GDF trap combined with erythropoietin receptor activator to increase red blood cell levels

Abstract

Provided are compositions for increasing red blood cell levels or treating anemia, comprising an erythropoietin receptor activator and a variant activin RIIB (ActRIIB) polypeptide having a leucine residue replaced by an acidic amino acid residue, wherein the variant ActRIIB polypeptide has a reduced affinity for activin B and can be referred to as a GDF trap. Provided are methods of using these compositions.

Description

[0001] This application is a divisional application of an invention patent application with an application date of August 13, 2010, application number 201510979291.8, and the same invention name as the present invention. Background of the Invention

[0002] Mature red blood cells, or erythrocytes, are responsible for oxygen transport in the vertebrate circulatory system. Red blood cells contain a high concentration of hemoglobin, a protein that binds oxygen in the lungs at a relatively high partial pressure of oxygen (pO2) and delivers oxygen to areas of the body with a relatively low pO2.

[0003] Mature red blood cells are produced by multipotent hematopoietic stem cells in a process called erythropoiesis. Postnatal erythropoiesis is mainly carried out in the red pulp of the bone marrow and spleen. The synergistic action of various signal transduction pathways controls the balance of cell proliferation, differentiation, survival and death. Under normal conditions, red blood cells are produced at a rate that maintains a constant amount of red blood cells in the body, and their production can be increased or decreased in response to various stimuli (including rising or falling oxygen tension or tissue demand). The process of erythropoiesis begins with the formation of lineage-directed precursor cells and proceeds through a series of different precursor cell types. The final stage of erythropoiesis appears as reticulocytes, which are released into the bloodstream and lose their mitochondria and ribosomes while assuming the morphology of mature red blood cells. The rising level of reticulocytes in the blood or the rising ratio of reticulocytes: red blood cells indicates that the rate of red blood cell production has increased.

[0004] Erythropoietin (EPO) is widely considered to be the most important positive regulator of postnatal erythropoiesis in vertebrates. EPO regulates the compensatory erythropoietic response to reduced tissue oxygen tension (hypoxia) and low red blood cell levels or low hemoglobin levels. In humans, rising EPO levels promote erythropoiesis by stimulating the production of erythroid progenitors in the bone marrow and spleen. In mice, EPO primarily increases erythropoiesis in the spleen.

[0005] The effect of EPO is mediated by cell surface receptors belonging to the cytokine receptor superfamily. The human EPO receptor gene encodes a transmembrane protein of 483 amino acids, and the active EPO receptor is considered to exist as a multimeric complex, even in the absence of a ligand (see U.S. Patent No. 6,319,499). The cloned full-length EPO receptor expressed in mammalian cells is combined with EPO with an affinity similar to that of the natural receptor on erythroid progenitor cells. The combination of EPO and its receptor causes conformational changes, leading to receptor activation and biological effects, including the increased proliferation of immature erythroblasts, the increased differentiation of immature erythroblasts, and the reduced apoptosis in erythroid progenitor cells (Liboi et al., 1993, Proc Natl Acad Sci USA90:11351-11355; Koury et al., 1990, Science 248:378-381).

[0006] Doctors use various forms of recombinant EPO to increase red blood cell levels in various clinical situations, particularly for the treatment of anemia. Anemia is a broadly defined condition characterized in that the hemoglobin or red blood cells in the blood are lower than normal levels. In some cases, anemia is caused by primary disorders in the generation or survival of red blood cells. More generally, anemia is secondary to diseases of other systems (Weatherall & Provan (2000) Lancet 355, 1169-1175). Anemia can be caused by a decrease in the rate of red blood cell production or an increase in the rate of destruction, or by the loss of red blood cells caused by bleeding. Anemia can be caused by a variety of disorders, including, for example, chronic renal failure, chemotherapy, myelodysplastic syndrome, rheumatoid arthritis and bone marrow transplantation.

[0007] Treatment with EPO usually results in a healthy person's hemoglobin rising by about 1-3 g / dL in a week. When applied to anemic individuals, the treatment regimen usually provides a substantial rise in hemoglobin and red blood cell levels, and improves quality of life and prolongs survival. EPO is not uniformly effective, and many individuals are refractory even at high doses (Horl et al. (2000) NephrolDial Transplant 15, 43-50). More than 50% of cancer patients have insufficient responses to EPO, and about 10% of patients with end-stage renal disease have low responsiveness (Glaspy et al. (1997) J Clin Oncol 15, 1218-1234; Demetri et al. (1998) J Clin Oncol 16, 3412-3425), and less than 10% of patients with myelodysplastic syndrome respond favorably (Estey (2003) Curr Opin Hematol 10, 60-67). Some factors, including inflammation, iron and vitamin deficiency, insufficient dialysis, aluminum toxicity and hyperparathyroidism can predict poor treatment response. The molecular mechanism of EPO resistance is still unclear. Recent evidence suggests that higher doses of EPO may be associated with increased risk of cardiovascular morbidity, tumor growth and mortality in certain patient groups (Krapf et al., 2009, Clin J Am Soc Nephrol 4:470-480; Glaspy, 2009, Annu Rev Med 60:181-192). Therefore, it has been suggested that EPO-based therapeutic compounds (erythropoietin-stimulating agents, ESAs) are administered with the minimum dose sufficient to avoid the need for red blood cell transfusion (Jelkmann et al., 2008, Crit Rev Oncol. Hematol 67:39-61).

[0008] It is therefore an object of the present disclosure to provide alternative methods of increasing a patient's red blood cell levels that would allow for the use of reduced doses of erythropoietin receptor activators. SUMMARY OF THE INVENTION

[0009] The present disclosure describes, in part, that GDF Traps can be administered in combination with EPO receptor activators (e.g., administered at the same time or at different times, but generally in a manner to achieve overlapping drug effects) to increase red blood cell levels (erythropoiesis) or treat anemia in patients in need thereof. The present disclosure describes, in part, that GDF Traps can be administered in combination with EPO receptor activators to synergistically increase red blood cell formation in patients. Thus, the effect of the combined treatment can be significantly greater than the sum of the effects of the GDF Traps and EPO receptor activators when administered alone in their respective amounts. In certain embodiments, this synergistic effect can be advantageous because it enables target red blood cell levels to be achieved with lower doses of EPO receptor activators, thereby avoiding potential side effects or other problems associated with higher levels of EPO receptor activation.

[0010] EPO receptor activators can stimulate erythropoiesis by directly contacting and activating EPO receptors. In certain embodiments, EPO receptor activators are one of a class of compounds based on the 165 amino acid sequence of natural EPO, and are generally referred to as erythropoiesis-stimulating agents (ESAs), examples of which are Epoetin α, Epoetin β, Epoetin δ, and Epoetin ω. In other embodiments, ESAs include synthetic EPO proteins (SEPs) and EPO derivatives with non-peptide modifications that confer desired pharmacokinetic properties (extended circulation half-life), examples of which are darbepoetin alfa and methoxy-polyethylene glycol Epoetin β. In certain embodiments, an EPO receptor activator may be an EPO receptor agonist that is not bound to an EPO polypeptide backbone or is not generally classified as an ESA. Such EPO receptor agonists may include, but are not limited to, peptide and non-peptide mimetics of EPO, agonistic antibodies targeting the EPO receptor, fusion proteins comprising an EPO mimetic domain, and erythropoietin receptor extended-duration limited agonists (EREDLA).

[0011] In certain embodiments, EPO receptor activators can indirectly stimulate erythropoiesis by enhancing endogenous EPO production without contacting the EPO receptor itself. For example, hypoxia-induced transcription factors (HIFs) are endogenous stimulators of EPO gene expression, which are inhibited (destabilized) by cellular regulatory mechanisms under normoxic conditions. The present disclosure provides increased erythropoiesis in patients in part by combining treatment with GDF traps and indirect EPO receptor activators with HIF stabilizing properties, such as prolyl hydroxylase inhibitors.

[0012] Variant ActRIIB polypeptides that have significantly reduced affinity for activins (e.g., activin A and / or activin B) relative to other ActRIIB ligands such as GDF11 and / or tubocurarine are referred to as GDF traps. The ActRIIB variants described herein are GDF traps unless otherwise specified. In particular, the present disclosure describes a GDF trap that is a soluble form of an ActRIIB polypeptide having an acidic residue at position 79 of SEQ ID NO: 1 that increases red blood cell levels in the blood when administered in vivo. Thus, in certain embodiments, the present disclosure provides methods for using GDF traps to increase red blood cell and hemoglobin levels in patients and to treat conditions associated with low red blood cell or hemoglobin levels in patients in need thereof. As described in U.S. Patent Application No. 12 / 012,652, which is incorporated herein by reference, GDF traps can be used to increase muscle mass and reduce fat mass.

[0013] In certain aspects, the present disclosure provides GDF traps that are variant ActRIIB polypeptides, including ActRIIB polypeptides with amino and carboxyl terminal truncations and sequence changes. Optionally, the GDF traps of the present invention can be designed to preferentially antagonize one or more ligands of the ActRIIB receptor, such as GDF8 (also known as tubocurarine), GDF11, Nodal, and BMP7 (also known as OP-1). Examples of GDF traps include a group of variants derived from ActRIIB that have greatly reduced affinity for activin. These variants exhibit the desired effects on red blood cells while reducing the effects on other tissues. Examples of such variants include those having an acidic amino acid (e.g., aspartic acid D or glutamic acid E) at a position corresponding to position 79 of SEQ ID NO.1. In certain embodiments, a GDF Trap polypeptide comprises an amino acid sequence comprising, consisting of, or consisting essentially of an amino acid sequence of SEQ ID NO: 7, 26, 28, 29, 32, 37, or 38, and a polypeptide at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any of the foregoing.

[0014] In certain aspects, the present disclosure provides a pharmaceutical formulation comprising a GDF trap that binds to an ActRIIB ligand such as GDF8, GDF11, an activin (e.g., activin B), BMP7, or nodal, and a pharmaceutically acceptable carrier. Optionally, the GDF trap binds to the ActRIIB ligand with a Kd of less than 10 micromolar, less than 1 micromolar, less than 100 nanomolar, less than 10 nanomolar, or less than 1 nanomolar. Optionally, the GDF trap inhibits ActRIIB signaling, such as intracellular signaling events triggered by an ActRIIB ligand. The GDF trap used in such a formulation can be any of those disclosed herein, including, for example, a GDF trap having an amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, or 40, or a GDF trap having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, or 40, or a GDF trap having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, or 40, wherein the position corresponding to L79 in SEQ ID NO: 1 is an acidic amino acid. Preferred GDF traps for use in such formulations consist of or consist essentially of the amino acid sequence of SEQ ID NO: 26. The GDF trap may comprise a functional fragment of a native ActRIIB polypeptide, such as one comprising at least 10, 20, or 30 amino acids of a sequence selected from SEQ ID NOs: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, or 40, or the sequence of SEQ ID NO: 2, lacking 1, 2, 3, 4, 5, or 10 to 15 amino acids at the C-terminus and lacking 1, 2, 3, 4, or 5 amino acids at the N-terminus. Preferred polypeptides will comprise a truncation of 2 to 5 amino acids at the N-terminus and no more than 3 amino acids at the C-terminus relative to SEQ ID NO: 2 or 40. The GDF trap may include one or more changes in the amino acid sequence of the ActRIIB polypeptide relative to a naturally occurring ActRIIB polypeptide (e.g., in the ligand binding domain). Changes in the amino acid sequence can, for example, alter the glycosylation of the polypeptide when produced in a mammalian, insect or other eukaryotic cell, or alter the proteolytic cleavage of the polypeptide relative to a naturally occurring ActRIIB polypeptide.

[0015] A GDF trap can be a fusion protein having an ActRIIB polypeptide as one domain (e.g., a ligand binding domain of ActRIIB with one or more sequence variations) and one or more other domains that provide desirable properties (e.g., improved pharmacokinetics, easier purification, targeting to a specific tissue, etc.). For example, the domains of the fusion protein can enhance one or more of the following: in vivo stability, in vivo half-life, uptake / administration, tissue localization or distribution, formation of protein complexes, multimerization and / or purification of the fusion protein. A GDF trap fusion protein can comprise an immunoglobulin Fc domain (wild type or mutant) or serum albumin. In some embodiments, a GDF trap fusion comprises a relatively unstructured linker between the Fc domain and the extracellular ActRIIB domain. The unstructured linker can correspond to an unstructured region of approximately 15 amino acids at the C-terminus ("tail") of the ActRIIB extracellular domain, or it can be an artificial sequence of 3 to 5, 15, 20, 30, 50 or more amino acids that are relatively free of secondary structure. The linker may be rich in glycine and proline residues and may, for example, contain repeating sequences of threonine / serine and glycine (e.g., single or repeating sequences of TG4 (SEQ ID NO: 13) or SG4 (SEQ ID NO: 14)) or a series of 3 glycines. The fusion protein may comprise a purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion. In certain embodiments, the GDF trap fusion comprises a leader sequence. The leader sequence may be a native ActRIIB leader sequence or a heterologous leader sequence. In certain embodiments, the leader sequence is a tissue plasminogen activator (TPA) leader sequence. In one embodiment, the GDF trap fusion protein comprises an amino acid sequence as set forth in formula ABC. Part B is an N- and C-terminally truncated ActRIIB polypeptide consisting of an amino acid sequence corresponding to amino acids 25-131 of SEQ ID NO: 2 or 40. Parts A and C may independently be absent, one, or more than one amino acid, and parts A and C are both heterologous to B. Part A and / or C may be linked to part B via a linker sequence.

[0016] Optionally, the GDF trap comprises a variant ActRIIB polypeptide having one or more modified amino acid residues selected from the group consisting of glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to a lipid moiety, and amino acids conjugated to an organic derivatizing agent. The pharmaceutical formulation may also comprise one or more additional compounds, such as compounds for treating an ActRIIB-related disorder. Preferably, the pharmaceutical formulation is substantially free of pyrogens. In general, it is preferred to express the GDF trap in a mammalian cell line, which appropriately mediates the native glycosylation of the GDF trap, thereby reducing the possibility of an adverse immune response in the patient. Human and CHO cell lines have been used successfully, and it is expected that other common mammalian expression vectors will be useful.

[0017] In certain aspects, the present disclosure provides a packaged pharmaceutical comprising a pharmaceutical formulation described herein and a label for increasing red blood cell levels in a human.

[0018] In certain aspects, the present disclosure provides a GDF trap, which is a soluble ActRIIB polypeptide comprising an altered ligand-binding (e.g., GDF8-binding) domain. A GDF trap with an altered ligand-binding domain may include, for example, one or more mutations in human ActRIIB amino acid residues such as E37, E39, R40, K55, R56, Y60, A64, K74, W78, L79, D80, F82, and F101 (numbered relative to SEQ ID NO: 1). Optionally, the altered ligand-binding domain may have increased selectivity for ligands such as GDF8 / GDF11 relative to the wild-type ligand-binding domain of the ActRIIB receptor. For illustration, these mutations are described herein to increase the selectivity of the altered ligand-binding domain over activin for GDF11 (and therefore, presumably, GDF8): K74Y, K74F, K74I, L79D, L79E, and D80I. The following mutations have the opposite effect, increasing the ratio of activin binding over GDF11: D54A, K55A, L79A, and F82A. Overall (GDF11 and activin) binding activity can be increased by inclusion of the "tail" region, or presumably unstructured linker region, and also by application of the K74A mutation. Other mutations that cause an overall decrease in ligand binding affinity include: R40A, E37A, R56A, W78A, D80K, D80R, D80A, D80G, D80F, D80M, and D80N. Mutations can be combined to achieve the desired effect. For example, many mutations that affect the GDF11:activin binding ratio have an overall negative effect on ligand binding, and therefore, they can be combined with mutations that generally increase ligand binding to produce improved binding proteins with ligand selectivity. In an exemplary embodiment, the GDF Trap is an ActRIIB polypeptide comprising a L79D or L79E mutation, optionally in combination with additional amino acid substitutions, additions or deletions.

[0019] Optionally, a GDF Trap comprising an altered ligand-binding domain has a K for activin binding of d and K for GDF8 binding d The ratio of the ligand-binding domain to the wild-type ligand-binding domain is at least 2, 5, 10 or even 100 times higher. Optionally, the GDF Trap comprising the altered ligand-binding domain has an IC for inhibiting activin of 50 With IC for inhibition of GDF8 / GDF11 50 The ratio of the ligand-binding domain to the wild-type ActRIIB is at least 2, 5, 10 or even 100 times higher. Optionally, the GDF trap containing the altered ligand-binding domain is used to inhibit the IC of activin. 50At least 2, 5, 10 or even 100 times lower IC 50 Inhibition of GDF8 / GDF11. These GDF Traps can be fusion proteins including an immunoglobulin Fc domain (wild type or mutant). In some cases, the subject soluble GDF Traps are antagonists (inhibitors) of GDF8 and / or GDF11.

