recombinant antibodies. The term "recombinant antibody" means an antibody or an antigen binding domain of an immunoglobulin, expressed from a nucleic acid that has been constructed using molecular biology techniques, such as a humanized antibody or a fully human antibody developed from of a single chain antibody. Single domain and single chain antibodies are also comprised by the expression "recombinant antibody."

In certain embodiments, an antibody of the invention to be used is a monoclonal antibody. A method of generating a monoclonal antibody that specifically binds an ActRIIa polypeptide, an ActRIIb polypeptide or an 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, obtain antibody-producing cells (e.g., spleen cells) from the mouse and fuse the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and analyze the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that specifically binds to the antigen. Once obtained, a hybridoma can be propagated in a cell culture, optionally under culture conditions in which the hybridoma derived cells produce the monoclonal antibody that specifically binds to the antigen. The monoclonal antibody can be purified from cell culture.

The term "specifically reactive with" as used herein in reference to an antibody means, as generally understood in the area, that the antibody is sufficiently selective among the antigen of interest (eg, an activin polypeptide, ActRIIa or ActRIIb) and other antigens that are not of interest so that the antibody is useful for at least detecting the presence of the antigen of interest in a particular type of biological sample. In certain methods that employ the antibody, as therapeutic applications, a higher degree of binding specificity may be desirable. Monoclonal antibodies generally have a greater tendency (compared to polyclonal antibodies) to effectively discriminate between desired antigens and cross-reaction polypeptides. A characteristic that influences the specificity of an antigen-antibody interaction is the affinity of the antibody for the antigen. Although the desired specificity can be achieved with a range of different affinities, the antibodies that are generally preferred will have an affinity (a dissociation constant) of about 10-6, 10-7, 10-8, 10-9 M or Minor.

In addition, 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 to bind an antigen in solution, it may be desirable to analyze the binding in solution. Several different techniques are available to analyze the interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISA, surface plasmon resonance binding assays (e.g., the BiacoreTM binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the IGEN International paramagnetic bead system, Inc ., Gaithersburg, Maryland), Western blots, immunoprecipitation and immunohistochemical assays.

Examples of categories of nucleic acid compounds that are activin or ActRII antagonists include antisense nucleic acids, RNAi constructs and catalytic nucleic acid constructs. A nucleic acid compound can be single stranded or double stranded. A double stranded compound may also include cantilever or non-complementarity regions, in which one or the other of the strands is single stranded. A single stranded compound may include regions of self-complementarity, which means that the compound forms a structure called "hairpin" or "hairpin loop", with a region of double helix structure. A nucleic acid compound may contain 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 complete nucleic acid sequence of ActRII or of the nucleic acid sequence of activin βA, βB, βC or βE. The complementarity region 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 non-coding sequence of the white transcript, as the portion of the coding sequence. Generally, a nucleic acid compound will have a length of about 8 to about 500 nucleotides or base pairs, and optionally the length will be about 14 to about 50 nucleotides. A nucleic acid can be a DNA (particularly for use as an antisense), RNA or an RNA: DNA hybrid. Any single chain can include a mixture of DNA and RNA, as well as modified forms that cannot be easily classified as DNA.

or RNA. Likewise, a double-stranded compound can be DNA: DNA, DNA: RNA or RNA: RNA, and any single strand can also include a mixture of DNA and RNA, as well as modified forms that cannot be easily classified as DNA or RNA. A nucleic acid compound may include one of several modifications, including one or modifications to the skeleton (the sugar-phosphate potion in a natural nucleic acid, including internucleotide bonds) or the base portion (the purine or pyrimidine portion of a natural nucleic acid ). An antisense nucleic acid compound will preferably have a length between about 15 and 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 compounds are likely to be administered, such as the stomach in the case of compounds administered orally and the lung for inhaled compounds. In the case of an RNAi construct, the strand complementary to the white transcript will generally be RNA or modifications thereof. The other strand can be RNA, DNA or any other variation. The duplex portion of the double-stranded or single-stranded "RNAi construct" will generally be 18 to 40 nucleotides in length and optionally approximately 21 to 23 nucleotides, provided it serves as a Dicer substrate. The catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also contain modified forms. Nucleic acid compounds can inhibit blank expression by approximately 50%, 75%, 90% or more when they come into contact with cells under physiological conditions and at a concentration in which a sense or antisense control has little or no effect. Preferred concentrations to test the effect of nucleic acid compounds are 1, 5 and 10 micromolar. Nucleic acid compounds can also be evaluated in relation to effects, for example, on red blood cell levels.

5. Screening tests

ActRII polypeptides (e.g., soluble ActRIIa or ActRIIb polypeptides) and activin polypeptides can be used to identify compounds (drugs) that are agonists or antagonists of the activin-ActRIIa and / or activin-ActRIIb signaling pathway. The compounds identified by this screening can be tested in order to evaluate their ability to modulate the levels of red blood cells, hemoglobin and / or reticulocytes in vivo or in vitro. These compounds can be tested, for example, in animal models.