[0020] Other GDF traps are contemplated as follows. A GDF trap fusion protein comprising a portion of an ActRIIB sequence derived from SEQ ID NO: 1 or 39 and a second polypeptide portion, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 21-29 of SEQ ID NO: 1 or 39 (optionally starting at 22-25 of SEQ ID NO: 1 or 39) and ending at any one of amino acids 109-134 of SEQ ID NO: 1 or 39, and wherein the GDF trap fusion protein inhibits signaling through activin, tubocurarine, and / or GDF11 in a cell-based assay. The above GDF trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 20-29 of SEQ ID NO: 1 or 39 (optionally starting at 22-25 of SEQ ID NO: 1 or 39) and ending at any one of amino acids 109-133 of SEQ ID NO: 1 or 39. The above-mentioned GDF trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 20-24 of SEQ ID NO: 1 or 39 (optionally starting at 22-25 of SEQ ID NO: 1 or 39) and ending at any one of amino acids 109-133 of SEQ ID NO: 1 or 39. The above-mentioned GDF trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 21-24 of SEQ ID NO: 1 or 39 and ending at any one of amino acids 109-134 of SEQ ID NO: 1 or 39. The above-mentioned GDF trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 20-24 of SEQ ID NO: 1 or 39 and ending at any one of amino acids 118-133 of SEQ ID NO: 1 or 39. The above-mentioned GDF trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 21-24 of SEQ ID NO: 1 or 39 and ending at any one of amino acids 118-134 of SEQ ID NO: 1 or 39. The above-mentioned GDF trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 20-24 of SEQ ID NO: 1 or 39 and ending at any one of amino acids 128-133 of SEQ ID NO: 1 or 39. The above-mentioned GDF trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 20-24 of SEQ ID NO: 1 or 39 and ending at any one of amino acids 128-133 of SEQ ID NO: 1 or 39.The above-mentioned GDF Trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 21-29 of SEQ ID NO: 1 or 39 and ending at any one of amino acids 118-134 of SEQ ID NO: 1 or 39. The above-mentioned GDF Trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 20-29 of SEQ ID NO: 1 or 39 and ending at any one of amino acids 118-133 of SEQ ID NO: 1 or 39. The above-mentioned GDF Trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 21-29 of SEQ ID NO: 1 or 39 and ending at any one of amino acids 128-134 of SEQ ID NO: 1 or 39. The above-mentioned GDF Trap fusion protein, wherein the portion derived from ActRIIB corresponds to a sequence starting at any one of amino acids 20-29 of SEQ ID NO: 1 and ending at any one of amino acids 128-133 of SEQ ID NO: 1 or 39. Surprisingly, the construct starting at 22-25 of SEQ ID NO: 1 or 39 has an activity level greater than that of a protein having the entire extracellular domain of human ActRIIB. In a preferred embodiment, the GDF Trap fusion protein comprises, consists essentially of, or consists of an amino acid sequence starting at amino acid position 25 of SEQ ID NO: 1 or 39 and ending at amino acid position 131 of SEQ ID NO: 1 or 39. In another preferred embodiment, the GDF Trap polypeptide consists of or consists essentially of an amino acid sequence of SEQ ID NO: 7, 26, 28, 29, 32, 37, or 38. Any of the above-mentioned GDF Trap fusion proteins can be produced as a homodimer. Any of the above GDF Trap fusion proteins may have a heterologous portion comprising a constant region from an IgG heavy chain, such as an Fc domain. Any of the above GDF Trap fusion proteins may comprise an acidic amino acid at a position corresponding to position 79 of SEQ ID NO: 1, optionally in combination with one or more additional amino acid substitutions, deletions or insertions relative to SEQ ID NO: 1.

[0021] Other GDF Trap proteins are contemplated as follows. A GDF Trap protein comprising an amino acid sequence having at least 80% identity to the sequence of amino acids 29-109 of SEQ ID NO: 1 or 39, wherein the position corresponding to 64 of SEQ ID NO: 1 is R or K, and wherein the GDF Trap protein inhibits signaling by activin, tubocurarine and / or GDF11 in a cell-based assay. The above GDF Trap protein, wherein at least one change relative to the sequence of SEQ ID NO: 1 or 39 is located outside the ligand binding pocket. The above GDF Trap protein, wherein at least one change relative to the sequence of SEQ ID NO: 1 or 39 is a conservative change located within the ligand binding pocket. The above GDF Trap protein, wherein at least one change relative to the sequence of SEQ ID NO: 1 or 39 is a change at one or more positions selected from the group consisting of: K74, R40, Q53, K55, F82, and L79. The GDF Trap protein described above, wherein the protein comprises at least one NXS / T sequence at a position different from the endogenous NXS / T sequence of ActRIIB and at a position outside the ligand binding pocket.

[0022] Other GDF traps are contemplated as follows. A GDF trap protein comprising an amino acid sequence having at least 80% identity to the sequence of amino acids 29-109 of SEQ ID NO: 1 or 39, and wherein the protein comprises at least one NXS / T sequence at a position different from the endogenous NXS / T sequence of ActRIIB, and at a position outside the ligand binding pocket. The above GDF trap, wherein the GDF trap protein comprises an N at a position corresponding to position 24 of SEQ ID NO: 1 or 39, and an S or T at a position corresponding to position 26 of SEQ ID NO: 1 or 39, and wherein the GDF trap inhibits signaling through activin, tubocurarine, and / or GDF11 in a cell-based assay. The above GDF trap, wherein the GDF trap protein comprises an R or K at a position corresponding to position 64 of SEQ ID NO: 1 or 39. The GDF trap described above, wherein the ActRIIB protein comprises a D or E at a position corresponding to position 79 of SEQ ID NO: 1 or 39, and wherein the GDF trap inhibits signaling through activin, tubocurarine and / or GDF11 in a cell-based assay. The GDF trap described above, wherein at least one change relative to the sequence of SEQ ID NO: 1 or 39 is a conservative change located within the ligand binding pocket. The GDF trap described above, wherein at least one change relative to the sequence of SEQ ID NO: 1 or 39 is a change at one or more positions selected from the group consisting of: K74, R40, Q53, K55, F82 and L79. The GDF trap described above, wherein the protein is a fusion protein further comprising a heterologous portion. Any of the above GDF trap fusion proteins can be produced as a homodimer. Any of the above GDF trap fusion proteins can have a heterologous portion comprising a constant region from an IgG heavy chain, such as an Fc domain.

[0023] In certain aspects, the present disclosure provides nucleic acids encoding GDF trap polypeptides. An isolated polynucleotide may comprise a coding sequence of a soluble GDF trap polypeptide, such as described above. For example, an isolated nucleic acid may comprise a sequence encoding a GDF trap comprising an extracellular domain (e.g., a ligand-binding domain) of an ActRIIB polypeptide having a sequence with one or more sequence variations, and a sequence encoding part or all of a transmembrane domain and / or a cytoplasmic domain of an ActRIIB polypeptide, except for a stop codon positioned in the transmembrane domain or cytoplasmic domain or positioned between the extracellular domain and the transmembrane domain or cytoplasmic domain. For example, an isolated polynucleotide encoding a GDF trap may comprise a full-length ActRIIB polynucleotide sequence, such as SEQ ID NO: 4 or a partially truncated form having one or more variations, the isolated polynucleotide further comprising a transcription stop codon at least six hundred nucleotides before the 3'-terminus or at other locations, such that translation of the polynucleotide produces an extracellular domain optionally fused to a truncated portion of the full-length ActRIIB. The nucleic acids disclosed herein can be operably linked to a promoter for expression, and the disclosure provides cells transformed with such recombinant polynucleotides. The cells are preferably mammalian cells, such as CHO cells.

[0024] In certain aspects, the present disclosure provides methods for preparing GDF Trap polypeptides. Such methods may include expressing any nucleic acid disclosed herein (e.g., SEQ ID NO: 5, 25, 27, 30, or 31) in a suitable cell, such as a Chinese hamster ovary (CHO) cell. Such methods may include: a) culturing the cells under conditions suitable for expression of the GDF Trap polypeptide, wherein the cells are transformed with a GDF Trap expression construct; and b) recovering the GDF Trap polypeptide so expressed. The GDF Trap polypeptide may be recovered as a crude, partially purified, or highly purified fraction using any known technique for obtaining proteins from cell culture.

[0025] In certain aspects, the GDF Trap polypeptides disclosed herein can be used in methods for promoting red blood cell production or increasing red blood cell levels in a subject. In certain embodiments, the disclosure provides methods for treating conditions associated with low red blood cell counts or low hemoglobin levels (e.g., anemia) or promoting erythropoiesis in patients in need thereof. The methods may include administering an effective amount of a GDF Trap polypeptide to a subject in need thereof. In certain aspects, the disclosure provides for the use of a GDF Trap polypeptide in the preparation of a medicament for treating a condition or condition described herein.

[0026] In certain aspects, the present disclosure provides methods for administering GDF Trap polypeptides to a patient. In part, the present disclosure illustrates that GDF Trap polypeptides can be used to increase red blood cell and hemoglobin levels. GDF Trap polypeptides can also be used to treat or prevent other therapeutic applications, such as promoting muscle growth. In some cases, when GDF Trap polypeptides are administered for promoting muscle growth, it may be desirable to monitor the effects on red blood cells during administration of the GDF Trap polypeptides, or to determine or adjust the dosing of the GDF Trap polypeptides in order to reduce undesirable effects on red blood cells. For example, an increase in red blood cell levels, hemoglobin levels, or hematocrit levels can cause an increase in blood pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0028] Figure 1 An alignment of the extracellular domains of human ActRIIA (SEQ ID NO: 15) and human ActRIIB (SEQ ID NO: 2) is shown, with residues indicated by boxes that are inferred herein to directly contact the ligand (ligand binding pocket) based on composite analysis of multiple ActRIIB and ActRIIA crystal structures.

[0029] Figure 2 Shown is a multiple sequence alignment of various vertebrate ActRIIB proteins and human ActRIIA (SEQ ID NOs: 16-23).

[0030] Figure 3 The entire amino acid sequence of the GDF trap ActRIIB (L79D 20-134)-hFc (SEQ ID NO: 11) is shown, including the TPA leader sequence (double underlined), ActRIIB extracellular domain (residues 20-134 in SEQ ID NO: 1; underlined), and hFc domain. The aspartic acid substituted at position 79 of the native sequence is double underlined and highlighted, as is the glycine shown by sequencing the N-terminal residue in the mature fusion protein.

[0031] Figure 4 The nucleotide sequence encoding ActRIIB(L79D 20-134)-hFc is shown. SEQ ID NO: 25 corresponds to the sense strand, and SEQ ID NO: 33 corresponds to the antisense strand. The TPA leader sequence (nucleotides 1-66) is double underlined, and the ActRIIB extracellular domain (nucleotides 76-420) is underlined.

[0032] Figure 5The entire amino acid sequence of the truncated GDF trap ActRIIB (L79D 25-131) -hFc (SEQ ID NO: 26) is shown, including the TPA leader sequence (double underlined), the truncated ActRIIB extracellular domain (residues 25-131 in SEQ ID NO: 1; underlined), and the hFc domain. The aspartic acid substituted at position 79 of the native sequence is double underlined and highlighted, as is the glutamic acid shown by sequencing the N-terminal residue in the mature fusion protein.

[0033] Figure 6 The nucleotide sequence encoding ActRIIB (L79D 25-131) -hFc is shown. SEQ ID NO: 27 corresponds to the sense strand, and SEQ ID NO: 34 corresponds to the antisense strand. The TPA leader sequence (nucleotides 1-66) is double underlined, and the truncated ActRIIB extracellular domain (nucleotides 76-396) is underlined. The amino acid sequence of the ActRIIB extracellular domain is also shown (residues 25-131 in SEQ ID NO: 1).

[0034] Figure 7 The amino acid sequence of the truncated GDF trap ActRIIB (L79D 25-131)-hFc without the leader sequence is shown (SEQ ID NO: 28). The truncated ActRIIB extracellular domain (residues 25-131 in SEQ ID NO: 1) is underlined. The aspartic acid substituted at position 79 of the native sequence is double underlined and highlighted, as is the glutamic acid shown by sequencing the N-terminal residue in the mature fusion protein.

[0035] Figure 8 The amino acid sequence of the truncated GDF trap ActRIIB (L79D 25-131) without the leader sequence, hFc domain and linker is shown (SEQ ID NO: 29). The aspartic acid substituted at position 79 of the native sequence is double underlined and highlighted, as is the glutamic acid shown by sequencing the N-terminal residue in the mature fusion protein.

[0036] Fig. 9 An alternative nucleotide sequence encoding ActRIIB(L79D 25-131)-hFc is shown. SEQ ID NO: 30 corresponds to the sense strand, and SEQ ID NO: 35 corresponds to the antisense strand. The TPA leader sequence (nucleotides 1-66) is double underlined, the truncated ActRIIB extracellular domain (nucleotides 76-396) is underlined, and the substitutions in the extracellular domain wild-type nucleotide sequence are double underlined and highlighted (compared to SEQ ID NO: 27, Figure 6). The amino acid sequence of the ActRIIB extracellular domain is also shown (residues 25-131 in SEQ ID NO: 1).

[0037] Fig.10 Show Fig. 9 Nucleotides 76-396 (SEQ ID NO: 31) of the alternative nucleotide sequence shown in (SEQ ID NO: 30). Fig. 9 The same nucleotide substitutions indicated in are also underlined and highlighted here. SEQ ID NO: 31 encodes only a truncated ActRIIB extracellular domain (corresponding to residues 25-131 in SEQ ID NO: 1) with a L79D substitution, e.g., ActRIIB(L79D 25-131).

[0038] Fig.11 Shown is the effect of ActRIIB(L79D 25-131)-hFc on hemoglobin concentration in a mouse model of chemotherapy-induced anemia. Data are mean ± SEM. **, P < 0.01, vs. paclitaxel, at the same time point. The GDF trap counteracted anemia induced by paclitaxel treatment.

[0039] Fig.12 Shown is the effect of ActRIIB(L79D 25-131)-hFc on red blood cell (RBC) levels in a unilateral nephrectomized (NEPHX) mouse model of chronic kidney disease. Data are mean ± SEM. ***, P < 0.001, relative to baseline. The GDF trap reversed the nephrectomy-induced anemia observed in control mice.

[0040] Fig.13 ActRIIB(L79D 25-131)-hFc effects on red blood cell (RBC), hemoglobin (HGB), and hematocrit (HCT) levels in a unilateral nephrectomized (NEPHX) mouse model of chronic kidney disease are shown. Data are mean changes (± SEM) from baseline over a 4-week period. *, P<0.05; **, P<0.01; ***, P<0.001, relative to NEPHX controls. The GDF trap prevented the nephrectomy-associated decrease in these red blood cell parameters, increasing each to a similar magnitude as in kidney-intact (sham) mice.

[0041] Fig.14Shown is the effect of ActRIIB(L79D 25-131)-hFc on red blood cell (RBC) levels in a rat model of acute blood loss-induced anemia. Blood removal occurred on day -1, and dosing was on days 0 and 3. Data are mean ± SEM. **, P < 0.01; ***, P < 0.001, relative to vehicle at the same time points. The GDF trap improved the speed and extent of recovery from blood loss-induced anemia.

[0042] Fig.15 Shown is the effect of treatment with ActRIIB(L79D 20-134)-hFc (grey) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change from baseline in red blood cell concentration in cynomolgus monkeys. VEH = vehicle. Data are means + SEM. n = 4-8 per group.

[0043] Fig.16 Shown is the effect of treatment with ActRIIB(L79D 20-134)-hFc (grey) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change from baseline in hematocrit in cynomolgus monkeys. VEH = vehicle. Data are means + SEM. n = 4-8 per group.

[0044] Fig.17 Shown are the effects of treatment with ActRIIB(L79D 20-134)-hFc (grey) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change from baseline in hemoglobin concentration in cynomolgus monkeys. VEH = vehicle. Data are mean values. + SEM. n = 4-8 per group.

[0045] Fig.18 Shown are the effects of treatment with ActRIIB(L79D 20-134)-hFc (grey) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change from baseline in circulating reticulocyte concentration in cynomolgus monkeys. VEH = vehicle. Data are means + SEM. n = 4-8 per group.

[0046] Fig.19The effect of combined treatment with erythropoietin (EPO) and ActRIIB (L79D 25-131) -hFc for 72 hours on hematocrit in mice is shown. Data are mean ± SEM (n = 4 per group), and means that are significantly different from each other (p < 0.05, unpaired t test) are designated by different letters. Compared with the vehicle, combined treatment increased hematocrit by 23%, which is a synergistic increase greater than the sum of the individual effects of EPO and ActRIIB (L79D 25-131) -hFc.

[0047] Fig. 20 The effect of combined treatment with EPO and ActRIIB (L79D 25-131)-hFc for 72 hours on hemoglobin concentration in mice is shown. Data are mean ± SEM (n = 4 per group), and means significantly different from each other (p < 0.05) are designated by different letters. Compared with the vehicle, combined treatment increased hemoglobin concentration by 23%, which is also a synergistic effect.

[0048] Fig.21 The effect of combined treatment with EPO and ActRIIB (L79D 25-131)-hFc for 72 hours on red blood cell concentration in mice is shown. Data are mean ± SEM (n = 4 per group), and means significantly different from each other (p < 0.05) are designated by different letters. Compared with the vehicle, combined treatment increased hemoglobin concentration by 20%, which is also a synergistic effect.

[0049] Fig. 22 The effect of combined treatment with EPO and ActRIIB (L79D 25-131)-hFc for 72 hours on the number of erythropoietic precursor cells in the spleen of mice is shown. Data are mean ± SEM (n = 4 per group), and means that are significantly different from each other (p < 0.01) are designated by different letters. Although EPO alone significantly increases the number of basophilic erythroblasts (BasoE) at the expense of late precursor maturation, combined treatment increases the number of BasoE to a lesser but still significant extent, while supporting maturation of late precursors without reduction. DETAILED DESCRIPTION OF THE INVENTION

[0050] 1. Overview

[0051] EPO is a glycoprotein hormone involved in the growth and maturation of erythrocyte progenitor cells into erythrocytes. EPO is produced by the liver during fetal life and by the kidneys in adults. The reduced production of EPO that usually occurs as a consequence of renal failure in adults causes anemia. EPO has been produced by genetic engineering techniques based on the expression and secretion of protein from host cells transfected with the EPO gene. The administration of this recombinant EPO is effective in the treatment of anemia. For example, Eschbach et al. (1987, NEngl J Med 316:73) describe the use of EPO to correct anemia caused by chronic renal failure.

[0052] The effects of EPO are mediated by its binding to and activation of cell surface receptors belonging to the cytokine receptor superfamily and designated as EPO receptors. Human and murine EPO receptors have been cloned and expressed (D'Andrea et al., 1989, Cell 57:277; Jones et al., 1990, Blood 76:31; Winkelman et al., 1990, Blood 76:24; WO90 / 08822 / US Patent No. 5,278,065). The human EPO receptor gene encodes a 483 amino acid transmembrane protein that contains an extracellular domain of approximately 224 amino acids and exhibits approximately 82% amino acid sequence identity with the murine EPO receptor (see US Patent No. 6,319,499). The cloned full-length EPO receptor (66-72 kDa) expressed in mammalian cells binds to erythroid progenitor cells with an affinity (K) similar to that of the native receptor on erythroid progenitor cells. D =100-300 nM) binds to EPO. Therefore, it is believed that this form contains the main EPO binding determinant and is called the EPO receptor. By analogy with other closely related cytokine receptors, it is believed that the EPO receptor dimerizes after agonist binding. However, the detailed structure of the EPO receptor, which can be a multimeric complex, and its specific activation mechanism are not completely clear (U.S. Patent No. 6,319,499).

[0053] EPO receptor activation causes several biological effects. These include increased proliferation of immature erythroblasts, increased differentiation of immature erythroblasts, and decreased apoptosis of erythroid progenitor cells (Liboi et al., 1993, Proc Natl Acad Sci USA 90:11351-11355; Koury et al., 1990, Science 248:378-381). The EPO receptor signal transduction pathways that mediate proliferation and differentiation appear to be different (Noguchi et al., 1988, Mol Cell Biol 8:2604; Patel et al., 1992, J Biol Chem 1992, 267:21300; Liboi et al., ibid). Some results suggest that accessory proteins may be required for mediation of differentiation signals (Chiba et al., 1993, Nature 362:646; Chiba et al., 1993, Proc Natl Acad Sci USA 90:11593); however, there is controversy regarding the role of accessory proteins in differentiation because constitutively activated forms of the receptor can stimulate both proliferation and differentiation (Pharr et al., 1993, Proc Natl Acad Sci USA 90:938).

[0054] EPO receptor activators include small molecule erythropoiesis-stimulating agents (ESAs) and compounds based on EPO. The former example is an agonist based on a dimeric peptide covalently linked to polyethylene glycol (proprietary name Hematide), which has been shown to have erythropoiesis-stimulating properties in healthy volunteers and patients with chronic kidney disease and endogenous anti-EPO antibodies (Stead et al., 2006, Blood 108: 1830-1834; Macdougall et al., 2009, N Engl J Med 361: 1848-1855). Other examples include non-peptide-based ESAs (Qureshi et al., 1999, Proc Natl Acad Sci USA 96: 12156-12161).

[0055] EPO receptor activators also include compounds that do not contact the EPO receptor itself and indirectly stimulate erythropoiesis by enhancing endogenous EPO production. For example, hypoxia-induced transcription factors (HIFs) are endogenous stimuli of EPO gene expression, which are inhibited (destabilized) by cell regulatory mechanisms under normoxic conditions. Therefore, HIF prolyl hydroxylase inhibitors are being studied for EPO-inducing activity in vivo. Other indirect activators of EPO receptors include GATA-2 transcription factor inhibitors (Nakano et al., 2004, Blood 104:4300-4307), which enhance the inhibition of EPO gene expression, and hematopoietic cell phosphatase (HCP or SHP-1) inhibitors, which work as negative regulators of EPO receptor signal transduction (Klingmuller et al., 1995, Cell 80:729-738).

[0056] The transforming growth factor-β (TGF-β) superfamily includes a variety of growth factors that have common sequence elements and structural motifs. These proteins are known to exert biological effects on a variety of cell types in vertebrates and invertebrates. Members of this superfamily perform important functions in pattern formation and tissue specialization during embryonic development and can affect a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. The family is divided into two major branches: the BMP / GDF and TGF-β / activin / BMP10 branches, whose members have diverse, usually complementary effects. By manipulating the activity of TGF-β family members, significant physiological changes in organisms can often be caused. For example, Piedmont and Belgian Blue cattle breeds carry loss-of-function mutations in the GDF8 (also known as tubocurarine) gene, which results in a significant increase in muscle mass. Grobet et al., Nat Genet. 1997, 17(1):71-4. Furthermore, in humans, the inactive allele of GDF8 is associated with increased muscle mass and, reportedly, superior strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

[0057] TGF-β signaling is mediated by a heteromeric complex of type I and type II serine / threonine kinase receptors that phosphorylate and activate downstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat. Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors are transmembrane proteins composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine specificity. Type I receptors are essential for signal transduction. Type II receptors are required for ligand binding and type I receptor expression. Type I and type II activin receptors form a stable complex upon ligand binding, resulting in phosphorylation of type I receptors by type II receptors.

[0058] Two related type II receptors (ActRII), ActRIIA and ActRIIB, have been identified as type II receptors for activins (Mathews and Vale, 1991, Cell 65:973-982; Attisano et al., 1992, Cell 68: 97-108). In addition to activins, ActRIIA and ActRIIB can biochemically interact with several other TGF-β family proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita et al., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is the major type I receptor for activins, particularly activin A, and ALK-7 can also serve as a receptor for activins, particularly activin B. In certain embodiments, the invention relates to ligands that antagonize the ActRIIB receptor (also referred to as ActRIIB ligands) with the subject GDF Trap polypeptides. Exemplary ligands for the ActRIIB receptor include some TGF-β family members, such as Activin, Nodal, GDF8, GDF11, and BMP7.

[0059] Activins are dimeric polypeptide growth factors that belong to the TGF-β superfamily. There are three major forms of activin (A, B, and AB), which are homo / heterodimers of two closely related β subunits (β A β A , β B β B and β A β B). The human genome also encodes activin C and activin E, which are expressed primarily in the liver and are also known to include heterodimeric forms of βc or βe. In the TGF-β superfamily, activin is a unique and multifunctional factor that stimulates hormone production in ovarian and placental cells, supports neuronal cell survival, affects cell cycle progression positively or negatively depending on the cell type, and induces mesoderm differentiation at least in amphibian embryos (DePaolo et al., 1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997, Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963). In addition, erythroid differentiation factor (EDF) isolated from stimulated human monocytic leukemia cells has been found to be identical to activin A (Murata et al., 1988, PNAS, 85:2434). Activin A has been shown to promote erythropoiesis in the bone marrow. In some 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 FSH secretion and synthesis, while inhibin prevents FSH secretion and synthesis. Other proteins that can regulate the biological activity of activin and / or bind to activin include follistatin (FS), follistatin-related protein (FSRP) and α2-macroglobulin.

[0060] Nodal protein has function in mesoderm and endoderm induction and formation and subsequent organization of axial structures such as heart and stomach in early embryogenesis. It has been shown that the dorsal tissue in the developing vertebrate embryo mainly contributes to the axial structure of the notochord and the prechordal plate, while it recruits surrounding cells to form non-axial embryonic structures. Nodal appears to signal through both type I and type II receptors and intracellular effectors called Smad proteins. Recent studies support the view that ActRIIA and ActRIIB play the role of type II receptors of Nodal (Sakuma et al., Genes Cell. 2002, 7: 401-12). It is suggested that Nodal ligands interact with their cofactors (e.g., cripto) to activate activin type I and type II receptors, which phosphorylate Smad2. Nodal protein is involved in many events important to early vertebrate embryos, including mesoderm formation, anterior patterning, and left-right axis specification. Experimental evidence has shown that Nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to specifically respond to activin and TGF-β. However, Nodal cannot induce pTlx2-Lux, a reporter that specifically responds to bone morphogenetic protein. Recent results provide direct biochemical evidence that Nodal signaling is mediated by the activin-TGF-β pathway Smads, Smad2, and Smad3. Other evidence has shown that the extracellular cripto protein is required for Nodal signaling, making it different from activin or TGF-β signaling.

[0061] Growth and differentiation factor-8 (GDF8) is also known as tubocurarine. GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highly expressed in developing and adult skeletal muscle. GDF8 null mutations in transgenic mice are characterized by significant hypertrophy and hyperplasia of skeletal muscle (McPherron et al., Nature, 1997, 387:83-90). Similar increases in skeletal muscle mass are evident in naturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974, Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994, 38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997, 94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and are striking in humans (Schuelke et al., N Engl J Med 2004;350:2682-8). Studies have also shown that muscle atrophy associated with HIV-infection in humans is accompanied by increased GDF8 protein expression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). In addition, GDF8 can regulate the production of muscle-specific enzymes (e.g., creatine kinase) and regulate myoblast cell proliferation (WO00 / 43781). GDF8 propeptide can non-covalently bind to mature GDF8 domain dimers, inactivating their biological activity (Miyazono et al. (1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol. Chem., 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3: 35-43). Other proteins that bind to GDF8 or structurally related proteins and inhibit their biological activity include follistatin, and potentially follistatin-related proteins (Gamer et al. (1999) Dev. Biol., 208: 222-232).

[0062] Growth and differentiation factor-11 (GDF11), also known as BMP11, is a secreted protein (McPherron et al., 1999, Nat. Genet. 22: 260-264). GDF11 is expressed in the tail bud, limb buds, maxillary and mandibular arches and dorsal root ganglia during mouse development (Nakashima et al., 1999, Mech. Dev. 80: 185-189). GDF11 plays a unique role in patterning mesoderm and neural tissue (Gamer et al., 1999, Dev Biol., 208: 222-32). GDF11 is shown to be a negative regulator of cartilage formation and myogenesis in developing chicken limbs (Gamer et al., 2001, Dev Biol. 229: 407-20). The expression of GDF11 in muscle also indicates its role in regulating muscle growth in a manner similar to GDF8. In addition, the expression of GDF11 in the brain suggests that GDF11 may also possess activities related to the function of neural tissue. Interestingly, GDF11 was found to inhibit neurogenesis in the olfactory epithelium (Wu et al., 2003, Neuron. 37: 197-207). Therefore, GDF11 may have in vitro and in vivo uses in the treatment of diseases such as muscle diseases and neurodegenerative diseases (e.g., amyotrophic lateral sclerosis).

[0063] Bone morphogenetic protein (BMP7), also known as osteogenic protein-1 (OP-1), is known to induce cartilage and bone formation. In addition, BMP7 regulates a wide range of physiological processes. For example, BMP7 may be an osteoinductive factor responsible for the phenomenon of epithelial bone formation. BMP7 has also been found to play a role in calcium regulation and bone homeostasis. Similar to activin, BMP7 binds to type II receptors ActRIIA and ActRIIB. However, BMP7 and activin recruit different type I receptors into heteromeric receptor complexes. The main BMP7 type I receptor observed is ALK2, while activin uniquely binds to ALK4 (ActRIIB). BMP7 and activin trigger different biological responses and activate different Smad pathways (Macias-Silva et al., 1998, J Biol Chem. 273:25628-36).

[0064] As described herein, GDF Trap polypeptides that are variant ActRIIB polypeptides (ActRIIB) are more effective at increasing red blood cell levels in vivo than wild-type soluble ActRIIB polypeptides and have beneficial effects in various anemia models. Additionally, it was shown that the use of GDF Trap polypeptides in combination with EPO receptor activators resulted in a substantial increase in red blood cell formation. It should be noted that hematopoiesis is a complex process that is regulated by a variety of factors, including erythropoietin, G-CSF, and iron homeostasis. The terms "increased red blood cell levels" and "promoting 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 as to the mechanism by which such changes occur.

[0065] In addition to stimulating red blood cell levels, GDF Trap polypeptides are useful for a variety of therapeutic purposes, including, for example, promoting muscle growth (see PCT Publication Nos. WO 2006 / 012627 and WO 2008 / 097541, which are incorporated herein by reference in their entireties). In some cases, when GDF Trap polypeptides are administered for the purpose of increasing muscle, it may be desirable to reduce or minimize the effects on red blood cells. By monitoring various hematological parameters in patients being treated with GDF Trap polypeptides or candidates for treatment with GDF Trap polypeptides, appropriate dosing (including administration amount and frequency) can be determined based on the needs of the individual patient, baseline hematological parameters, and the purpose of the treatment. Moreover, the progress of treatment and the effects on one or more hematological parameters over time can be used to manage patients administered GDF Trap polypeptides by facilitating patient care, determining appropriate maintenance doses (amounts and frequency), and the like.

[0066] The terms used in this specification generally have their ordinary meaning in the art in the context of the present invention and in the specific context in which each term is used. Certain terms are discussed below and in other parts of the specification to provide additional guidance to practitioners in describing the compositions and methods of the present invention and how to make and use them. Any scope of use or meaning of a term will be apparent from the specific context in which the term is used.

[0067] "About" and "approximately" should generally indicate an acceptable degree of error in the measured amount, taking into account the nature and precision of the measurement method. Typically, exemplary degrees of error are within 20% of a given value or range of values, preferably within 10%, and more preferably within 5%.

[0068] Alternatively, particularly in biological systems, the terms "about" and "approximately" may refer to values ​​within an order of magnitude, preferably within 5 times, more preferably within 2 times, of a given value. Unless otherwise indicated, the quantities given herein are approximate, meaning that the term "about" or "approximately" can be inferred when not expressly indicated.

[0069] The method of the present invention may include a step of comparing sequences to each other, including comparing the wild-type sequence with one or more mutants (sequence variants). Such comparisons typically include alignments of polymer sequences, for example, using sequence alignment programs and / or algorithms well known in the art (e.g., BLAST, FASTA, and MEGALIGN, to name a few). Those skilled in the art will readily appreciate that in such alignments, when a mutation includes an insertion or deletion of a residue, the sequence alignment will introduce a "space" (usually represented by a dash or "A") in the polymer sequence that does not include the residue of the insertion or deletion.

[0070] "Homology" in all its grammatical forms and spelling variations refers to the relationship between two proteins with a "common evolutionary origin" (including proteins from superfamilies of the same biological species, as well as homologous proteins from different biological species). Such proteins (and their encoding nucleic acids) have sequence homology, which is reflected in their sequence similarity, whether in terms of percentage identity or in terms of the presence of specific residues or motifs and conserved positions.

[0071] The term "sequence similarity" in all of 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.

[0072] However, in common usage and in this application, when the term "homology" is modified by an adverb such as "highly", it may indicate sequence similarity and may or may not involve a common evolutionary origin.

[0073] 2. GDF capture peptides

[0074] In certain aspects, the present invention relates to GDF trap polypeptides, such as soluble variant ActRIIB polypeptides, including, for example, fragments, functional variants and modified forms of ActRIIB polypeptides. In certain embodiments, the GDF trap polypeptide has at least one biological activity similar or identical to the corresponding wild-type ActRIIB polypeptide. For example, the GDF trap polypeptide of the present invention can bind to and inhibit the function of an ActRIIB ligand (e.g., activin A, activin AB, activin B, Nodal, GDF8, GDF11 or BMP7). Optionally, the GDF trap polypeptide increases red blood cell levels. Examples of GDF trap polypeptides include human ActRIIB precursor polypeptides (SEQ ID NO: 1 or 39) with one or more sequence changes, and soluble human ActRIIB polypeptides (e.g., SEQ ID NOs: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, 40 and 41) with one or more sequence changes. A GDF Trap refers to an ActRIIB polypeptide that has reduced affinity for activin relative to other ActRIIB ligands, including, for example, GDF11 and / or tubocurarine.

[0075] The term "ActRIIB" as used herein refers to a family of type IIb activin receptor (ActRIIB) proteins from any species and variants derived from the ActRIIB proteins by mutagenesis or other modifications. References to ActRIIB herein should be understood as references to any of the currently identified forms. Members of the ActRIIB family are typically transmembrane proteins consisting of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase activity. The amino acid sequences of the human ActRIIA soluble extracellular domain (provided for comparison) and the ActRIIB soluble extracellular domain are in Figure 1 Medium illustration.

[0076] The term "ActRIIB polypeptide" includes polypeptides comprising any naturally occurring polypeptide of an ActRIIB family member and any variants thereof (including mutants, fragments, fusions, and peptido-mimetic forms) that retain useful activity. See, e.g., WO / 2006 / 012627. For example, ActRIIB polypeptides include polypeptides derived from the sequence of any known ActRIIB, having a sequence that is at least about 80% identical, optionally at least 85%, 90%, 95%, 97%, 99% or more identical to the sequence of an ActRIIB polypeptide. For example, an ActRIIB polypeptide can bind to an ActRIIB protein and / or activin and inhibit its function. ActRIIB polypeptides that are selected as GDF traps can be selected for their activity in promoting erythrocyte formation in vivo. Examples of ActRIIB polypeptides include human ActRIIB precursor polypeptides (SEQ ID NOs: 1 and 39) and soluble human ActRIIB polypeptides (e.g., SEQ ID NOs: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, 40, and 41). The amino acid numbering for all ActRIIB-related polypeptides described herein is based on the numbering of SEQ ID NO: 1, unless otherwise specifically indicated.

[0077] The sequence of human ActRIIB precursor protein is as follows:

[0078]

[0079] The signal peptide is single underlined; the extracellular domain is in bold, and potential N-linked glycosylation sites are boxed.

[0080] A form with alanine at position 64 is also reported in the literature as follows:

[0081]

[0082] The sequence of the soluble (extracellular), processed polypeptide of human ActRIIB is as follows:

[0083]

[0084] The optional forms with A64 are as follows:

[0085]

[0086] Under some conditions, proteins can be produced with the "SGR..." sequence at the N-terminus. The C-terminal "tail" of the extracellular domain is underlined. The sequence without the "tail" (Δ15 sequence) is shown below:

[0087]

[0088] The optional forms with A64 are as follows:

[0089]

[0090] Under some conditions, the protein can be produced with an "SGR..." sequence at the N-terminus. The nucleic acid sequence encoding the human ActRIIB precursor protein is as follows: (nucleotides 5-1543 of Genbank entry NM_001106) (the sequence shown provides an alanine at position 64 and can be modified to provide an arginine instead)

[0091]

[0092]

[0093] The nucleic acid sequence encoding the human ActRIIB soluble (extracellular) polypeptide is as follows (the sequence shown provides an alanine at position 64, and may be modified to provide an arginine instead):

[0094]

[0095] In a particular embodiment, the present invention relates to a GDF trap polypeptide that is a soluble ActRIIB polypeptide in a variant form. The term "soluble ActRIIB polypeptide" as described herein generally refers to a polypeptide comprising an extracellular domain of an ActRIIB protein. The term "soluble ActRIIB polypeptide" as used herein includes any naturally occurring extracellular domain of an ActRIIB protein and any variant thereof (including mutants, fragments, and peptide mimetic forms) that retains useful activity. For example, the extracellular domain of an ActRIIB protein binds to a ligand and is generally soluble. Examples of soluble ActRIIB polypeptides include ActRIIB soluble polypeptides (e.g., SEQ ID NOs: 22, 3, 7, 11, 26, 28, 29, 32, 37, 38, 40, and 41). Other examples of soluble ActRIIB polypeptides include a signal sequence in addition to the extracellular domain of an ActRIIB protein, see Example 1. The signal sequence may be a natural signal sequence of ActRIIB, or a signal sequence from another protein, such as tissue plasminogen activator (TPA). signal sequence or honey bee melittin (HBM) signal sequence.