There are numerous methods for screening therapeutic agents that increase red blood cell or hemoglobin levels by targeting activin signaling and ActRII signaling. High-performance screening of compounds can be carried out to identify drugs that alter activin-mediated effects.

or ActRII on a chosen cell line. In certain embodiments, the assay is carried out to screen and identify compounds that specifically inhibit or decrease the binding of an ActRIIa or ActRIIb polypeptide to activin. Alternatively, the assay can be used to identify compounds that increase the binding of an ActRIIa or ActRIIb polypeptide to activin. The compounds can be identified by their ability to interact with an activin, an ActRIIb polypeptide or an ActRIIa polypeptide.

A variety of test formats will be sufficient and, in the light of this disclosure, those that are not expressly described in this document will however be understood by a technician with experience in the subject. As described herein, the test compounds (drugs) of the invention can be created by any combinatorial chemistry method. Alternatively, the subject compounds may be natural biomolecules synthesized in vivo or in vitro. The compounds (drugs) to be tested for their ability to act as tissue growth modulators can be, for example, produced by bacteria, yeasts, plants or other organisms (e.g., natural products), chemically produced (by eg, small molecules, including peptidomimetics), or recombinantly produced. Test compounds include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones and nucleic acid molecules. Such a test agent is a small organic molecule with a molecular weight less than about 2000 Dalton.

Test compounds may be provided as discrete, individual entities, or provided in more complex collections, such as those made by combinatorial chemistry. Such collections may comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other kinds of organic compounds. The presentation of the test compound to the test system can be in isolation or as mixtures of compounds, especially in the initial screening steps. Optionally, the compounds can be derivatized with other compounds and have derivatization groups that facilitate the isolation of the compounds. Non-limiting examples of derivatization groups include biotin, fluorescein, digoxigenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S transferase (GST), photoactivatable crosslinkers or any combination thereof.

In many drug screening programs that analyze collections of natural compounds and extracts, high performance test assays are desirable to maximize the number of compounds studied over a given period of time. Assays performed on cell-free systems, such as those that can be obtained with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to allow rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. On the other hand, in general the effects of cellular toxicity or bioavailability of the test compound in the in vitro system can be ignored, instead the test focuses mainly on the effect of the drug on the molecular target since it can be manifested in an alteration of binding affinity between an ActRIIa polypeptide and activin or between an ActRIIb polypeptide and activin.

Just to illustrate, in an example screening test, the compound of interest is contacted with an isolated and purified ActRIIa polypeptide that is normally capable of binding to activin. A composition containing an ActRIIa ligand is then added to the mixture of the compound and the ActRIIa polypeptide. The detection and quantification of ActRIIa / activin complexes provides a means to determine the efficacy of the compound for

inhibit (or enhance) the formation of the complex between the ActRIIa polypeptide and activin. The efficacy of the compound can be evaluated by generating dose-response curves from the data obtained using various concentrations of the test compound. In addition, a control test can also be carried out to establish a baseline for comparison. For example, in a control assay, isolated and purified activin is added to a composition containing the ActRIIa polypeptide, and the formation of ActRIIa / activin complex is quantified in the absence of the test compound. It will be understood that, in general, the order in which the reagents can be mixed can be varied and that they can be mixed simultaneously. On the other hand, instead of purified proteins, cell extracts and lysates can be used to provide a suitable test system that lacks cells. Compounds that affect ActRIIb signaling can be similarly identified using an ActRIIb polypeptide and an ActRIIb ligand.

Complex formation between the ActRII polypeptide and activin can be detected by various techniques. For example, modulation of complex formation can be quantified using, for example, proteins detectably labeled as radiolabeled (e.g., 32P, 35S, 14C or 3H), fluorescently labeled (e.g., FITC), or the polypeptide ActRIIa or ActRIIb or activin labeled enzymatically, by immunoassay, or by chromatographic detection.

Fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays can be used to measure, directly or indirectly, the degree of interaction between an ActRII polypeptide and its binding protein. In addition, other detection modes, such as those based on optical waveguides (PCT Publication WO 96/26432 and US Pat. No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors and sensors may be used. of surface strength. On the other hand, an interaction trap assay, also known as the "two hybrid assay," can be used to identify agents that disrupt or enhance the interaction between an ActRII polypeptide and its binding protein. See for example, U.S. Patent 5,283,317; Zervos et al., (1993) cell 72: 223-232; Madura et al., (1993) J Biol Chem 268: 12046-12054; Bartel et al., (1993) Biotechniques 14: 920-924; and Iwabuchi et al. (1993) Oncogene 8: 1693-1696). Two reverse hybrid systems can be used to identify compounds (eg, small molecules or peptides) that dissociate the interactions between an ActRII polypeptide and its binding protein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res 27: 919-29; Vidal and Legrain, (1999) Trends Biotechnol 17: 374-81; and United States patents No. 5,525,490; 5,955,280; and 5,965,368.

The subject compounds can be identified by their ability to interact with an ActRII polypeptide or activin of the invention. The interaction between the compound and the ActRIIa, ActRIIb or activin polypeptide can be covalent or non-covalent. For example, such interaction can be identified at the protein level by in vitro biochemical methods, including photo-crosslinking, radiolabeled ligand binding and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46: 1). In certain cases, the compounds can be screened in a mechanism-based assay, such as an assay to detect compounds that bind to a polypeptideactivin or ActRII. This may include a solid or liquid phase binding episode. Alternatively, the gene encoding an activin or ActRII polypeptide can be transinfected with a reporter system (e.g., β-galactosidase, luciferase or green fluorescent protein) in a cell and screened against the collection, optionally by screening assay. High performance or with individual members of the collection. Other binding assays based on mechanisms can be used, for example, binding assays that detect changes in free energy. Binding assays can be performed with the objective fixed to a well, pearl or chip or captured by an antibody immobilized or resolved by capillary electrophoresis. Usually bound compounds can be detected using colorimetry or fluorescence or surface plasmon resonance.

6. Therapeutic uses

Activin-ActRII antagonists (for example, ActRIIa or ActRIIb polypeptides) can be used to increase red blood cell levels in mammals such as rodents and primates, and especially in human patients. The present invention provides certain activin-ActRII antagonists for use in a method of treatment or prevention of anemia in an individual in need thereof. The formation of red blood cells can be promoted in an individual by administration to the individual of a therapeutically effective amount of an activin-ActRII antagonist, particularly an ActRII polypeptide. These methods can be used for therapeutic and prophylactic treatments of mammals and especially humans.

As used herein, a therapeutic agent that "prevents", a disorder or condition refers to a compound that, in a statistical sample, reduces the incidence of the disorder or condition in the treated sample with respect to a control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder

or the condition in relation to the control sample. The term "treat" as used herein includes the prophylaxis of the aforementioned condition or the improvement or elimination of the condition once it has been installed. In either case, prevention or treatment can be discerned from the diagnosis of a doctor or other medical care provider and the expected outcome of the administration of the therapeutic agent.

As shown herein, the activin-ActRIIa antagonists and the activin-ActRIIb antagonists can be used to increase the levels of red blood cells, hemoglobin or reticulocytes in healthy individuals and such antagonists can be used in populations of selected patients. Examples of patient populations

Suitable include those with undesirably low levels of red blood cells or hemoglobin, such as patients who have anemia, and those at risk of undesirably low levels of red blood cells

or hemoglobin, such as patients who are about to undergo major surgery or other procedures that can cause considerable blood loss. A patient with adequate levels of red blood cells can be treated with an activin-ActRIIa antagonist to increase blood cell levels red, and then draw blood and store it for later use in blood transfusions. A patient with adequate levels of red blood cells can be treated with an activin-ActRIIb antagonist to increase red blood cell levels, and then draw blood and store it for later use in blood transfusions.

The activin-ActRII antagonists disclosed herein, and especially the ActRIIa-Fc and ActRIIb proteins, can be used to increase red blood cell levels in patients with anemia. When observing hemoglobin levels in humans, a lower than normal level for the appropriate gender and age category may be indicative of anemia, although individual variations are taken into account. For example, a hemoglobin level of 12 g / dl is usually considered the lower limit of normal in the general adult population. Possible causes can be blood loss, nutritional deficits, reaction to medications, various problems with the bone marrow and many diseases. More particularly, anemia has been associated with various disorders that include, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis and bone marrow transplantation. Anemia can also be associated with the following conditions: solid tumors (for example, breast cancer, lung cancer, colon cancer); tumors of the lymphatic system (for example, chronic lymphocytic leukemia, Hodgkin's and non-Hodgkin's lymphomas); tumors of the hematopoietic system (eg leukemia, myelodysplastic syndrome, multiple myeloma); radiotherapy; chemotherapy (eg platinum containing regimens); inflammatory and autoimmune diseases, including, but not limited to, rheumatoid arthritis, other inflammatory arthritis, systemic lupus erythematosus (SLE), acute or chronic skin diseases (eg, psoriasis), inflammatory bowel disease (for example, Crohn's disease and ulcerative colitis ); acute or chronic kidney disease or insufficiency, including congenital or idiopathic conditions; acute or chronic liver disease; acute or chronic bleeding; situations in which the transfusion of red blood cells is not possible due to allo - or autoantibodies of the patient or for religious reasons (for example, some Jehovah's Witnesses); infections (for example, malaria, osteomyelitis); hemoglobinopathies, including, for example, sickle cell anemia, thalassemias; drug use or drug addiction, for example, alcohol abuse; Pediatric patients with anemia due to any cause to avoid transfusion; and in elderly patients or in patients with underlying cardiopulmonary disease with anemia who cannot receive transfusions due to risk of circulatory overload.

Patients can be treated with a dosage regimen intended to restore a hemoglobin level in the patient, generally between about 10 g / dl and about 12.5 g / dl and usually about 11.0 g / dl (see also Jacobs et al (2000 ) Nephrol Dial transplant 15, 15-19), although lower end levels may cause fewer cardiovascular side effects. Alternatively, hematocrit levels (percentage of the volume of a blood sample occupied by cells) can be used as a measure of the state of red blood cells. Hematocrit levels for healthy individuals vary from 41 to 51% for adult men and 35 to 45% for adult women. The hematocrit levels sought are usually around 30 to 33%. On the other hand, hemoglobin / hematocrit levels vary from person to person. Therefore, under optimal conditions, the desired hemoglobin / hematocrit level can be individualized for each patient.