[0096] The present disclosure identifies functionally active portions and variants of ActRIIB. Applicants have determined that an Fc fusion protein having a sequence disclosed by Hilden et al. with an alanine (A64) at a position corresponding to amino acid 64 of SEQ ID NO: 1 (Blood. 1994 Apr 15; 83(8): 2163-70) has a relatively low affinity for activin and GDF-11. In contrast, the same Fc fusion protein with an arginine (R64) at position 64 has an affinity for activin and GDF-11 in the low nanomolar to high picomolar range. Therefore, in the present disclosure, the sequence with R64 is used as a wild-type reference sequence for human ActRIIB.

[0097] Attisano et al. (Cell. 1992 Jan 10; 68(1): 97-108) showed that the deletion of the C-terminal proline knot in the extracellular domain of ActRIIB reduces the affinity of the receptor for activin. The ActRIIB-Fc fusion protein "ActRIIB(20-119)-Fc" containing amino acids 20-119 of SEQ ID NO: 1 has reduced binding to GDF-11 and activin, the reduction being relative to ActRIIB(20-134)-Fc, which includes the proline knot region and the complete membrane-proximal domain. However, the ActRIIB(20-129)-Fc protein retains similar but somewhat reduced activity relative to the wild type, even if the proline knot region is destroyed. Therefore, ActRIIB extracellular domains that stop at amino acids 134, 133, 132, 131, 130, and 129 are all expected to be active, but constructs that stop at 134 or 133 may be most active. Similarly, mutations at any of residues 129-134 are not expected to change ligand binding affinity to large limits. In support of this, mutations at P129 and P130 do not substantially reduce ligand binding. Thus, a GDF trap polypeptide that is an ActRIIB-Fc fusion protein may terminate as early as amino acid 109 (the last cysteine), but forms that terminate at or between 109 and 119 are expected to have reduced ligand binding. Amino acid 119 is poorly conserved and therefore easily altered or truncated. Forms that terminate at 128 or later retain ligand binding activity. Forms that terminate at 119 and 127 or between 119 and 127 will have moderate binding capacity. Any of these forms may be desired for use depending on the clinical or experimental situation.

[0098] At the N-terminus of ActRIIB, it is expected that proteins starting at or before amino acid 29 will maintain ligand binding activity. Amino acid 29 represents the initial cysteine. The alanine to asparagine mutation at position 24 introduces the N-linked glycosylation sequence and does not substantially affect ligand binding. This confirms that mutations in the region between the signal cleavage peptide and the cysteine ​​cross-linking region corresponding to amino acids 20-29 are fully tolerated. In particular, constructs starting at positions 20, 21, 22, 23 and 24 will maintain activity, and constructs starting at positions 25, 26, 27, 28 and 29 are also expected to maintain activity. The data shown in the examples illustrate that, surprisingly, constructs starting at 22, 23, 24 or 25 will have the greatest activity.

[0099] In general, the active portion of ActRIIB comprises amino acids 29-109 of SEQ ID NO: 1, and a GDF Trap construct can, for example, comprise a portion of ActRIIB starting at residues corresponding to amino acids 20-29 of SEQ ID NO: 1 or 39 and ending at positions corresponding to amino acids 109-134 of SEQ ID NO: 1 or 39. Other examples include constructs that start at positions 20-29 or 21-29 of SEQ ID NO: 1 or 39 and end at positions 119-134, 119-133, 129-134, or 129-133 of SEQ ID NO: 1 or 39. Other examples include constructs that start at positions 20-24 (or 21-24 or 22-25) of SEQ ID NO: 1 or 39 and end at positions 109-134 (or 109-133), 119-134 (or 119-133), or 129-134 (or 129-133) of SEQ ID NO: 1 or 39. Variants within these ranges are also contemplated, particularly those that are at least 80%, 85%, 90%, 95%, or 99% identical to the corresponding portion of SEQ ID NO: 1 or 39. In certain embodiments, the GDF Trap polypeptide comprises, consists essentially of, or consists of a polypeptide having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid residues 25-131 of SEQ ID NO: 1 or 39. In certain embodiments, the GDF Trap polypeptide comprises, consists essentially of, or consists of a polypeptide having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 7, 26, 28, 29, 32, 37 or 38. In preferred embodiments, the GDF Trap polypeptide consists of, or consists essentially of, the amino acid sequence of SEQ ID NOs: 7, 26, 28, 29, 32, 37 or 38.

[0100] The present disclosure includes the results of the analysis of the composite ActRIIB structure, which is Figure 1, indicating that the ligand binding pocket is limited by residues Y31, N33, N35, L38 to T41, E47, E50, Q53 to K55, L57, H58, Y60, S62, K74, W78 to N83, Y85, R87, A92 and E94 to F101. At these positions, it is expected that conservative mutations will be tolerated, although the K74A mutation is fully tolerated, such as R40A, K55A, F82A and the mutation of position L79. R40 is K in clawed frogs, indicating that the basic amino acid at this position will be tolerated. Q53 is R in cattle ActRIIB and K in clawed frogs ActRIIB, and therefore the amino acid including R, K, Q, N and H will be tolerated at this position. Thus, a general formula for a GDF Trap protein is one that includes amino acids 29-109 of SEQ ID No. 1 or 39, but optionally begins at positions in the range 20-24 or 22-25, and ends at positions in the range 129-134, and contains no more than 1, 2, 5, 10, or 15 conservative amino acid changes in the ligand binding pocket, and 0, 1, or more non-conservative changes at positions 40, 53, 55, 74, 79, and / or 82 in the ligand binding pocket. Such a protein may retain greater than 80%, 90%, 95%, or 99% sequence identity to the sequence of amino acids 29-109 of SEQ ID NO: 1 or 39. Sites outside the binding pocket where variability may be particularly well tolerated include the amino and carboxyl termini of the extracellular domain (as described above), and positions 42-46 and 65-73. The asparagine to alanine change (N65A) at position 65 actually improves ligand binding in the A64 background, and is therefore expected to have no deleterious effect on ligand binding in the A64 background. This change has the potential to eliminate glycosylation at N65 in the A64 background, thus suggesting that significant changes in this region may be tolerated. While the R64A change is poorly tolerated, R64K is well tolerated, and therefore other basic residues such as H may be tolerated at position 64.

[0101] ActRIIB is fully conserved in almost all vertebrates, with a large, completely conserved extracellular domain extension. Many ligands that bind to ActRIIB are also highly conserved. Accordingly, comparisons of ActRIIB sequences from various vertebrate organisms provide insights into residues that can be altered. Therefore, an active human ActRIIB variant polypeptide used as a GDF trap may include one or more amino acids at a position corresponding to the sequence of another vertebrate ActRIIB, or may include residues similar to those in a human or other vertebrate sequence. The following examples illustrate this method for determining active ActRIIB variants. L46 is valine in ActRIIB of the clawed frog species, and therefore this position can be changed and optionally can be changed to another hydrophobic residue such as V, I or F, or a non-polar residue such as A. E52 is K in the clawed frog species, indicating that this site can tolerate a variety of changes, including polar residues such as E, D, K, R, H, S, T, P, G, Y and possibly A. T93 is K in Xenopus, indicating that a wide range of structural variations are tolerated at this position, and polar residues are favored, such as S, K, R, E, D, H, G, P, G, and Y. F108 is Y in Xenopus, and thus Y or other hydrophobic groups such as I, V, or L should be tolerated. E111 is K in Xenopus, indicating that charged residues will be tolerated at this position, including D, R, K, and H, as well as Q and N. R112 is K in Xenopus, indicating that basic residues are tolerated at this position, including R and H. The A at position 119 is relatively poorly conserved, and behaves as P in rodents and V in Xenopus, so essentially any amino acid should be tolerated at this position.

[0102] The present disclosure demonstrates that the addition of an additional N-linked glycosylation site (NXS / T) increases the serum half-life of ActRIIB-Fc fusion proteins relative to the ActRIIB(R64)-Fc form. By introducing an asparagine at position 24 (A24N construct), a NXT sequence conferring a longer half-life was generated. Other NX(T / S) sequences are found at 42-44 (NQS) and 65-67 (NSS), although the latter may not be efficiently glycosylated with an R at position 64. NXS / T sequences may typically be found at Figure 1The NXS / T sequence may be introduced at a position outside the ligand binding pocket defined in . Particularly suitable sites for introducing non-endogenous NXS / T sequences include amino acids 20-29, 20-24, 22-25, 109-134, 120-134 or 129-134. The NXS / T sequence may also be introduced into the linker between the ActRIIB sequence and the Fc or other fusion component. Such sites may be introduced with minimal effort by introducing an N at the correct position relative to a pre-existing S or T, or by introducing an S or T at a position corresponding to a pre-existing N. Thus, the desired changes that would result in an N-linked glycosylation site are: A24N, R64N, S67N (possibly in combination with an N65A change), E106N, R112N, G120N, E123N, P129N, A132N, R112S and R112T. It is predicted that any S that is glycosylated can be changed to a T without creating an immunogenic site because of the protection provided by glycosylation. Likewise, any T predicted to be glycosylated may be changed to an S. Thus, variations S67T and S44T are contemplated. Likewise, in the A24N variant, a S26T variation may be applied. Accordingly, a GDF Trap may be an ActRIIB variant having one or more additional non-endogenous N-linked glycosylation consensus sequences.

[0103] Position L79 of ActRIIB can be altered to confer altered activin-tubocurarine (GDF-11) binding properties. L79A or L79P reduces GDF-11 binding to a greater extent than activin binding. L79E or L79D maintains GDF-11 binding. Significantly, the L79E and L79D variants have greatly reduced activin binding. In vivo experiments have shown that these non-activin receptors retain a significant ability to increase red blood cells, but show a reduced effect on other tissues. These data illustrate the desirability and feasibility of obtaining polypeptides with a reduced effect on activin. In an exemplary embodiment, the methods described herein utilize a GDF trap polypeptide that is a variant ActRIIB polypeptide, which comprises an acidic amino acid (e.g., D or E) at a position corresponding to position 79 of SEQ ID NO: 1 or 39, optionally in combination with one or more additional amino acid substitutions, additions, or deletions.

[0104] The described variations can be combined in various ways. Additionally, the results of the mutagenesis program described herein indicate that there are amino acid sequences in ActRIIB that are generally beneficial for conservation. These include position 64 (basic amino acid), position 80 (acidic or hydrophobic amino acid), position 78 (hydrophobic, and especially tryptophan), position 37 (acidic, and especially aspartic acid or glutamic acid), position 56 (basic amino acid), position 60 (hydrophobic amino acid, especially phenylalanine or tyrosine). Therefore, in each variant disclosed herein, the disclosure provides a framework of amino acids that can be conserved. Other positions that may be expected to be conserved are as follows: position 52 (acidic amino acid), position 55 (basic amino acid), position 81 (acidic), 98 (polar or charged, especially E, D, R or K).

[0105] In certain embodiments, isolated fragments of ActRIIB polypeptides can be obtained by screening polypeptides recombinantly produced from corresponding fragments of nucleic acids encoding ActRIIB polypeptides (e.g., SEQ ID NOs: 4 and 5). In addition, fragments can be chemically synthesized using techniques known in the art (e.g., conventional Merrifield solid phase f-Moc or t-Boc chemistry). Fragments can be produced (by recombinant or chemical synthesis) and tested to identify those peptidyl fragments that can act as antagonists (inhibitors) or agonists (activators) of, for example, ActRIIB proteins or ActRIIB ligands.

[0106] In certain embodiments, the GDF Trap polypeptide is a variant ActRIIB polypeptide having an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NO: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, 40, or 41. In certain instances, the GDF Trap has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, 40, or 41. In certain embodiments, the GDF Trap comprises, consists essentially of, or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, 40, or 41, wherein the position corresponding to L79 of SEQ ID NO: 1 is an acidic amino acid (e.g., a D or E amino acid residue).

[0107] In certain embodiments, the present invention contemplates the preparation of functional variants by modifying the structure of a GDF Trap polypeptide for the purpose of enhancing therapeutic efficacy or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). GDF Trap polypeptides may also be produced by amino acid substitutions, removals, or additions. For example, it is reasonable to expect that substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar amino acid substitutions with structurally related amino acids (e.g., conservative mutations) alone will not have a significant effect on the biological activity of the resulting molecule. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of a GDF Trap polypeptide produces a functional variant can be readily determined by assessing the ability of the GDF Trap polypeptide to generate a response in a cell relative to an unmodified GDF Trap polypeptide or a wild-type ActRIIB polypeptide or to bind one or more ligands, such as activin, GDF-11, or tubocurarine, as compared to an unmodified GDF Trap polypeptide or a wild-type ActRIIB polypeptide.

[0108] In certain specific embodiments, the present invention contemplates making mutations in the extracellular domain (also referred to as the ligand-binding domain) of an ActRIIB polypeptide such that the ActRIIB polypeptide has an altered ligand-binding activity (e.g., binding affinity or binding specificity). In some cases, such GDF Trap polypeptides have an altered (increased or decreased) binding affinity for a particular ligand. In other cases, the GDF Trap polypeptides have an altered binding specificity for an ActRIIB ligand.

[0109] For example, the present disclosure provides GDF Trap polypeptides that preferentially bind to GDF8 / GDF11 relative to activin. The present disclosure further establishes the desirability of such polypeptides to reduce off-target effects, although such selection variants may be less desirable for treating serious diseases in which very large increases in red blood cell levels may be required for therapeutic effects and some levels of off-target effects are acceptable. For example, amino acid residues such as E39, K55, Y60, K74, W78, D80, and F101 of the ActRIIB protein are in the ligand-binding pocket and mediate binding to its ligands such as activin and GDF8. Therefore, the present invention provides GDF traps comprising an altered ligand-binding domain (e.g., a GDF8-binding domain) of an ActRIIB receptor, comprising one or more mutations at these amino acid residues. Optionally, the altered ligand-binding domain may have increased selectivity for ligands such as GDF8 relative to the wild-type ligand-binding domain of the ActRIIB receptor. For example, these mutations increase the selectivity of the altered ligand-binding domain for GDF8 relative to activin. Optionally, the altered ligand-binding domain has a K for activin binding.d and K for GDF8 binding d The ratio of the ligand-binding domain to the wild-type ligand-binding domain is at least 2, 5, 10 or even 100 times higher. Optionally, the altered ligand-binding domain has an IC for inhibiting activin of 50 With IC for inhibition of GDF8 50 The ratio of the ligand-binding domain to the wild-type ligand-binding domain is at least 2, 5, 10 or even 100 times higher. Optionally, the altered ligand-binding domain is used to inhibit the IC 50 At least 2, 5, 10 or even 100 times lower IC 50 Inhibition of GDF8.

[0110] As a specific example, the positively charged amino acid residue Asp (D80) of the ligand-binding domain of ActRIIB can be mutated to a different amino acid residue to produce a GDF Trap polypeptide that preferentially binds GDF8 rather than activin. Preferably, the D80 residue is changed to an amino acid residue selected from the following: an uncharged amino acid residue, a negatively charged amino acid residue, and a hydrophobic amino acid residue. As a further specific example, the hydrophobic residue L79 can be changed to the acidic amino acids aspartic acid or glutamic acid to greatly reduce activin binding while maintaining GDF11 binding. As known to those skilled in the art, most of the described mutations, variants or modifications can be made at the nucleic acid level or in some cases by post-translational modification or chemical synthesis. Such techniques are well known in the art.

[0111] In certain embodiments, the present invention contemplates GDF Trap polypeptides having specific mutations in ActRIIB, thereby altering the glycosylation of the ActRIIB polypeptide. Exemplary glycosylation sites in GDF Trap polypeptides are Figure 1exemplified in (e.g., underlined NX(S / T) sites). Such mutations may be selected to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites typically comprise a tripeptide sequence, i.e., asparagine-X-threonine (where "X" is any amino acid), which is specifically recognized by appropriate cellular glycosylation enzymes. The wild-type ActRIIB polypeptide sequence may also be altered by the addition or substitution (for O-linked glycosylation sites) of one or more serine or threonine residues. A variety of amino acid substitutions or removals at one or both of the first or third amino acid positions of the glycosylation recognition site (and / or removal of the amino acid at the second position) result in non-glycosylation at the modified tripeptide sequence. Another way to increase the number of carbohydrate moieties on a GDF trap polypeptide is by chemically or enzymatically coupling glycosides to the GDF trap polypeptide. Depending on the coupling mode used, sugars can be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline; (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan; or (f) amide groups in glutamine. These methods are described in WO 87 / 05330 and Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, which are incorporated herein by reference. Removal of one or more carbohydrate moieties present on a GDF Trap polypeptide can be achieved chemically and / or enzymatically. Chemical deglycosylation can involve, for example, exposure of a GDF Trap polypeptide to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact. Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and Edge et al. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on GDF Trap polypeptides can be achieved by using a variety of endo- and exo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350. Where appropriate, the sequence of the GDF Trap polypeptide can be adjusted depending on the type of expression system used, as mammalian, yeast, insect, and plant cells can all introduce different glycosylation patterns that can be affected by the amino acid sequence of the peptide. In general, GDF Trap polypeptides for use in humans will be expressed in a mammalian cell line (e.g., HEK293 or CHO cell line) that provides appropriate glycosylation, although other mammalian expression cell lines are also expected to be useful.

[0112] The present disclosure further contemplates methods of generating variants, particularly methods of generating combinatorial variant sets of GDF Trap polypeptides, including optionally truncated variants; combinatorial mutant collections are particularly useful for identifying GDF Trap sequences. The purpose of screening such combinatorial libraries can be to generate GDF Trap polypeptide variants with altered properties, such as altered pharmacokinetics or altered ligand binding, for example. Various screening assays are provided below, and such assays can be used to evaluate variants. For example, GDF Trap polypeptide variants can be screened for the ability to bind to an ActRIIB polypeptide, prevent an ActRIIB ligand from binding to an ActRIIB polypeptide, or interfere with signaling caused by an ActRIIB ligand.