The rapid effect on red blood cell levels of the activin-ActRIIa antagonists disclosed in this document indicates that these agents act by a mechanism different from that of Epo. Consequently, these antagonists may be useful for increasing the levels of red blood cells and hemoglobin in patients who do not respond well to Epo. For example, an activin-ActRIIa antagonist may be beneficial for a patient in whom the administration of a dose of normal to increased (> 300 IU / kg / week) of Epo does not result in an increase in the hemoglobin level until desired level Patients with an inadequate response to Epo are found in all types of anemia, but particularly higher numbers of non-response have been observed in patients with cancer and patients with end-stage renal disease. An inadequate response to Epo may be constitutive (that is, observed after the first treatment with Epo) or acquired (for example, observed when repeating Epo treatment).

Activin-ActRII antagonists can also be used to treat patients who are prone to the adverse effects of Epo. The main adverse effects of Epo are an excessive increase in hematocrit or hemoglobin levels and polycythemia. Elevated hematocrit levels can cause hypertension (more particularly worsening of hypertension) and vascular thrombosis. Other adverse effects of Epo that have been reported, some of which were related to hypertension, are headache, flu-like syndrome, shunt obstruction, myocardial infarction and brain seizures due to thrombosis, hypertensive encephalopathy and red blood cell aplasia ( Singibarti, (1994) J. Clin Investig 72 (suppl 6), S36-S43; Horl et al. (2000) Nephrol Dial Transplant 15 (suppl 4), 51-56; Delanty et al. (1997) Neurology 49, 686 -689; Bunn (2002) N Engl J Med 346 (7), 522523).

7. Pharmaceutical compositions

Activin-ActRII antagonists (for example, ActRIIa and ActRIIb polypeptides) can be formulated with a pharmaceutically acceptable excipient. For example, an ActRII 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 manner for use in human or veterinary medicine.

The composition can be administered systemically or locally as an implant or device. When the therapeutic composition for use in this invention is administered, it is, of course, in a physiologically acceptable, non-pyrogenic form. Other therapeutically useful agents other than the activin-ActRII antagonists that can also be included in the composition as described above, can be administered simultaneously or consecutively with the compounds of the subject (for example, ActRIIa and ActRIIb polypeptides) in the invention.

Normally, activin-ActRII antagonists will be administered parenterally. Pharmaceutical compositions suitable for parenteral administration may contain one or more ActRII polypeptides in combination with one or more sterile, isotonic, aqueous or non-aqueous pharmaceutically acceptable solutions, dispersions, suspensions or emulsions, or sterile powders that may be reconstituted into solutions or dispersions. Sterile injectables just before use, which may contain antioxidants, pH absorbers, bacteriostats, solutes that make the isotonic formulation with the recipient's blood, or suspending or thickeners. Examples of suitable aqueous and non-aqueous vehicles that can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and their suitable mixtures, vegetable oils, such as olive oil and injectable organic esters, such as ethyl oleate. Adequate 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.

In addition, the composition can be encapsulated or injected into a form for release at the desired site of the tissue (eg, bone marrow). In certain embodiments, the compositions for use in the present invention may include a matrix capable of releasing one or more therapeutic compounds to a desired tissue site (eg, bone marrow), providing a structure for the developing tissue and optimally capable of be reabsorbed in the body. For example, the matrix can provide a slow release of ActRII polypeptides. Such matrices can be made of materials currently in use for other implantable medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the subject compositions will define the appropriate formulation. Possible matrices for the compositions may be calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and biodegradable and chemically defined polyanhydrides. Other possible materials are biodegradable and biologically well defined, such as bone or dermal collagen. Other matrices are composed of pure proteins or components of the extracellular matrix. Other possible matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates or other ceramics. The matrices may be composed of combinations of any of the above types of materials, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics composition can be altered as in calcium phosphate aluminate and processed to modify the pore size, particle size, particle shape and biodegradability.

The compositions can be administered orally, for example, in the form of capsules, wafers, pills, tablets, tablets (using a flavored base, generally sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion or as an elixir or syrup, or as tablets (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouthwashes, and the like, each with a predetermined amount of a drug as active ingredient. A drug can also be administered as a bolus, sugary remedy or paste.

In solid pharmaceutical forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), one or more therapeutic compounds can be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate, dicalcium phosphate and / or any of the following: (1) fillers

or diluents, such as starches, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) humectants, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) dyes. In the case of capsules, tablets and pills, pharmaceutical compositions may also include pH buffers. Solid compositions of similar type can also be used as filler of soft and hard gelatin capsules, using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Liquid pharmaceutical forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid pharmaceutical forms may contain inert diluents commonly used in the area, such as water or other solvents, solubilizers 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, peanut, corn, wheat germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan esters of fatty acids and their mixtures In addition to inert diluents, oral compositions may also include adjuvants such as humectants, emulsifiers and suspensions, sweeteners, flavorings, colorants, perfumes and preservatives.

The suspensions, in addition to the active substances, may contain suspensions such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

The compositions may also contain adjuvants, such as preservatives, humectants, emulsifiers and dispersants. The action of microorganisms can be prevented by the inclusion of various antibacterials and antifungals, for example, paraben, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include in the compositions isotonic agents, such as sugars, sodium chloride and the like. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

It is understood that the dosage regimen will be determined by the physician taking into account various factors that modify the action of the compounds used in the invention.

The various factors include, but not exclusively, the red blood cell count, the hemoglobin level or other diagnostic assessments of the patient, the red blood cell count of the intended destination, the age, gender and diet of the patient, the severity of any disease that may be contributing to a depressed level of red blood cells, time of administration and other clinical factors. The addition of other known growth factors to the final composition may also affect the dose. The evolution can be monitored by periodic evaluation of the levels of red blood cells and hemoglobin, as well as by evaluations of reticulocyte levels and other indicators of the hematopoietic process.

Experiments with primates and humans have shown that the effects of ActRIIa-Fc on red blood cell levels are detectable when the compound is administered at intervals and in sufficient quantities to reach serum concentrations of around 100 ng / ml or greater during a period of at least about 20 to 30 days. A dosage can also be used to obtain serum levels of 200 ng / ml, 500 ng / ml, 1000 ng / ml or greater for a period of at least 20 to 30 days. Bone effects can be observed at serum levels of approximately 200 ng / ml, with substantial effects starting at around 1000 ng / ml or more, over a period of at least approximately 20 to 30 days. Therefore, if desired to achieve effects on red blood cells but with little effect on bone, a dosage scheme can be designed to provide a serum concentration between about 100 and 1000 ng / ml over a period of approximately 20 to 30 days. In humans, serum levels of 200 ng / ml can be achieved with a single dose of 0.1 mg / kg

or higher and serum levels of 1000 ng / ml can 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 therefore an effective sustained serum level can be achieved, for example, by administering around 0, 05 to 0.5 mg / kg weekly or biweekly, or higher doses may be used at longer intervals between administrations. For example, doses of 0.1 to 1 mg / kg could be used monthly or bimonthly.

Genotherapy can be used for the in vivo production of ActRII polypeptides. Such therapy would achieve its therapeutic effect by introducing the ActRIIa or ActRIIb polynucleotide sequences into cells or tissues that have the disorders mentioned above. The administration of ActRII polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. The use of targeted liposomes is preferred for therapeutic administration of ActRII polynucleotide sequences.

Several viral vectors that can be used for the genotherapy taught herein include adenovirus, herpes virus, vaccinia or an RNA virus such as a retrovirus. The retroviral vector may be a derivative of an avian or murine retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but not exclusively: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV) and Rous sarcoma virus (RSV). A series of additional retroviral vectors can incorporate multiple genes. All these vectors can transfer or incorporate a gene for a selectable marker so that the transduced cells can be identified and generated. Retroviral vectors can be made target specific by linking them, for example, a sugar, a glycolipid or a protein. Preferred targeted administration is achieved through the use of an antibody. Those skilled in the area will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow specific administration in the target of the retroviral vector containing the ActRII polynucleotide.

Alternatively, tissue culture cells can be directly transinfected with plasmids encoding the gag, pol and env retroviral structural genes, by conventional transfection with calcium phosphate. Then, those cells are transinfected with the vector plasmid that contains the genes of interest. The resulting cells release the retroviral vector in the culture medium.

Another directed administration system for ActRII polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems that include oil-in-water emulsions, micelles, mixed micelles and liposomes. A preferred colloidal system is a liposome. Liposomes are artificial membrane vesicles, useful as vehicles for administration in vitro and in vivo. RNA, DNA and intact virions can be encapsulated in the aqueous interior and administered to the cells in a biologically active form (see e.g., Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfer using a liposome vehicle are known in the area, see for example, Mannino, et al., Biotechniques, 6: 682, 1988. The liposome composition is generally a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

Examples of lipids useful in the production of liposomes include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. Targeted administration of liposomes is also possible based on, for example, organ specificity, cell specificity and organelle specificity, as is known in the area.

EXAMPLES

The invention that is being described in general will be more readily understood by reference to the following examples, which are included only for illustrative purposes of certain embodiments of the present invention, and are not intended to limit the invention.

Example 1: ActRIIa-Fc fusion proteins

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 between them. The constructs are called ActRIIa-hFc and mFc-ActRIIa, respectively.

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

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

(i)

 Honey honey melitin (HBML): MKFLVNVALVFMVYISYIYA (SEQ ID NO: 8)

(ii)

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

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

5 Both ActRIIa-hFc and ActRIIa-mFc were remarkably prone to recombinant expression. As shown in Figure 1, the protein was purified as a unique and well-defined protein peak. Nterminal sequencing showed a single ILGRSTQE sequence (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, sepharose Q chromatography, phenylsepharose chromatography, chromatography of

10 size exclusion and cation exchange chromatography. Purification could be completed with viral filtration and exchange of buffer solution. The ActRIIa-hFc protein was purified to a purity> 98% as determined by size exclusion chromatography and> 95% as determined by SDS PAGE.

ActRIIa-hFc and ActRIIa-mFc showed high affinity for ligands, particularly for activin A. GDF-11 or activin A ("ActA") was immobilized on a Biacore CM5 chip using a standard amine coupling procedure. ActRIIa-hFc and ActRIIa-mFc proteins were loaded into the system and binding was measured. ActRIIa-hFc bound the activin with a dissociation constant (KD) of 5 x 1012 and the protein bound to GDF11 with a KD of 9.96 x 10-9. See figure 2 ActRIIa-mFc behaved similarly.