[0113] The activity of a GDF Trap polypeptide or variant thereof can also be tested in a cell-based assay or in vivo assay. For example, the effect of a GDF Trap polypeptide variant on the expression of genes involved in hematopoiesis can be assessed. This can be performed in the presence of one or more recombinant ActRIIB ligand proteins (e.g., activin), and cells can be transfected to produce a GDF Trap polypeptide and / or variant thereof, and optionally an ActRIIB ligand. Similarly, a GDF Trap polypeptide can be administered to a mouse or other animal, and one or more blood measurements, such as RBC count, hemoglobin level, hematocrit level, iron storage, or reticulocyte count, can be assessed using methods known in the art.

[0114] Combinatorially derived variants having selective potency relative to a reference GDF Trap polypeptide can be generated. Such variant proteins can be used in gene therapy protocols when expressed from a recombinant DNA construct. Likewise, mutagenesis can produce variants whose intracellular half-life is significantly different from that of the corresponding unmodified GDF Trap polypeptide. For example, the altered protein can become more stable or less stable to proteolytic degradation or other processes that result in the destruction or otherwise inactivation of the unmodified GDF Trap polypeptide. These variants and the genes encoding them can be used to alter GDF Trap polypeptide levels by modulating the half-life of the GDF Trap polypeptide. For example, a short half-life can produce a more transient biological effect and, as part of an inducible expression system, can allow for tighter control of the levels of recombinant GDF Trap polypeptides in cells. In Fc fusion proteins, mutations can be made in the linker (if any) and / or the Fc portion to alter the half-life of the protein.

[0115] In certain embodiments, the GDF Trap polypeptides of the present invention may further include post-translational modifications in addition to any modifications naturally present in the ActRIIB polypeptide. These modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the GDF Trap polypeptides may include non-amino acid elements, such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The effects of such non-amino acid elements on the functionality of the GDF Trap polypeptides may be tested as described herein for other GDF Trap polypeptide variants. When the GDF Trap polypeptides are produced in cells by cleaving the nascent form of the GDF Trap polypeptide, post-translational processing may also be important for the correct folding and / or function of the protein. Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3, or HEK293) have specific cellular structures and characteristic mechanisms for such post-translational activities and may be selected to ensure the correct modification and processing of the GDF Trap polypeptides.

[0116] In certain aspects, the GDF trap polypeptide comprises a fusion protein having at least a portion of an ActRIIB polypeptide and one or more fusion domains. Examples of such fusion domains are well known, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. The fusion domain may be selected to impart desired properties. For example, some fusion domains are particularly useful for isolating the fusion protein by affinity chromatography. For the purpose of affinity purification, affinity chromatography related matrices 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 QIAexpress™ system (Qiagen) that can be used with the (HIS6) fusion partner. As another example, the fusion domain may be selected to facilitate detection of the GDF trap polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and "epitope tags," which are generally short peptide sequences for which specific antibodies can be obtained. Well-known epitope tags for which specific monoclonal antibodies can be readily obtained include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain has a protease cleavage site, such as for factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein, thereby releasing the recombinant protein therefrom. The released protein can then be separated from the fusion domain by subsequent chromatographic separation. In certain preferred embodiments, the GDF trap polypeptide is fused to a domain that stabilizes the GDF trap polypeptide in vivo (a "stabilizer" domain). "Stabilization" refers to any situation that increases serum half-life, whether it is caused by reduced destruction, reduced renal clearance, or other pharmacokinetic effects. Fusions to the Fc portion of immunoglobulins are known to confer desired pharmacokinetic properties to a variety of proteins. Similarly, fusions to human serum albumin can confer desired properties. Other types of fusion domains that may be selected include multimerization (eg, dimerization, tetramerization) domains and functional domains (which confer additional biological functions, such as further increasing erythrocyte levels).

[0117] As a specific example, the present invention provides a GDF trap that is an ActRIIB-Fc fusion protein comprising an extracellular domain of an ActRIIB polypeptide fused to a (eg, ligand-binding) Fc domain. The sequence of an exemplary Fc domain is shown below (SEQ ID NO: 6).

[0118]

[0119] Optionally, the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In some cases, the ability of a mutant Fc domain having one or more of the mutations (e.g., Asp-265 mutation) to bind to an Fcγ receptor is reduced relative to a wild-type Fc domain. In other cases, the ability of a mutant Fc domain having one or more of the mutations (e.g., Asn-434 mutation) to bind to a class I MHC-associated Fc receptor (FcRN) is increased relative to a wild-type Fc domain.

[0120] It will be appreciated that the various elements of the fusion protein may be arranged in any manner consistent with the desired functionality. For example, a GDF Trap polypeptide may be placed at the C-terminus of a heterologous domain, or alternatively, a heterologous domain may be placed at the C-terminus of a GDF Trap polypeptide. The GDF Trap polypeptide domain and the heterologous domain need not be adjacent to each other in the fusion protein, and additional domains or amino acid sequences may be included at the C- or N-terminus of either domain or between domains.

[0121] In certain embodiments, the GDF Trap fusion protein comprises the amino acid sequence set forth in Formula ABC. Portion B is an N- and C-terminally truncated ActRIIB polypeptide consisting of an amino acid sequence corresponding to amino acids 26-132 of SEQ ID NO: 26. Portions A and C may independently be 0, 1, or more than 1 amino acid, and both portions A and C are heterologous to B when present. Portions A and / or C may be linked to portion B via a linker sequence. Exemplary linkers include short polypeptide linkers such as 2-10, 2-5, 2-4, 2-3 glycine residues, e.g., a Gly-Gly-Gly linker. Other suitable linkers are described herein above. In certain embodiments, the GDF Trap fusion protein comprises the amino acid sequence set forth in Formula ABC, wherein A is a leader sequence, B consists of amino acids 26-132 of SEQ ID NO: 26, and C is a polypeptide portion that enhances one or more of in vivo stability, in vivo half-life, uptake / administration, tissue localization or distribution, protein complex formation, and / or purification. In certain embodiments, the GDF Trap fusion protein comprises the amino acid sequence set forth in Formula ABC, wherein A is the TPA leader sequence, B consists of amino acids 26-132 of SEQ ID NO: 26, and C is an immunoglobulin Fc domain. A preferred GDF Trap fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 26.

[0122] In certain embodiments, the GDF Trap polypeptides of the present invention comprise one or more modifications that stabilize the GDF Trap polypeptide. For example, these modifications increase the in vitro half-life of the GDF Trap polypeptide, increase the circulating half-life of the GDF Trap polypeptide, or reduce the proteolytic degradation of the GDF Trap polypeptide. These stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising a GDF Trap polypeptide and a stabilizer domain), modifications of glycosylation sites (including, for example, adding glycosylation sites to the GDF Trap polypeptide), and modifications of carbohydrate moieties (including, for example, removing carbohydrate moieties from the GDF Trap polypeptide). In the case of a fusion protein, the GDF Trap polypeptide is fused to a stabilizer domain such as an IgG molecule (e.g., an Fc domain). As used herein, the term "stabilizer domain" refers not only to a fusion domain (e.g., Fc) in the case of a fusion protein, but also includes non-protein modifications, such as carbohydrate moieties, or non-protein polymers, such as polyethylene glycol.

[0123] In certain embodiments, the present disclosure enables the availability of isolated and / or purified forms of GDF Trap polypeptides that are separated from, or otherwise substantially free of, other proteins.

[0124] In certain embodiments, the GDF Trap polypeptides of the invention (unmodified or modified) can be produced by various well-known techniques. For example, such GDF Trap polypeptides can be synthesized using standard protein chemistry techniques, such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant GA (ed.), Synthetic Peptides: A User's Guide, WH Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen / Biosearch 9600). Alternatively, GDF Trap polypeptides, fragments or variants thereof can be recombinantly produced using various expression systems known in the art (e.g., E. coli, Chinese hamster ovary (CHO) cells, COS cells, baculovirus). In yet another embodiment, modified or unmodified GDF Trap polypeptides can be generated by digesting a recombinantly produced full-length GDF Trap polypeptide using, for example, a protease, such as trypsin, thermolysin, chymotrypsin, pepsin, or paired alkaline amino acid converting enzyme (PACE). Computer analysis (using commercially available software, such as MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to identify proteolytic cleavage sites. Alternatively, such GDF Trap polypeptides can be generated from recombinantly produced full-length GDF Trap polypeptides, such as by standard techniques known in the art, such as by chemical cleavage (e.g., cyanogen bromide, hydroxylamine).

[0125] 3. Nucleic Acids Encoding GDF Trap Polypeptides

[0126] In certain aspects, the present invention provides isolated and / or recombinant nucleic acids encoding any of the GDF Trap polypeptides disclosed herein. SEQ ID NO: 4 encodes a naturally occurring ActRIIB precursor polypeptide, while SEQ ID NO: 5 encodes a soluble ActRIIB polypeptide, and SEQ ID NOs: 25, 27, 30, and 31 encode soluble GDF traps. The subject nucleic acids can be single-stranded or double-stranded. Such nucleic acids can be DNA or RNA molecules. These nucleic acids can be used, for example, in methods of making GDF Trap polypeptides, or as direct therapeutic agents (e.g., in gene therapy methods).

[0127] In certain aspects, the subject nucleic acids encoding GDF Trap polypeptides are further understood to include nucleic acids that are variants of SEQ ID NOs: 5, 25, 27, 30, and 31. Variant nucleotide sequences include sequences that vary in one or more nucleotide substitutions, additions, or deletions, such as allelic variants; and will therefore include coding sequences that differ from the nucleotide sequences of the coding sequences specified in SEQ ID NOs: 5, 25, 27, 30, and 31.

[0128] In certain embodiments, the present invention provides an isolated or recombinant nucleic acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 5, 25, 27, 30 or 31. One of ordinary skill in the art will appreciate that nucleic acid sequences complementary to SEQ ID NO: 5, 25, 27, 30 or 31, as well as variants of SEQ ID NO: 5, 25, 27, 30 or 31 are also within the scope of the present invention. In further embodiments, the nucleic acid sequences of the present invention may be isolated, recombinant and / or fused to heterologous nucleotide sequences, or in a DNA library.

[0129] In other embodiments, the nucleic acids of the invention also include nucleotide sequences that hybridize to the nucleotide sequences set forth in SEQ ID NO: 5, 25, 27, 30 or 31, the complementary sequences of SEQ ID NO: 5, 25, 27, 30 or 31, or fragments thereof under highly stringent conditions. As discussed above, it will be readily appreciated by those of ordinary skill in the art that suitable stringent conditions for promoting DNA hybridization can be varied. For example, hybridization can be performed at 6.0 x sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing at 2.0 x SSC at 50°C. For example, the salt concentration in the washing step can be selected from about 2.0 x SSC at 50°C for low stringency to about 0.2 x SSC at 50°C for high stringency. In addition, the temperature in the washing step can be raised from room temperature, i.e., about 22°C, for low stringency conditions to about 65°C for high stringency conditions. Both temperature and salt can be varied, or the temperature or salt concentration can be kept constant while the other variable is varied. In one embodiment, the invention provides nucleic acids that hybridize under low stringency conditions of 6 x SSC, room temperature, followed by a wash at 2 x SSC, room temperature.

[0130] Isolated nucleic acids that differ from the nucleic acids set forth in SEQ ID NOs: 5, 25, 27, 30, or 31 due to the degeneracy of the genetic code are also within the scope of the present invention. For example, many amino acids are specified by more than one triplet codon. Codons that specify the same amino acid, or synonymous codons (e.g., CAU and CAC are synonymous codons for histidine), can result in "silent" mutations that do not affect the amino acid sequence of the protein. In certain embodiments, the GDF Trap polypeptide will be encoded by an alternative nucleotide sequence. The alternative nucleotide sequence is generated relative to the native GDF Trap nucleic acid sequence, but still encodes the same fusion protein. In certain embodiments, a GDF Trap having SEQ ID NO: 26 is encoded by an alternative nucleic acid sequence comprising SEQ ID NO: 30. However, it is expected that DNA sequence polymorphisms exist in mammalian cells that do result in changes in the amino acid sequence of the subject protein. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) in a nucleic acid encoding a particular protein may exist in individuals of a particular species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of the present invention.

[0131] In certain embodiments, the recombinant nucleic acid of the present invention can be operably connected to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences are generally suitable for host cells for expression. Suitable expression vectors and suitable regulatory sequences for various types of host cells known in the art are known in the art. The one or more regulatory nucleotide sequences generally may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancer or activator sequences. The present invention also contemplates constitutive or inducible promoters known in the art. The promoter may be a naturally occurring promoter, or a hybrid promoter that incorporates the elements of more than one promoter. The expression construct may be present on an episome (e.g., plasmid) in the cell, or the expression construct may be inserted into a chromosome. In a preferred embodiment, the expression vector comprises a selectable marker gene, thereby allowing the selection of transformed host cells. Selectable marker genes are well known in the art and will change with the host cell used.

[0132] In certain aspects of the invention, a subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a GDF trap polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are known in the art and are selected to direct the expression of a GDF trap polypeptide. Thus, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of a variety of expression control sequences that control the expression of a DNA sequence when operably linked to the DNA sequence can be used in these vectors to express a DNA sequence encoding a GDF trap polypeptide. These useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the immediate early promoters of adenovirus and cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter that directs expression by T7 RNA polymerase, the major operator and promoter region of bacteriophage lambda, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatases, such as Pho5, the promoter of yeast α-mating factor, the polyhedral promoter of the baculovirus system, and other sequences known to control gene expression in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It 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. In addition, the copy number of the vector, the ability to control the copy number, and the expression of any other proteins encoded by the vector (e.g., antibiotic markers) should also be considered.

[0133] The recombinant nucleic acids of the present invention can be produced by ligating the cloned gene or portion thereof into a vector suitable for expression in prokaryotes, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vectors for producing recombinant GDF Trap polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotes (e.g., E. coli).

[0134] Some mammalian expression vectors contain both prokaryotic sequences that promote vector propagation in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian cell expression vectors suitable for eukaryotic cell transfection. Some of these vectors are modified with sequences from bacterial plasmids (such as pBR322) to promote replication and drug resistance selection in both prokaryotes and eukaryotic cells. Alternatively, viral derivatives such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. Various methods for preparing plasmids and transforming host organisms are well known in the art. Other suitable prokaryotic and eukaryotic cell expression systems and general recombination procedures are described in Molecular Cloning A Laboratory Manual, 2nd Ed., ed. Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some cases, it may be necessary to use a baculovirus expression system to express the recombinant polypeptide. Examples of these baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., pBlueBac III containing β-gal).

[0135] In a preferred embodiment, vectors designed for producing the subject GDF Trap polypeptides in CHO cells, such as the pcmv-Script vector (Stratagene, La Jolla, Calif), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wisc.) will be used. Obviously, the subject gene constructs can be used to cause expression of the subject GDF Trap polypeptides in cells propagated in culture, for example, to produce proteins, including fusion proteins or variant proteins, for purification.

[0136] The present disclosure also relates to host cells transfected with a recombinant gene comprising one or more coding sequences of a subject GDF Trap polypeptide (e.g., SEQ ID NO: 4, 5, 25, 27, 30, or 31). The host cell can be any prokaryotic or eukaryotic cell. For example, the GDF Trap polypeptides of the present invention can be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are well known to those skilled in the art.

[0137] Thus, the present invention further relates to methods for producing the subject GDF trap polypeptides. For example, host cells transfected with an expression vector encoding a GDF trap polypeptide can be cultured under appropriate conditions to allow expression of the GDF trap polypeptide to occur. The GDF trap polypeptide can be secreted and separated from a mixture of cells and a culture medium containing the GDF trap polypeptide. Alternatively, the GDF trap polypeptide can be retained in the cytoplasm or in a membrane fraction, and the cells can be harvested, lysed, and the protein isolated. The cell culture comprises host cells, culture medium, and other byproducts. Suitable culture media for cell culture are well known in the art. The subject GDF trap polypeptide can be isolated from the cell culture medium, the host cells, or both using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification using antibodies specific for specific epitopes of the GDF trap polypeptide. In a preferred embodiment, the GDF trap polypeptide is a fusion protein comprising a domain that facilitates its purification.

[0138] In another embodiment, a fusion gene encoding a purification leader sequence (e.g., a polyhistidine / enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant GDF Trap polypeptide) can allow for the purification of the desired portion of the recombinant GDF Trap polypeptide by using Ni. 2+ The expressed fusion protein can be purified by affinity chromatography with a metal resin. The purification leader sequence can then be removed by enterokinase treatment to provide a purified GDF Trap polypeptide (e.g., see Hochuli et al., (1987) J. Chromatography 411: 177; and Janknecht et al., PNAS USA 88: 8972).

[0139] The techniques for preparing fusion genes are well known. In essence, various DNA fragments encoding different polypeptide sequences are connected according to conventional techniques using blunt or staggered ends for connection, restriction enzyme digestion to provide suitable ends, filling of cohesive ends as needed, alkaline phosphatase treatment to avoid undesired connection, and enzymatic connection. In another embodiment, the fusion gene can be synthesized by conventional techniques including an automated DNA synthesizer. Alternatively, PCR amplification of the gene fragments can be performed using anchor primers that produce complementary overhangs between two consecutive gene fragments, which can then be annealed to form a chimeric gene sequence (e.g., see Current Protocols in Molecular Biology , eds. Ausubel et al., John Wiley & Sons: 1992).

[0140] 4. Screening Assay

[0141] In certain aspects, the invention relates to the use of subject GDF Trap polypeptides (e.g., soluble variant ActRIIB polypeptides) for identifying compounds (agents) that are agonists or antagonists of ActRIIB polypeptides. Compounds identified by this screening can be tested to assess their ability to modulate red blood cell, hemoglobin and / or reticulocyte levels in vivo or in vitro. These compounds can be tested, for example, in animal models.

[0142] There are a variety of methods for screening therapeutic agents that increase red blood cell or hemoglobin levels by targeting ActRIIB signaling. In certain embodiments, high-throughput screening of compounds can be performed to identify agents that interfere with ActRIIB-mediated effects on selected cell lines. In certain embodiments, the assay is performed to screen and identify compounds that specifically inhibit or reduce the binding of an ActRIIB polypeptide to its binding partner, such as an ActRIIB ligand (e.g., activin, Nodal, GDF8, GDF11, or BMP7). Alternatively, the assay can be used to identify compounds that increase the binding of an ActRIIB polypeptide to its binding partner, such as an ActRIIB ligand. In a further embodiment, a compound can be identified by its ability to interact with an ActRIIB polypeptide.