ActRIIa-hFc was very stable in pharmacokinetic studies. Rats were given 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, 1 mg / kg, 10 mg / kg or 30 mg / kg were administered to rats. In rats, ActRIIa-hFc had a serum half-life of 11 to 14 days and the circulating levels of the drug were quite high after two weeks (11 µg / ml, 110 µg / ml or 304 µg / ml for initial administrations of 1 mg / kg, 10 mg / kg or 30 mg / kg, respectively.) In Filipino monkeys, the plasma half-life was substantially longer than 14 days and the circulating levels of the drug were 25 µg / ml, 304 µg / ml or 1440 µg / ml for initial administrations of 1 mg / kg, 10 mg / kg or 30 mg / kg, respectively.

Example 2: ActRIIa-hFc increases red blood cell levels in non-human primates

The study employed four groups of five male Filipino monkeys and five female Filipino monkeys each, with three per sex per group with a scheduled termination on the 29th and two per sex per group with a scheduled termination on the 57th. Each animal received a vehicle ( group I) or ActRIIa-Fc at doses of 1, 10 or 30 mg / kg (groups 2, 3 and 4, respectively) by intravenous (IV) injection on days 1, 8, 15 and 22. The dose volume was maintained at 3 mL / kg. Several measures of red blood cell levels were evaluated two days before the first administration and on days 15, 29 and 57 (for the remaining two animals) after the first administration.

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

Statistical significance was calculated for each treatment group with respect to the mean for the treatment group at baseline (baseline).

Notably, increases in red blood cell counts and hemoglobin levels are approximately equivalent in magnitude to the effects reported with erythropoietin. The appearance of these effects is faster with ActRIIa-Fc than with erythropoietin.

Similar results were observed with rats and mice.

Example 3: ActRIIa-hFc increases red blood cell levels 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 fundamentally assess the safety of the protein in healthy, postmenopausal women. Forty-eight subjects were randomized in cohorts of 6 to receive a single dose of ActRIIa-hFc or placebo (5 active: 1 placebo). Dose levels varied between 0.01 and 3.0 mg / kg intravenously (IV) and between 0.03 to 0.1 mg / kg subcutaneously (SC). All subjects were followed for 120 days. In addition to PK) pharmacokinetic analyzes, the biological activity of ActRIIa-hFc was also evaluated by measurement of biochemical markers of bone formation and resorption and FSH levels.

To look for possible changes, hemoglobin numerical values and red blood cell counts for all subjects during the study were examined in detail and compared with the levels at baseline. Platelet counts were compared during the same time as the control. There were no clinically significant changes in initial values over time for platelet counts.

The pharmacokinetic analysis of ActRIIa-hFc showed a linear profile with the dose and a half-life of approximately 25 to 32 days. The area under the curve (AUC) for ActRIIa-hFc was linearly related to the dose, and absorption after SC administration was essentially complete (see Figures 7 and 8). These data indicate that the SC pathway is a desirable dosage method because it provides equivalent bioavailability and serum half-life for the drug, while avoiding the peak in serum concentrations of the drug associated with the first days of IV dosing (see Figure 8). ActRHa-hFc caused a rapid, sustained and dose-dependent increase in serum levels of bone-specific alkaline phosphatase (BAP), which is a marker of anabolic bone growth, and a dose-dependent decrease in the collagen telopeptide Cterminal type 1 and 5b tartrate-resistant acid phosphatase levels, which are markers of bone resorption. Other markers, such as PINP, showed inconclusive results. BAP levels showed effects close to saturation at the highest dose of the drug, indicating that half the maximum effects can be achieved in this anabolic bone biomarker at a dose of 0.3 mg / kg, with increases ranging up to 3 mg / kg Calculated as a ratio of the pharmacodynamic effect for AUC for the drug, the EC50 is 51,465 (day * ng / ml). See Figure 9. These changes in the bone radiolabel were maintained for approximately 120 days at dose levels.

Highest tested. There was also a dose-dependent decrease in serum FSH levels compatible with activin inhibition.

In general, there was a very small decrease in hemoglobin, unrelated to the drug, during the first week of the study probably related to phlebotomy of the study in the 0.01 and 0.03 mg / kg dose groups already administered IV or SC. The hemoglobin results corresponding to 0.1 mg / kg SC and IV were stable or showed modest increases by days 8 to 15. At the dose level of 0.3 mg / kg IV there was a clear increase in the levels of HGB seen as early as day 2 and often with a maximum on days 15 to 29 that was not seen in placebo subjects. At this point in the study, this change did not reach statistical significance.

Overall, ActRIIa-hFc showed a dose-dependent effect on red blood cell and reticulocyte counts. For a summary of the hematological changes, see Figures 10-13.