[0143] A variety of assay formats can meet the needs, and according to the present disclosure, assays that are not clearly described herein can be understood by those of ordinary skill in the art. As described herein, the test compounds (agents) of the present invention can be generated by any combination of chemical methods. Alternatively, the subject compound can be a naturally occurring biomolecule synthesized in vivo or in vitro. The compound to be tested (agent) for testing its ability as a tissue growth regulator can be produced by, for example, bacteria, yeast, plants or other organisms (e.g., natural products), produced by chemical methods (e.g., small molecules, including peptide mimetics) or recombinant production. The test compounds considered by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptide mimetics, sugars, hormones and nucleic acid molecules. In a specific embodiment, the test agent is a small organic molecule with a molecular weight less than about 2000 Daltons.

[0144] The test compounds of the present invention can be provided as separate, discrete entities, or in more complex libraries, for example prepared by combinatorial chemistry. These libraries can contain, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. The test compounds can be displayed in the test system (particularly in the initial screening step) in isolated form or as a mixture of compounds. Optionally, the compounds can be derivatized with other compounds and have derivatizing groups that facilitate the separation of the compounds. Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxigenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S-transferase (GST), photoactivatable cross-linking agents or any combination thereof.

[0145] In many drug screening programs where libraries of compounds and natural extracts are tested, it is desirable to use high-throughput assays to maximize the number of compounds investigated in a given time frame. Assays conducted in cell-free systems (e.g., as can be derived with purified or semi-purified proteins) are often preferred as "primary" screens because they can be generated, allowing for rapid development and relatively easy detection of changes in molecular targets mediated by test compounds. In addition, the cytotoxicity or bioavailability of a test compound can often be ignored in an in vitro system, allowing the assay to instead focus primarily on the effect of the drug on the molecular target, which can be demonstrated by changes in the binding affinity between an ActRIIB polypeptide and its binding partner (e.g., an ActRIIB ligand).

[0146] For illustrative purposes only, in an exemplary screening assay of the present invention, a compound of interest is contacted with an isolated and purified ActRIIB polypeptide (which is generally capable of binding an ActRIIB ligand, such as is suitable for analytical purposes). A composition containing an ActRIIB ligand is then added to the mixture of the compound and the ActRIIB polypeptide. Detection and quantification of the ActRIIB / ActRIIB ligand complex provides a means for determining the effectiveness of a compound in inhibiting (or enhancing) the formation of a complex between an ActRIIB polypeptide and its binding protein. The effectiveness of the compound can be evaluated by generating a dose-response curve from data obtained from different concentrations of the test compound. In addition, a control assay can also be performed to provide a baseline for comparison. For example, in a control assay, an isolated and purified ActRIIB ligand is added to a composition containing an ActRIIB polypeptide, and the formation of the ActRIIB / ActRIIB ligand complex is quantified in the absence of the test compound. It should be understood that in general, the order of mixing the reactants can be varied and can be mixed simultaneously. In addition, cell extracts and lysates can be substituted for purified proteins to produce suitable cell-free assay systems.

[0147] Complex formation between an ActRIIB polypeptide and its binding protein can be detected by a variety of techniques. For example, a detectably labeled protein such as a radiolabeled protein (e.g., 32 P. 35 S. 14 C or 3 H), fluorescently labeled (e.g., FITC) or enzyme-labeled ActRIIB polypeptide or its binding protein, and modulation of complex formation is quantified by immunoassay or by chromatographic detection.

[0148] In certain embodiments, the present invention contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays to directly or indirectly measure the degree of interaction between an ActRIIB polypeptide and its binding protein. In addition, other detection modes, such as those based on optical waveguides (PCT Publication WO 96 / 26432 and U.S. Pat. No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, may be suitable for use in various embodiments of the present invention.

[0149] In addition, the present invention contemplates the use of interaction capture assays (also referred to as "two-hybrid assays") to identify agents that disrupt or enhance the interaction between an ActRIIB polypeptide and its binding partner. See, for example, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment, the present invention contemplates the use of a reverse two-hybrid system to identify compounds (e.g., small molecules or peptides) that dissociate the interaction between an ActRIIB polypeptide and its binding protein. See, for example, Vidal and Legrain, (1999) Nucleic acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; and US Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

[0150] In certain embodiments, the subject compound can be identified by its ability to interact with the ActRIIB polypeptide. The interaction between the compound and the ActRIIB polypeptide can be covalent or non-covalent. For example, this interaction can be identified at the protein level using in vitro biochemical methods, including photocrosslinking, radiolabeled ligand binding, and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46: 1). In some cases, compounds can be screened in mechanism-based assays (e.g., assays for detecting compounds that bind to ActRIIB polypeptides). This can include solid phase or liquid phase binding events. Alternatively, genes encoding ActRIIB polypeptides can be transfected into cells using a reporter system (e.g., β-galactosidase, luciferase, or green fluorescent protein), and the library is preferably screened by high-throughput screening or screened with individual members of the library. Other mechanism-based binding assays can be used, for example, binding assays that detect free energy changes. Binding assays can be performed with the target immobilized to a well, bead or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. Bound compounds are typically detected colorimetrically or fluorescently or by surface plasmon resonance.

[0151] 5. Exemplary Therapeutic Uses

[0152] In certain embodiments, the GDF Trap polypeptides of the present invention can be used to increase red blood cell levels in mammals, such as rodents and primates, and particularly in human patients. Additionally, as shown herein, GDF Trap polypeptides can be used in combination with EPO receptor activators to achieve an increase in red blood cells at a lower dose range. This can be beneficial in reducing known off-target effects and risks associated with high doses of EPO receptor activators. In certain embodiments, the present invention provides methods for treating or preventing anemia in an individual in need thereof by administering to the individual a therapeutically effective amount of a GDF Trap polypeptide or a combination (or combination therapy) of a GDF Trap polypeptide and an EPO receptor activator. These methods can be used for therapeutic or prophylactic treatment of mammals, particularly humans.

[0153] GDF Trap polypeptides can be used in combination with EPO receptor activators to reduce the required dose of these activators in patients susceptible to EPO side effects. The main side effects of EPO are excessive increases in hematocrit or hemoglobin levels and polycythemia. Elevated hematocrit levels can cause hypertension (more particularly worsening hypertension) and vascular thrombosis. Other side effects of EPO that have been reported, some of which are related to hypertension, are headache, flu-like syndrome, shunt obstruction, myocardial infarction and cerebral convulsions due to thrombosis, hypertensive encephalopathy and erythrocyte applasia (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) NEngl J Med 346(7), 522-523).

[0154] The rapid effects of the GDF Trap polypeptides disclosed herein on red blood cell levels suggest that these agents act through a different mechanism than EPO. Accordingly, these antagonists can be used to increase red blood cell and hemoglobin levels in patients who do not respond adequately to EPO. For example, GDF Trap polypeptides can benefit patients in which administration of normal to increased (>300 IU / kg / week) doses of EPO does not result in an increase in hemoglobin levels to target levels. Patients with an inadequate EPO response are found in all types of anemia, but higher numbers of non-responders have been observed particularly frequently in patients with cancer and patients with end-stage renal disease. An inadequate response to EPO can be constitutive (i.e., observed after initial treatment with EPO) or acquired (e.g., observed after repeated treatment with EPO).

[0155] As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound that reduces the occurrence of the disorder or condition in a treated sample relative to an untreated control sample in a statistical sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample. As used herein, the term "treat" includes preventing a specified condition or ameliorating or eliminating the condition after it has developed. In either case, prevention or treatment can be distinguished in the diagnosis provided by a physician or other health care provider and the expected results of the administration of the therapeutic agent.

[0156] As shown herein, the GDF Trap polypeptides disclosed herein, optionally in combination with an EPO receptor activator, can be used to increase red blood cell, hemoglobin, or reticulocyte levels in healthy individuals, and these GDF Trap polypeptides can be used in selected patient populations. Examples of suitable patient populations include patients with undesirably low red blood cell or hemoglobin levels, such as patients with anemia, and patients at risk for developing undesirably low red blood cell or hemoglobin levels, such as patients about to undergo major surgery or other procedures that may result in significant blood loss. In one embodiment, a patient with adequate red blood cell levels is treated with a GDF Trap polypeptide to increase red blood cell levels, and then blood is drawn and stored for later use in a transfusion.

[0157] GDF Trap polypeptides disclosed herein, optionally in combination with an EPO receptor activator, can be used to increase red blood cell levels in patients suffering from anemia. When hemoglobin levels are observed in humans, levels below normal values ​​for appropriate age and gender classifications can indicate anemia, although individual variation is taken into account. For example, a hemoglobin level of 12 g / dl is generally considered to be the lower limit of normal values ​​in the general adult population. Potential causes include blood loss, nutritional deficiencies, drug reactions, various problems with the bone marrow and various diseases. More specifically, anemia is associated with a variety of conditions, including, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis, and bone marrow transplantation. Anemia may also be associated with the following conditions: solid tumors (e.g., breast cancer, lung cancer, colon cancer); tumors of the lymphatic system (e.g., chronic lymphocytic leukemia, non-Hodgkin's lymphoma, and Hodgkin's lymphoma); tumors of the hematopoietic system (e.g., leukemia, myelodysplastic syndrome, multiple myeloma); radiation therapy; chemotherapy (e.g., platinum-containing regimens); inflammatory and autoimmune diseases, including but not limited to rheumatoid arthritis, other inflammatory arthritis rashes, systemic lupus erythematosus (SLE), acute or chronic skin diseases (e.g., psoriasis), inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); acute or Chronic renal disease or failure, including idiopathic or congenital conditions; acute or chronic liver disease; acute or chronic bleeding; conditions in which red blood cells cannot be transfused due to the patient's allo- or autoantibodies and / or for religious reasons (e.g., certain Jehovah's Witnesses); infections (e.g., malaria, osteomyelitis); hemoglobinopathies, including, for example, sickle cell disease, thalassemia; drug use or abuse, such as alcohol abuse; pediatric patients with anemia due to any reason to avoid transfusions; and elderly patients with anemia or patients with underlying cardiopulmonary disease who cannot receive transfusions due to concerns about circulatory load.

[0158] GDF Trap polypeptides, optionally in combination with EPO receptor activators, are suitable for treating anemias of hypoproliferative bone marrow, which are typically associated with little change in red blood cell (RBC) morphology. Hypoproliferative anemias include: 1) anemias of chronic disease, 2) anemias of renal disease, and 3) anemias associated with a low metabolic state. In each of these types, endogenous erythropoietin levels are inappropriately low for the degree of anemia observed. Other hypoproliferative anemias include: 4) early iron deficiency anemia, and 5) anemias caused by bone marrow damage. In these types, endogenous erythropoietin levels are appropriately elevated for the degree of anemia observed.

[0159] The most common type is anemia of chronic disease, which includes inflammation, infection, tissue damage, and conditions such as cancer, and is distinguished by low erythropoietin levels and an insufficient response to erythropoietin in the bone marrow (Adamson, 2008, Harrison's Principles of Internal Medicine, 17th ed.; McGraw Hill, New York, pp 628-634). Many factors can lead to cancer-related anemia. Some are related to the disease process itself and the production of inflammatory cytokines such as interleukin-1, interferon-γ, and tumor necrosis factor (Bron et al., 2001, Semin Oncol 28(Suppl 8):1-6). Among its effects, inflammation induces the key iron-regulating peptide hepcidin, which inhibits iron transport from macrophages and generally limits iron availability for erythropoiesis (Ganz, 2007, J Am Soc Nephrol 18:394-400). Blood loss through various routes can also lead to cancer-related anemia. The prevalence of anemia due to cancer progression varies with cancer type, ranging from 5% in prostate cancer to 95% in multiple myeloma. Cancer-related anemia has significant consequences for patients, including fatigue and decreased quality of life, reduced efficacy, and increased mortality.

[0160] Chronic kidney disease is associated with hypoproliferative anemia, and the severity of the anemia changes with the degree of renal damage. The anemia is mainly due to insufficient production of erythropoietin and reduced survival of red blood cells. Chronic kidney disease usually progresses to the terminal (5th phase) disease in a few years or decades, at which time dialysis or kidney transplantation is required to survive the patient. Anemia usually occurs in the early stages of this process and worsens with disease progression. The clinical consequences of anemia in renal disease have been fully documented, including development of left ventricular hypertrophy, impaired cognitive function, decreased quality of life and changes in immune function (Levin et al., 1999, Am J Kidney Dis 27:347-354; Nissenson, 1992, Am J Kidney Dis 20(Suppl1):21-24; Revicki et al., 1995, Am J Kidney Dis 25:548-554; Gafter et al., 1994, Kidney Int 45:224-231). As Applicants have demonstrated in a mouse model of chronic kidney disease (see Examples below), GDF Trap polypeptides, optionally in combination with an EPO receptor activator, can be used to treat anemia of kidney disease.

[0161] Many conditions that result in a low metabolic rate can produce mild to moderate hypoproliferative anemia. Among the conditions are endocrine deficiency conditions. For example, anemia can occur in Addison's disease, hypothyroidism, hyperparathyroidism, or in men who are castrated or treated with estrogen. Mild to moderate anemia can also occur with reduced dietary protein intake, a condition that is particularly common in the elderly. Finally, anemia can also develop in patients with chronic liver disease from almost any cause (Adamson, 2008, Harrison's Principles of Internal Medicine, 17th ed.; McGraw Hill, New York, pp 628-634).

[0162] Anemia caused by massive acute blood loss, such as trauma or postpartum hemorrhage, is called acute post-hemorrhagic anemia. Acute blood loss initially causes hypovolemia without anemia because there is a proportional depletion of RBCs along with other blood components. However, hypovolemia quickly triggers physiological mechanisms that transfer fluid from the extravascular space to the vascular space, which leads to hemodilution and anemia. If chronic, blood loss gradually depletes the body's iron stores and eventually leads to iron deficiency. As described by the applicant in a mouse model (see Examples below), GDF trap polypeptides optionally combined with EPO receptor activators can be used to quickly recover from anemia from acute blood loss.

[0163] Iron deficiency anemia is the final stage in the hierarchical progression of increasing iron deficiency (it includes negative balance of iron and iron deficiency erythropoiesis as the intermediate stage). Iron deficiency can be caused by increased iron demand, reduced iron intake or increased iron loss, which is exemplified in the following conditions: such as pregnancy, insufficient diet, intestinal malabsorption, acute or chronic inflammation, and acute or chronic blood loss. With this type of mild to moderate anemia, the bone marrow maintains low proliferation, and the RBC morphology is roughly normal; but even mild anemia can also cause some microcytic hypochromic RBC, and, to the transition of severe iron deficiency anemia, the high proliferation of bone marrow and the increasingly ubiquitous microcytic and hypochromic RBC (Adamson, 2008, Harrison's Principles of Internal Medicine, 17th ed.; McGraw Hill, NewYork, pp 628-634) are accompanied. The appropriate treatment of iron deficiency anemia depends on its cause and severity, and oral iron preparations, parenteral iron preparations and RBC transfusions are the main conventional options. GDF Trap polypeptides, optionally in combination with an EPO receptor activator, can be used alone or in combination with conventional treatments to treat chronic iron deficiency anemia, particularly for the treatment of anemia of multifactorial origin.

[0164] Hypoproliferative anemia can be caused by primary dysfunction or failure of the bone marrow (rather than dysfunction secondary to inflammation, infection, or cancer progression). Prominent examples are bone marrow suppression caused by cancer chemotherapy drugs or cancer radiotherapy. A comprehensive review of clinical trials found that mild anemia occurs in 100% of patients after chemotherapy, while more severe anemia occurs in up to 80% of these patients (Groopman et al., 1999, J Natl Cancer Inst 91: 1616-1634). Myelosuppressive drugs include: 1) alkylating agents, such as nitrogen mustards (such as melphalan) and nitrosoureas (such as streptozotocin); 2) antimetabolites, such as folic acid antagonists (such as methotrexate), purine analogs (such as thioguanine) and pyrimidine analogs (such as gemcitabine); 3) cytotoxic antibiotics such as anthracyclines (such as doxorubicin); 4) kinase inhibitors (such as gefitinib); 5) mitotic inhibitors such as taxanes (such as paclitaxel) and vinca alkaloids (such as vinorelbine); 6) monoclonal antibodies (such as rituximab); and 7) topoisomerase inhibitors (such as topotecan and etoposide). As demonstrated in a mouse model of chemotherapy-induced anemia (see Examples below), GDF Trap polypeptides, optionally in combination with EPO receptor activators, can be used to treat anemia caused by chemotherapeutic agents and / or radiation therapy.

[0165] GDF Trap polypeptides, optionally in combination with an EPO receptor activator, are also suitable for treating anemias of RBC maturation disorders, characterized in part by undersized (microcytic), oversized (macrocytic), oddly shaped or abnormally colored (hypochromic) RBCs.

[0166] Although lower target levels may cause fewer cardiovascular side effects, patients may be treated with a dosing regimen designed to restore the patient to a target hemoglobin level, which is generally between about 10 g / dl and about 12.5 g / dl, typically about 11.0 g / dl (see also Jacobs et al. (2000) Nephrol Dial Transplant 15, 15-19). Alternatively, the hematocrit level (the percentage of cells in the volume of a blood sample) may be used as a measure of the condition of the red blood cells. Hematocrit levels for healthy individuals range from 41-51% for adult males and 35-45% for adult females. Target hematocrit levels are generally about 30-33%. In addition, hemoglobin / hematocrit levels vary from individual to individual. Thus, optimally, the target hemoglobin / hematocrit level may be individualized for each patient.