Example 4: Alternative ActRIIa-Fc Proteins

Various variants of ActRIIa that can be used in accordance with the methods described herein are described in the international patent application published as WO2006 / 012627 (see, e.g., pp. 55-58). An alternative construct can have a deletion of the C-terminal tail (the final 15 amino acids of the extracellular domain of ActRIIa. The sequence for that construct (underlined Fc portion) is presented below (SEQ. ID NO: 12):

Example 5: ActRIIb-Fc fusion proteins

Applicants constructed a soluble ActRIIb fusion protein that has the extracellular domain of human ActRIIb fused to a human Fc domain. A co-crystalline structure of extracellular activin and ActRIIb showed no role for the final (C-terminal) 15 amino acids (referred to as the "tail" in this document) of the extracellular domain of the binding ligand. This sequence could not be resolved in the crystalline structure, suggesting that these residues are present in a flexible loop that was not packed evenly in the crystal. Thompson et al. EMBO J. 2003 Apr 1; 22 (7): 15555-66. This sequence is also poorly conserved between ActRIIb and ActRIIa. Consequently, these residues were omitted in the basic, or background, fusion merger, ActRIIb-Fc. In addition, position 64 in the bottom form is occupied by an alanine, which is generally considered the "natural" form, although an A64R allele occurs naturally. Therefore, the ActRIIb-Fc background fusion has the sequence (underlined Fc portion) (SEQ ID NO: 20):

Surprisingly, it was found that the C-terminal tail increases the binding of activin and GDF-1, therefore a preferred version of ActRIIb-Fc has a sequence (underlined Fc portion) (SEQ ID NO: 21): 5

Various variants of ActRIIb that can be used in accordance with the methods described herein are described in the international patent application published as WO2006 / 012627 (see for example, pp. 59-60).

Example 6: ActRIIb-hFc stimulates erythropoiesis in nonhuman primates

ActRIIb-hFc (IgG1) was administered once a week for 1 month to male and female Filipino monkeys by subcutaneous injection. Forty-eight Filipino monkeys (24 / sex) were assigned to one of four treatment groups (6 animals / sex / group) and were given subcutaneous vehicle injections or ActRIIb-hFc at doses of 3, 10 or 30 mg / kg once a week for 4 weeks (a total of 5 doses). The parameters evaluated included general analytical tests (hematology, clinical biochemistry, coagulation and urine analysis). ActRIIbhFc caused a statistically significant high mean average of reticulocyte values by day 15 in treated animals. By day 36, ActRIIb-hFc caused several hematologic changes, such as a high absolute mean of reticulocyte values and red blood cell distribution width values, and a mean lower corpuscular hemoglobin concentration. All treated groups and both sexes were affected.

These effects are compatible with a positive effect of ActRIIb-hFc on the release of immature reticulocytes from the bone marrow. This effect was reversed after the pharmacological rest period of the treated animals (towards day 56 of the study). Consequently, we conclude that ActRIIb-hFc stimulates erythropoiesis.

While specific accomplishments of the topic have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to experts in the area after reviewing this specification and the following claims. The total scope of the invention should be determined by reference to the claims and the specification.

SEQUENCE LIST

[0151]

<110> ACCELERON PHARMA INC.

<120> ACTIVTA-ACTRII ANTAGONISTS AND USES TO INCREASE THE LEVELS OF RED BLOODS

<130> P47729EP3 / NJL

<140> EP 11195130.7

<141> 2007-12-18

<150> US 60 / 875,682

<151> 2006-12-18

<160> 23

<170> PatentIn Ver. 3.3

<210> 1

<211> 513

<212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 115

<212> PRT

<213> Homo sapiens

<400> 2

<210> 3 10 <211> 100

<212> PRT

<213> Artificial sequence

<220> 15 <223> Description of the artificial sequence: Synthetic polypeptide

<400> 3

<210> 4

<211> 1542

<212> DNA

<213> Homo sapiens

<400> 4

<210> 5

<211> 345

<212> DNA

<213> Homo sapiens

<400> 5

<210> 6

<211> 225 10 <212> PRT

<213> Artificial sequence

<220>

<223>

Description of the artificial sequence: Synthetic polypeptide 15

<220>

<221> MOD_RES

<222> (43)

<223>

Asp or Ala 20

<220>

<221> MOD_RES

<222> (100)

<223>

Lys or Ala 25

<220>

<221> MOD_RES

<222> (212)

<223>

His or Ala 30

<400> 6

<210> 7

<211> 344 5 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic construct 10

<400> 7

<210> 8

<211> 21

<212> PRT

<213> Apis mellifera

<400> 8

<210> 9 10 <211> 22

<212> PRT

<213> Unknown body

<220> 15 <223> Description of the unknown organism: Lead sequence of plasminogen tissue activator

<400> 9

<210> 10

<211> 20

<212> PRT

<213> Unknown body 25

<220>

<223> Description of the unknown organism: Natural leader sequence

<400> 10 30

<210> 11

<211> 8 35 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide 40

<400> 11

45 <210> 12

<211> 329

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic construct

<400> 12

<210> 13

<211> 369

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic construct

<400> 13

<210> 14

<211> 1114 5 <212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic polynucleotide 10

<400> 14

<210> 15

<211> 512

<212> PRT

<213> Homo sapiens 20

<400> 15

<210> 16

<211> 116

<212> PRT

<213> Homo sapiens

<400> 16

<210> 17

<211> 101

<212> PRT 15 <213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic polypeptide

<400> 17

<210> 18

<211> 1539

<212> DNA

<213> Homo sapiens 10

<400> 18

15 <210> 19

<211> 348

<212> DNA

<213> Homo sapiens

<400> 19

10 <210> 20

<211> 329

<212> PRT

<213> Artificial sequence

15 <220>

<223> Description of the artificial sequence: Synthetic polypeptide

<400> 20

<210> 21

<211> 344 5 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic polypeptide 10

<400> 21

<210> 22

<211> 5 5 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide 10

<400> 22

<210> 23 15 <211> 5

<212> PRT

<213> Artificial sequence

<220> 20 <223> Description of the artificial sequence: Synthetic peptide

<400> 23

25 LIST OF SEQUENCES

[0152]

30 <110> ACCELERON PHARMA INC.