[0167] In certain embodiments, the present invention provides methods for managing a patient who has been treated with a GDF Trap polypeptide or is a candidate for treatment with a GDF Trap polypeptide by measuring one or more hematological parameters in the patient. The hematological parameters can be used to evaluate appropriate dosing for a patient who is a candidate for treatment with a GDF Trap polypeptide, monitor hematological parameters during treatment with a GDF Trap polypeptide, evaluate whether to adjust the dose during treatment with a GDF Trap polypeptide, and / or evaluate the appropriate maintenance dose of a GDF Trap polypeptide. If one or more of the hematological parameters are outside of normal levels, dosing with the GDF Trap polypeptide can be reduced, delayed, or stopped.

[0168] Hematological parameters that can be measured according to the methods provided herein include, for example, red blood cell levels, blood pressure, iron storage, and other agents found in body fluids associated with increased red blood cell levels, using methods known in the art. Such parameters can be determined using a blood sample from a patient. Increases in red blood cell levels, hemoglobin levels, and / or hematocrit levels can cause increases in blood pressure.

[0169] In one embodiment, if one or more hematological parameters are outside the normal range or on the high side of normal, in a patient who is a candidate for GDF Trap polypeptide treatment, the initiation of administration of a GDF Trap polypeptide may be delayed until the hematological parameter has returned to normal or acceptable levels either naturally or through therapeutic intervention. For example, if the candidate patient is hypertensive or prehypertensive, the patient may be treated with an antihypertensive drug to lower the patient's blood pressure. Any antihypertensive drug suitable for the individual patient's condition may be used, including, for example, diuretics, adrenergic inhibitors (including alpha blockers and beta blockers), vasodilators, calcium channel blockers, angiotensin converting enzyme (ACE) inhibitors, or angiotensin II receptor blockers. Blood pressure may optionally be treated with a diet and exercise regimen. Similarly, if the candidate patient has iron stores that are below normal or on the low side of normal, the patient may be treated with an appropriate diet and / or iron supplementation regimen until the patient's iron stores return to normal or acceptable levels. For patients with higher than normal red blood cell levels and / or hemoglobin levels, administration of a GDF Trap polypeptide may be delayed until the levels return to normal or acceptable levels.

[0170] In certain embodiments, if one or more hematological parameters are outside the normal range, or on the high side of normal, initiation of administration may be delayed in a patient who is a candidate for GDF Trap polypeptide treatment. However, the dose or dosing frequency of the GDF Trap polypeptide may be set to an amount that will reduce the risk of an unacceptable increase in an increased hematological parameter upon administration of the GDF Trap polypeptide. Alternatively, a treatment regimen may be developed for a patient who combines a GDF Trap polypeptide with a therapeutic agent that addresses the undesirable level of a hematological parameter. For example, if a patient has elevated blood pressure, a treatment regimen involving administration of a GDF Trap polypeptide and a blood pressure-lowering drug may be designed. For patients with less than desired iron stores, a treatment regimen of GDF Trap polypeptides and iron supplementation may be developed.

[0171] In one embodiment, baseline parameters for one or more hematological parameters may be established for a patient who is a candidate for GDF Trap polypeptide treatment, and an appropriate dosing regimen may be established for the patient based on the baseline values. Alternatively, the baseline parameters established based on the patient's medical history may be used to inform an appropriate GDF Trap polypeptide dosing regimen for the patient. For example, if a healthy patient has an established baseline blood pressure reading that is above a defined normal range, it may not be necessary to bring the patient's blood pressure to a range considered normal for the general population prior to GDF Trap polypeptide treatment. The patient's baseline values ​​for one or more hematological parameters prior to GDF Trap polypeptide treatment may also be used as relevant comparative values ​​to monitor any changes in the hematological parameters during GDF Trap polypeptide treatment.

[0172] In certain embodiments, one or more hematological parameters are measured in a patient treated with a GDF Trap polypeptide. The hematological parameters can be used to monitor the patient during treatment and allow for adjustment or discontinuation of GDF Trap polypeptide administration or additional administration of another therapeutic agent. For example, if administration of a GDF Trap polypeptide results in an increase in blood pressure, red blood cell levels, or hemoglobin levels, or a decrease in iron stores, the dose of the GDF Trap polypeptide can be reduced in amount or frequency in order to reduce the effect of the GDF Trap polypeptide on one or more hematological parameters. If administration of a GDF Trap polypeptide results in a change in one or more hematological parameters that is detrimental to the patient, administration of the GDF Trap polypeptide can be temporarily, until the hematological parameters return to an acceptable level, or permanently discontinued. Similarly, if one or more hematological parameters are not within an acceptable range after reducing the dose or frequency of administration of the GDF Trap polypeptide, administration can be discontinued. As an alternative or in addition to reducing or discontinuing administration of a GDF Trap polypeptide, the patient can be given an additional therapeutic agent to address the undesirable level of a hematological parameter, for example, a blood pressure-lowering drug or an iron supplement. For example, if a patient treated with a GDF Trap polypeptide has elevated blood pressure, GDF Trap polypeptide administration can be continued at the same level and a blood pressure lowering medication added to the treatment regimen, GDF Trap polypeptide administration can be reduced (e.g., amount and / or frequency) and a blood pressure lowering medication added to the treatment regimen, or GDF Trap polypeptide administration can be stopped and the patient can be treated with the blood pressure lowering medication.

[0173] In certain embodiments, the patient being treated with a GDF Trap polypeptide or a candidate patient to be treated with a GDF Trap polypeptide is a patient in need of muscle growth, such as a patient suffering from or at risk of developing a neuromuscular disorder or a muscle production disorder. For example, the patient or candidate patient may suffer from or be at risk of developing Lou Gehrig's disease (ALS), cancer anorexia-cachexia syndrome, muscular dystrophy, muscle atrophy, congestive obstructive pulmonary disease (and muscle atrophy associated with COPD), muscle wasting syndrome, sarcopenia, or cachexia. Muscular dystrophy refers to a group of degenerative muscle diseases characterized by the gradual weakening and deterioration of skeletal muscle and sometimes cardiac and respiratory muscles. Exemplary muscular dystrophies that can be treated with regimens comprising a subject GDF Trap polypeptide include: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emory-Dreyfuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSH or FSHD) (also known as Landouzy-Dejerine), myotonia multiforme (MMD) (also known as Steinert's disease), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital muscular dystrophy (CMD).

[0174] 6. Pharmaceutical Compositions

[0175] In certain embodiments, the compounds of the invention (e.g., GDF Trap polypeptides) are formulated with a pharmaceutically acceptable carrier. For example, the GDF Trap polypeptides can be administered alone or as a component of a pharmaceutical formulation (therapeutic composition). The subject compounds can be formulated so as to be administered in any convenient manner for use in humans or veterinary medicine.

[0176] In certain embodiments, the therapeutic methods of the invention include systemic or local administration of the composition as an implant or device. When administered, the therapeutic compositions used in the invention are, of course, in a pyrogen-free, physiologically acceptable form. Therapeutically useful agents other than GDF Trap polypeptides that may also be optionally included in the above compositions may be administered simultaneously or sequentially with the subject compounds (e.g., GDF Trap polypeptides) in the methods of the invention.

[0177] Typically, the compound will be administered parenterally. Pharmaceutical compositions suitable for parenteral administration may comprise one or more GDF Trap polypeptides in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions; or sterile powders that may be reconstituted into sterile injectable solutions or dispersions immediately prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending agents or thickening agents. Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof; vegetable oils, such as olive oil; and injectable organic esters, such as ethyl oleate. For example, appropriate fluidity may be maintained by the use of a coating material such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by the use of a surfactant.

[0178] In addition, the compositions can be encapsulated or injected in a form for delivery to a target tissue site, such as the bone marrow. In certain embodiments, the compositions described herein can include a matrix capable of delivering one or more therapeutic compounds, such as a GDF Trap polypeptide, to a target tissue site, such as the bone marrow, providing structure to the developing tissue and optimally capable of being reabsorbed into the body. For example, the matrix can provide a slow release of the GDF Trap polypeptide. Such matrices can be formed from materials currently used in other implant medical applications.

[0179] The selection of matrix materials is based on biocompatibility, biodegradability, mechanical properties, aesthetic appearance and interface properties. The specific application of the subject composition will define the appropriate formulation. Possible matrices for the composition can be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other possible materials are biodegradable and biologically fully defined, such as bone or dermal collagen. Further matrices include pure proteins or extracellular matrix components. Other possible matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates or other ceramics. The matrix can include a combination of any of the above-mentioned types of materials, such as a combination of polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can be changed in composition (such as calcium-aluminate-phosphate) and processed to change pore size, particle size, particle shape and biodegradability.

[0180] In certain embodiments, the methods of the present invention can be administered orally, for example in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and gum arabic or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a lozenge (using an inert base, such as gelatin and glycerin or sucrose and gum arabic) and / or as a mouthwash, etc., each containing a predetermined amount of the agent as the active ingredient. The agent can also be administered in the form of a bolus, electuary, or paste.

[0181] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more therapeutic compounds of the invention may be mixed with one or more pharmaceutically acceptable carriers (e.g., sodium citrate or dicalcium phosphate) and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or gum arabic; ; (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 retardants such as paraffin; (6) absorption promoters such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glyceryl monostearate; (8) adsorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical composition may also contain a buffer. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols and the like.

[0182] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, colorants, fragrances and preservatives.

[0183] Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0184] The composition of the present invention may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, etc.) are introduced to ensure protection against the effects of microorganisms. It may also be desirable to include isotonic agents (e.g., sugars, sodium chloride, etc.) in the composition. In addition, the absorption of injectable drug forms may be extended by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0185] It will be appreciated that the dosage regimen may be determined by the attending physician taking into account various factors that alter the effects of the subject compounds of the invention (e.g., GDF Trap polypeptides). Such factors include, but are not limited to, the patient's red blood cell count, hemoglobin level or other diagnostic assessment, the desired target red blood cell count, the patient's age, sex and diet, the severity of any disease that may result in reduced red blood cell levels, the time of administration, and other clinical factors. The addition of other known growth factors to the final composition may also affect the dosage. Progress may be monitored by periodically assessing red blood cell and hemoglobin levels, as well as assessing reticulocyte levels and other indicators of the hematopoietic process.

[0186] In certain embodiments, the present invention also provides gene therapy for in vivo production of GDF trap polypeptides. Such therapy can be achieved by introducing a GDF trap polynucleotide sequence into cells or tissues having the above-listed conditions. The delivery of the GDF trap polynucleotide sequence can be achieved using a recombinant expression vector (e.g., a chimeric virus or a colloidal dispersion system). Preferably, targeted liposomes are used for therapeutic delivery of the GDF trap polynucleotide sequence.

[0187] Various viral vectors that can be used for gene therapy as taught herein include adenovirus, herpes virus, vaccinia virus or RNA virus such as retrovirus. The retroviral vector can be a derivative of a mouse or avian retrovirus. Examples of retroviral vectors into which a single exogenous gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV) and Rous sarcoma virus (RSV). Many other retroviral vectors can incorporate a variety of genes. All of these vectors can transfer or incorporate a selection marker gene so that transduced cells can be identified and generated. Retroviral vectors can have target specificity by attaching, for example, sugars, glycolipids or proteins. Targeting is preferably achieved by using antibodies. Those skilled in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to the viral envelope to allow target-specific delivery of retroviral vectors containing GDF capture polynucleotides.

[0188] Alternatively, plasmids encoding the retroviral structural genes gag, pol, and env can be used to directly transfect tissue culture cells by conventional calcium phosphate transfection. These cells are then transfected with a vector plasmid containing the gene of interest. The resulting cells release the retroviral vector into the culture medium.

[0189] Another targeted delivery system for GDF Trap polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). The preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane vesicles that can be used as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated inside the aqueous phase and delivered to cells in a biologically active form (e.g., see Fraley et al., Trends Biochem. Sci., 6:77, 1981). Methods for effectively transferring genes using liposome vectors are known in the art, for example, see Mannino et al., Biotechniques, 6:682, 1988. The composition of liposomes is generally a combination of phospholipids, and is often combined with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0190] Examples of lipids for producing liposomes include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. The targeting of liposomes may also be based on, for example, organ specificity, cell specificity and organelle specificity, and is known in the art. Example

[0191] The present invention has been generally described above. The present invention will be more readily understood with reference to the following examples, which are merely intended to illustrate certain embodiments and embodiments of the present invention and are not intended to limit the present invention.

[0192] Example 1: Generation of GDF Trap

[0193] Applicants constructed GDF traps as follows. A polypeptide having a modified ActRIIB extracellular domain with greatly reduced activin A binding relative to GDF11 and / or tubocurarine (as a consequence of a leucine-to-aspartic acid substitution at position 79 in SEQ ID NO: 1) was fused to a human or mouse Fc domain with a minimal linker (3 glycine amino acids) in between. The constructs were referred to as ActRIIB(L79D 20-134)-hFc and ActRIIB(L79D 20-134)-mFc, respectively. An alternative form (L79E) with glutamic acid instead of aspartic acid at position 79 was similarly implemented. An alternative form with alanine instead of valine at position 226 relative to SEQ ID NO: 7 below was also generated and equivalently implemented in all aspects of the test. The aspartic acid at position 79 (relative to SEQ ID NO: 1, or relative to position 60 of SEQ ID NO: 7) is highlighted in gray below. The valine at position 226 relative to SEQ ID NO: 7 is also highlighted in grey below.

[0194] The GDF Trap ActRIIB(L79D 20-134)-hFc is shown below as purified from a CHO cell line (SEQ ID NO: 7).

[0195]

[0196] The ActRIIB-derived portion of the GDF Trap has the amino acid sequence shown below (SEQ ID NO: 32), and this portion will function as a monomer, or as a non-Fc fusion protein that is a monomer, dimer, or higher order complex.

[0197]

[0198] GDF Trap protein was expressed in a CHO cell line. Three different leader sequences were considered:

[0199] (i) Honey bee melittin (HBML):

[0200] (ii) Tissue plasminogen activator (TPA):

[0201] (iii) Natural: .

[0202] The selected form utilizes the TPA leader sequence and has the following unprocessed amino acid sequence:

[0203]

[0204] The polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 12):

[0205]

[0206] Purification can be by a series of column chromatography steps, including for example 3 or more of the following to realize in any order: protein A chromatography, Q agarose chromatography, phenyl agarose chromatography, size exclusion chromatography and cation exchange chromatography. Purification can be completed with virus filtration and buffer exchange. In an example of purification scheme, cell culture medium is passed through protein A column, washed in 150 mM Tris / NaCl (pH 8.0), then washed in 50 mM Tris / NaCl (pH 8.0) and eluted with 0.1 M glycine pH 3.0. Low pH eluate was maintained at room temperature for 30 minutes, as a virus removal step. Then the eluate is neutralized and washed in 50 mM Tris pH 8.0, 50 mM NaCl, and eluted in 50 mMTris pH 8.0 (with 150 mM to 300 mM NaCl concentration). The eluate was then changed to 50 mM Tris pH 8.0, 1.1 M ammonium sulfate and passed through a phenyl sepharose column, washed and eluted in 50 mM Tris pH 8.0 with 150 mM to 300 mM ammonium sulfate. The eluate was dialyzed and filtered for use.

[0207] Additional GDF Traps (ActRIIB-Fc fusion proteins modified to reduce the ratio of activin A binding relative to tubocurarine or GDF11) are described in PCT / US2008 / 001506 and WO 2006 / 012627, which are incorporated herein by reference.

[0208] Example 2. Bioassays for GDF-11 and Activin-mediated signaling.

[0209] A-204 reporter gene assay was used to evaluate the effects of ActRIIB-Fc protein and GDF trap on signaling through GDF-11 and activin A. Cell line: human rhabdomyosarcoma (derived from muscle). Reporter vector: pGL3(CAGA)12 (described in Dennler et al., 1998, EMBO 17: 3091-3100). The CAGA12 motif is present in TGF-β responsive genes (PAI-1 gene), so this vector is often used for factors that signal through Smad2 and 3.

[0210] Day 1: A-204 cells were split into 48-well plates.

[0211] Day 2: A-204 cells were transfected with 10 ug pGL3(CAGA)12 or pGL3(CAGA)12 (10 ug) + pRLCMV (1 ug) and Fugene.

[0212] Day 3: Add factors (diluted in culture medium + 0.1% BSA). Inhibitors need to be pre-incubated with factors for 1 hour before adding to cells. After 6 hours, cells are rinsed with PBS and lysed.

[0213] Luciferase assay was then performed. In the absence of any inhibitor, Activin A showed 10-fold stimulation of reporter gene expression and ED50 ~ 2 ng / ml. GDF-11: 16-fold stimulation, ED50: ~ 1.5 ng / ml.

[0214] ActRIIB(20-134) is a potent inhibitor of activin, GDF-8 and GDF-11 activity in this assay. Variants were also tested in this assay.

[0215] Example 3. GDF-11 Inhibition by N-terminal and C-terminal Truncations

[0216] ActRIIB(20-134)-hFc variants with N-terminal and / or C-terminal truncations were generated and tested for their activity as inhibitors of GDF-11 and activin. The activities are shown below (as measured in conditioned medium):

[0217] C-terminal ActRIIB-hFc truncation:

[0218]

[0219] As can be seen, truncation of the C-terminal 3 (ending with ... PPT), 6 (ending with ... YEP) or more amino acids causes a 3-fold or greater decrease in the activity of the molecule. Truncation of the last 15 amino acids of the ActRIIB portion causes a greater loss of activity (see WO2006 / 012627).

[0220] The amino terminal truncations were made in the context of ActRIIB(20-131)-hFc protein. The activities are shown below (as measured in conditioned medium):

[0221] N-terminal ActRIIB-hFc truncation:

[0222]

[0223] Accordingly, truncations of 2, 3, or 4 amino acids from the N-terminus resulted in production of more active proteins than forms with full-length extracellular domains. Additional experiments showed that a 5 amino acid truncation of ActRIIB(25-131)-hFc had equivalent activity to the untruncated form, and that additional deletions at the N-terminus continued to reduce the activity of the protein. Thus, the optimal construct would have a C-terminus terminating between amino acids 133-134 of SEQ ID NO: 1 and an N-terminus starting at amino acids 22-24 of SEQ ID NO: 1. An N-terminus corresponding to amino acids 21 or 25 would result in activity similar to that of the ActRIIB(20-134)-hFc construct. These truncations may also be used in the context of GDF traps, such as the L79D or L79E variants.