<120> ACTIVTA-ACTRII ANTAGONISTS AND USES TO INCREASE THE LEVELS OF RED BLOODS

<130> P47729EP3 / NJL

<140> EP 11195130.7 5 <141> 2007-12-18

<150> US 60 / 875,682

<151> 2006-12-18

10 <160> 23

<170> PatentIn Ver. 3.3

<210> 1

<211> 513 15 <212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 115

<212> PRT

<213> Homo sapiens

<400> 2

10 <210> 3

<211> 100

<212> PRT

<213> Artificial sequence

15 <220>

<223> Description of the artificial sequence: Synthetic polypeptide

<400> 3

<210> 4

<211> 1542

<212> DNA

<213> Homo sapiens

<400> 4

<210> 5

<211> 345

<212> DNA

<213> Homo sapiens

<400> 5

<210> 6 10 <211> 225

<212> PRT

<213> Artificial sequence

<220> 15 <223> Description of the artificial sequence: Synthetic polypeptide

<220>

<221> MOD_RES

<222>

(43) 20 <223> Asp or Ala

<220>

<221> MOD_RES

<222>

(100) 25 <223> Lys or Ala

<220>

<221> MOD_RES

<222>

(212) 30 <223> His or Ala

<400> 6

<210> 7

<211> 344 5 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic construct 10

<400> 7

<210> 8

<211> 21

<212> PRT

<213> Apis mellifera

<400> 8

<210> 9 10 <211> 22

<212> PRT

<213> Unknown body

<220> 15 <223> Description of the unknown organism: Lead sequence of plasminogen tissue activator

<400> 9

<210> 10

<211> 20

<212> PRT

<213> Unknown body 25

<220>

<223> Description of the unknown organism: Natural leader sequence

<400> 10 30

<210> 11

<211> 8 35 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide 40

<400> 11

45 <210> 12

<211> 329

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic construct

<400> 12

<210> 13

<211> 369

<212> PRT 5 <213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic construct

10 <400> 13

<210> 14

<211> 1114 5 <212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic polynucleotide 10

<400> 14

15 <210> 15

<211> 512

<212> PRT

<213> Homo sapiens

20 <400> 15

<210> 16

<211> 116

<212> PRT

<213> Homo sapiens

<400> 16

<210> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic polypeptide

<400> 17

<210> 18

<211> 1539

<212> DNA 15 <213> Homo sapiens

<400> 18

<210> 19

<211> 348

<212> DNA

<213> Homo sapiens

<400> 19

<210> 20

<211> 329 15 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic polypeptide 20

<400> 20

<210> 21

<211> 344 5 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic polypeptide 10

<400> 21

<210> 22

<211> 5 5 <212> PRT

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide 10

<400> 22

15 <210> 23

<211> 5

<212> PRT

<213> Artificial sequence

20 <220>

<223> Description of the artificial sequence: Synthetic peptide

<400> 23

Claims (12)

1. An activin-ActRII antagonist for use in a method of treatment or prevention of anemia in a human patient in need thereof, wherein said antagonist is chosen from: 5 an antibody that binds to activin and destabilizes ActRIIa binding , an antibody that binds to activin and destabilizes binding to ActRIIb, an anti-activin antibody βA, βB, βC or βE, an anti-ActRIIa antibody, an anti-ActRIIb antibody, 10 folistatin, and inhibin. 2. The antagonist for use according to claim 1, wherein the antagonist is an antibody that binds to activin and destabilizes ActRIIa binding. 3. The antagonist for use according to claim 1, wherein the antagonist is an antibody that binds to activin and destabilizes ActRIIb binding.

Four.

 The antagonist for use according to claim 1, wherein the antagonist is an anti-activin antibody A.

5.

 The antagonist for use according to claim 1, wherein the antagonist is a B anti-activin antibody.

6.

 The antagonist for use according to claim 1, wherein the antagonist is a activC anti-activin antibody.

7.

 The antagonist for use according to claim 1, wherein the antagonist is an anti-activin antibody E.

The antagonist for use according to claim 1, wherein the antagonist is an anti-ActRIIa antibody.

9.

 The antagonist for use according to claim 1, wherein the antagonist is an anti-ActRIIb antibody.

10.

 The antagonist for use according to claim 1, wherein the antagonist is folistatin.

eleven.

The antagonist for use according to claim 10, wherein the antagonist is folistatin-288.

12. The antagonist for use according to claim 10, wherein the antagonist is folistatin-315. 13. The antagonist for use according to claim 1, wherein the antagonist is inhibin. 14. The antagonist for use according to any of the preceding claims, wherein the anemia is associated with multiple myeloma, chronic renal disease or insufficiency, acute renal disease or insufficiency, chemotherapy treatment of the patient, a myelodysplastic syndrome or thalassemia. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13