[0224] Example 4. ActRIIB-Fc variants, cell-based activity.

[0225] The activity of ActRIIB-Fc protein and GDF traps were tested in cell-based assays, as described above. The results are summarized in the table below. Some variants were tested in different C-terminal truncation constructs. As discussed above, truncation of 5 or 15 amino acids caused reduced activity. GDF traps (L79D and L79E variants) showed a substantial loss of activin binding while maintaining almost wild-type inhibition of GDF-11.

[0226] Soluble ActRIIB-Fc binds to GDF11 and Activin A:

[0227]

[0228] + Poor activity (roughly 1×10 -6 K I )

[0229] ++ Moderate activity (approximately 1×10 -7 K I )

[0230] +++ (wild type) good activity (approximately 1×10 -8 K I )

[0231] ++++ Higher than wild-type activity.

[0232] Several variants have been evaluated for serum half-life in rats. ActRIIB (20-134) -Fc has a serum half-life of approximately 70 hours. ActRIIB (A24N 20-134) -Fc has a serum half-life of approximately 100-150 hours. The A24N variant has activity equivalent to that of the wild-type molecule in cell-based assays (described above) and in vivo assays (below). Combined with a longer half-life, this means that over time the A24N variant will produce a greater effect per unit of protein than the wild-type molecule. The A24N variant and any other variant tested above can be combined with a GDF capture molecule, such as the L79D or L79E variant.

[0233] Example 5. GDF-11 and Activin A Binding.

[0234] In BiaCore TM Certain ActRIIB-Fc proteins and GDF traps were tested for binding to ligands in the assay.

[0235] ActRIIB-Fc variants or wild-type proteins were captured in the system using anti-hFc antibodies. Ligands were injected and flowed over the captured receptor proteins. The results are summarized in the table below.

[0236] Ligand Binding Specificity IIB Variants.

[0237]

[0238] These data demonstrate that the A24N variant retains ligand-binding activity similar to the ActRIIB(20-134)-hFc molecule, and that the L79D or L79E molecules retain tubocurarine and GDF11 binding but show significantly reduced (unquantifiable) binding to activin A based on cell-based assay data.

[0239] Other variants have been generated and tested, as reported in WO2006 / 012627 (incorporated herein by reference in its entirety), see, e.g., pages 59-60, which employ ligands coupled to the device and mobile receptors on the coupled ligands. Significantly, K74Y, K74F, K74I (and presumably other hydrophobic substitutions at K74, such as K74L) and D80I resulted in a reduction in the ratio of activin A binding to GDF11 binding relative to the wild-type K74 molecule. The data tables for these variants are reproduced below:

[0240] Soluble ActRIIB-Fc variant binding to GDF11 and Activin A (BiaCore analysis)

[0241]

[0242] * No observed combination

[0243] -- < 1 / 5 WT combined

[0244] - ~ 1 / 2 WT combined

[0245] + WT

[0246] ++ < 2× increased binding

[0247] +++ ~5× increased bonding

[0248] ++++ ~10× increased binding

[0249] +++++ ~ 40× increased bindings.

[0250] Example 6. ActRIIB-hFc stimulates erythropoiesis in non-human primates

[0251] ActRIIB (20-134) -hFc (IgG1) was administered to male and female cynomolgus monkeys by subcutaneous injection once a week for 1 month. 48 cynomolgus monkeys (24 / sex) were assigned to 1 of 4 treatment groups (6 animals / sex / group) and administered vehicle or 3, 10 or 30 mg / kg of ActRIIB-hFc by subcutaneous injection once a week for 4 weeks (5 doses in total). The parameters evaluated included general clinical pathology (hematology, clinical chemistry, coagulation and urine tests). By day 15, ActRIIB-hFc caused a statistically significant increase in the mean absolute reticulocyte value in treated animals. By day 36, ActRIIB-hFc caused several hematological changes, including increased mean absolute reticulocyte and red cell distribution width values ​​and lower mean microgranular hemoglobin concentrations. All treatment groups and both sexes were affected. These effects are consistent with the positive effect of ActRIIB-hFc on the release of immature reticulocytes from the bone marrow. This effect was reversed after washout of the drug from treated animals (by study day 56). Accordingly, we concluded that ActRIIB-hFc stimulates erythropoiesis.

[0252] Example 7. ActRIIB-mFc promotes erythropoiesis in mice by stimulating spleen erythropoietic activity

[0253] In this study, the effect of in vivo administration of ActRIIB (20-134) -mFc on the frequency of hematopoietic progenitors in the bone marrow and spleen was analyzed. One group of C57BL / 6 mice were injected with PBS as a control and the second group of mice were administered two doses of 10 mg / kg of ActRIIB-mFc, and both groups were killed after 8 days. Peripheral blood was used for complete blood counts, and femurs and spleens were used for in vitro clonal analysis to evaluate the lymph, erythrocyte and bone marrow progenitor cell content in each organ. In the short time frame of this study, no significant changes in the levels of erythrocytes, hemoglobin or leukocytes in the treated mice were seen. In the femur, there was no difference in the number of nucleated cells or the content of ancestors between the control and treatment groups. In the spleen, the compound treatment group experienced a statistically significant increase in the number of mature erythrocyte ancestors (CFU-E) colonies / dish, frequency and total number of ancestors / spleen. In addition, an increase was seen in the number of bone marrow (CFU-GM), immature erythrocytes (BFU-E) and total number of ancestors / spleen.

[0254] animal:

[0255] 16 C57BL / 6 female mice of 6-8 weeks old were used in the study. Test compound ActRIIB-mFc was injected subcutaneously to 8 mice at the dosage of 10 mg / kg on the 1st and 3rd days, and phosphate buffered saline (PBS) was injected subcutaneously to 8 mice with a volume of 100 μL per mouse. All mice were killed according to the relevant animal management guidelines 8 days after the first injection. Peripheral blood (PB) samples from individual animals were collected by cardiac puncture, and used for complete blood count and differential count (CBC / Diff). Femur and spleen were harvested from each mouse.

[0256] Tests performed:

[0257] CBC / Diff Count

[0258] PB from each mouse was collected by cardiac puncture and placed in an appropriate microtainer. The samples were sent to CLV for analysis on a CellDyn 3500 counter.

[0259] Colony production assay

[0260] The myeloid, erythroid, and lymphoid lineages were evaluated for clonogenic progenitors using the in vitro methylcellulose-based culture system described below.

[0261] Mature erythrocyte progenitors

[0262] The clonogenic progenitors of the mature erythroid (CFU-E) line were cultured in MethoCultTM 3334, a methylcellulose-based medium containing recombinant human (rh) erythropoietin (3 U / mL).

[0263] Lymphoid Ancestors

[0264] The clonogenic progenitors of the lymphoid (CFU-pre-B) lineages were cultured in MethoCult® 3630, a methylcellulose-based medium containing rhIL-7 (10 ng / mL).

[0265] Myeloid and Immature Erythroid Progenitors

[0266] The clonogenic progenitors of granulocyte-monocyte (CFU-GM), erythroid (BFU-E), and pluripotent (CFU-GEMM) lines were cultured in MethoCultTM 3434, a methylcellulose-based medium containing recombinant murine (rm) stem cell factor (50 ng / mL), rh interleukin-6 (10 ng / mL), rm interleukin-3 (10 ng / mL), and rh erythropoietin (3 U / mL).

[0267] method:

[0268] Mouse femur and spleen were processed by standard protocol. In brief, by using 21 gauge needles and 1 cc syringes, the femoral cavity was rinsed with the Dulbecco's culture medium modified by Iscove containing 2% fetal bovine serum (IMDM 2% FBS), and bone marrow was obtained. Spleen was squeezed by 70 μM filters and the filter was rinsed with IMDM 2% FBS to obtain splenocytes. The nucleated cell count in 3% glacial acetic acid was then carried out on a single cell suspension with a Neubauer counting chamber, so that the total cells of each organ can be calculated. In order to remove contaminated red blood cells, total splenocytes were subsequently diluted with 3 times the volume of ammonium chloride lysis buffer, and incubated on ice for 10 minutes. The cells were then washed and resuspended in IMDM 2% FBS, and a second cell count was performed to determine the cell concentration of the cells after the cracking.

[0269] Cell stocks were prepared and added to each methylcellulose-based media formulation to obtain the optimal plating concentration for each tissue in each media formulation. Bone marrow cells were plated at 1 × 10 cells per dish. 5 To assess mature erythroid progenitors, plate 2 x 10 cells per dish in MethoCultTM 3334. 5 Cells were plated into MethoCultTM 3630 to assess lymphoid progenitors and 3 × 10 cells were plated per dish. 4Cells were plated into MethoCultTM 3434 to assess immature erythroid and myeloid progenitors. Splenocytes were plated at 4 × 10 per dish. 5 Cells were plated into MethoCultTM 3334 to assess mature erythroid progenitors at 4 × 10 cells per dish. 5 Cells were plated into MethoCultTM 3630 to assess lymphocyte progenitors and 2×10 cells were plated per dish. 5 Cells were plated into MethoCult™ 3434 to assess immature erythroid and myeloid progenitors. Cultures plated into triplicate dishes were incubated at 37°C, 5% CO2 until colony counts and evaluation were performed by trained personnel. Mature erythroid progenitors were cultured for 2 days, lymphoid progenitors for 7 days, and mature erythroid and myeloid progenitors for 12 days.

[0270] analyze

[0271] The mean + / - 1 standard deviation was calculated for triplicate cultures for clonogenic assays and for all data sets for control and treatment groups.

[0272] The frequency of colony forming cells (CFC) in each tissue was calculated as follows:

[0273] Cells plated per dish

[0274] The average CFC scored per dish

[0275] The total CFC for each femur or spleen was calculated as follows:

[0276] Total CFC scored × nucleated cell count per femur or spleen (after RBC lysis)

[0277] Number of nucleated cells in culture

[0278] Standard t-test was performed to assess whether there was a difference in the mean number of cells or hematopoietic progenitors between PBC control mice and compound-treated mice. Because colony counting may be subjective, p values ​​less than 0.01 were considered significant. The mean values ​​(+ / - SD) of each group are shown in the table below.

[0279] Table: Hematological parameters

[0280]

[0281] Table: CFC from femur and spleen

[0282]

[0283] *Preliminary analysis showed p < 0.005.

[0284] Treating mice with ActRIIB (20-134)-mFc did not result in a significant increase in red blood cell or hemoglobin content within the short time frame of this study. However, the effect on progenitor cell content was significant. In the femur, there was no difference in the number of nucleated cells or progenitor cell content between the control and treatment groups. In the spleen, the compound treatment group experienced a statistically significant increase in the number of nucleated cells before red blood cell lysis and the number of mature red blood cell progenitors (CFU-E) colonies per culture dish, the frequency of each spleen, and the total number of progenitors. In addition, an increase was observed in the bone marrow progenitors (CFU-GM), immature red blood cell progenitors (BFU-E), and the total number of progenitors per spleen. Accordingly, it is expected that ActRIIB (20-134)-mFc treatment can result in increased red blood cell and hemoglobin content over a longer period of time.

[0285] Example 8: GDF Trap Increases Red Blood Cell Levels in Vivo

[0286] Twelve-week-old male C57BL / 6NTac mice were assigned to one of two treatment groups (N=10). Mice were given vehicle or variant ActRIIB polypeptide ("GDF trap") [ActRIIB(L79D20-134)-hFc] at 10 mg / kg by subcutaneous injection (SC), twice a week for 4 weeks. At study cessation, whole blood was collected by cardiac puncture into EDTA-containing tubes and analyzed for cell distribution using an HM2 hematology analyzer (Abaxis, Inc).

[0287] Group Assignment

[0288]

[0289] Treatment with GDF traps had no statistically significant effect on white blood cell (WBC) numbers compared to vehicle controls. Red blood cell (RBC) numbers were increased in the treated groups compared to controls (see table below). Hemoglobin content (HGB) and hematocrit (HCT) were also increased due to the extra red blood cells. The mean width of the red blood cells (RDWc) was higher in treated animals, indicating an increase in the immature red blood cell population. Therefore, treatment with GDF traps resulted in an increase in red blood cells with no discernible effect on the white blood cell population.

[0290] Hematology results

[0291]

[0292] *=p<0.05, **= p<0.01.

[0293] Example 9: GDF Trap is superior to ActRIIB-Fc for increasing red blood cell levels in vivo

[0294] Male C57BL / 6NTac mice aged 19 weeks were randomly assigned to one of three groups. Mice were given vehicle (10 mM Tris-buffered saline, TBS), wild-type ActRIIB (20-134)-mFc or GDF trap ActRIIB (L79D 20-134)-hFc by subcutaneous injection twice a week for 3 weeks. Blood was collected by buccal bleeding at baseline and after 3 weeks of dosing and analyzed for cell distribution using a hematology analyzer (HM2, Abaxis, Inc.).

[0295] Treatment with ActRIIB-Fc or GDF traps had no significant effect on white blood cell (WBC) numbers compared to vehicle controls. Red blood cell counts (RBC), hematocrit (HCT), and hemoglobin levels were all increased in GDF trap-treated mice compared to controls or wild-type constructs (see table below). Thus, in direct comparison, GDF traps promoted an increase in red blood cells to a significantly greater extent than wild-type ActRIIB-Fc protein. In fact, in this experiment, wild-type ActRIIB-Fc protein did not cause a statistically significant increase in red blood cells, suggesting that longer or higher doses are required to exhibit this effect.

[0296] Hematological results after 3 weeks of dosing

[0297]

[0298] **=p<0.01.

[0299] Example 10. Generation of GDF Traps with Truncated ActRIIB Extracellular Domains

[0300] As described in Example 1, a GDF Trap termed ActRIIB(L79D 20-134)-hFc was generated by fusion of the TPA leader sequence to the N-terminus of the ActRIIB extracellular domain (residues 20-134 in SEQ ID NO: 1) containing a leucine-to-aspartate substitution (residue 79 in SEQ ID NO: 1) and the human Fc domain to the C-terminus with a minimal linker (3 glycine residues) ( FIG. 3 ). The nucleotide sequence corresponding to this fusion protein is Figure 4 Shown in.

[0301] A GDF trap with a truncated ActRIIB ectodomain, termed ActRIIB(L79D 25-131)-hFc, was generated by fusion of the TPA leader sequence to the N-terminus of the truncated ectodomain (residues 25-131 in SEQ ID NO: 1) containing a leucine-to-aspartate substitution (residue 79 in SEQ ID NO: 1) and the human Fc domain to the C-terminus with a minimal linker (3 glycine residues). Figure 5 ). The nucleotide sequence corresponding to the fusion protein is Figure 6 Shown in.

[0302] Example 11. Selective ligand binding by GDF traps with double-truncated ActRIIB ectodomains

[0303] The affinity of GDF traps and other ActRIIB-hFc proteins for several ligands was evaluated in vitro using a Biacore™ instrument. The results are summarized in the table below. Kd values ​​were obtained by fitting the steady-state affinity due to the very fast association and dissociation of the complex, which prevents k 结合 and k 解离 accurate determination.

[0304] Ligand selectivity of ActRIIB-hFc variants:

[0305]

[0306] Compared to the ActRIIB-hFc counterpart lacking the L79D substitution, the GDF trap ActRIIB(L79D 25-131)-hFc with a truncated extracellular domain equals or exceeds the ligand selectivity exhibited by the longer variant ActRIIB(L79D 20-134)-hFc, with a significant loss of activin A and activin B binding and almost complete retention of GDF11 binding. Note that truncation alone (without the L79D substitution) does not alter the inter-ligand selectivity exhibited here [compare ActRIIB(L79 25-131)-hFc vs. ActRIIB(L79 20-134)-hFc].

[0307] Example 12. Generation of ActRIIB(L79D 25-131)-hFc with alternative nucleotide sequences

[0308] To generate ActRIIB(L79D 25-131)-hFc, the human ActRIIB ectodomain with an aspartic acid substitution at native position 79 (SEQ ID NO: 1) and with N-terminal and C-terminal truncations (residues 25-131 in SEQ ID NO: 1) was fused at the N-terminus to a TPA leader sequence instead of the native ActRIIB leader sequence and at the C-terminus to a human Fc domain via a minimal linker (3 glycine residues) ( Figure 5 ). A nucleotide sequence encoding the fusion protein is Figure 6 (SEQ ID NO: 27), and alternative nucleotide sequences encoding identical fusion proteins are shown in Fig. 9 The protein was expressed and purified using the methodology described in Example 1.

[0309] Example 13. GDF Trap with Truncated ActRIIB Extracellular Domain Increases Proliferation of Erythroid Progenitors in Mice

[0310] ActRIIB(L79D 25-131)-hFc was evaluated to determine its effect on erythroid progenitor proliferation. Male C57BL / 6 mice (8 weeks old) were treated with ActRIIB(L79D 25-131)-hFc (10 mg / kg, subcutaneous; n = 6) or vehicle (TBS; n = 6) on days 1 and 4 and then euthanized on day 8 to collect spleen, tibia, femur, and blood. Cells from spleen and bone marrow were isolated, diluted in Iscove's modified Dulbecco's medium containing 5% fetal bovine serum, suspended in a specialized methylcellulose-based medium, and cultured for 2 or 12 days to assess the levels of clonal progenitors generated at the colony-forming unit-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) stages, respectively. The methylcellulose-based medium used for BFU-E determination (MethoCult M3434, Stem Cell Technologies) includes recombinant murine stem cell factor, interleukin-3 and interleukin-6, which are absent in the methylcellulose medium used for CFU-E determination (MethoCult M3334, Stem Cell Technologies), while both mediums contain other components such as erythropoietin. For both BFU-E and CFU-E, the number of clones was determined in two culture plates derived from each tissue sample, and the statistical analysis of the results was based on the number of mice / treatment group.

[0311] Spleen-derived cultures from mice treated with ActRIIB (L79D 25-131) -hFc had twice the number of CFU-E clones as the corresponding cultures from control mice (P < 0.05), while the number of BFU-E clones was not significantly different from the in vivo treatment. The number of CFU-E or BFU-E clones from bone marrow cultures was also not significantly different from the treatment. As expected, in mice treated with ActRIIB (L79D 25-131) -hFc, the increased number of CFU-E clones in spleen-derived cultures was accompanied by significant (P < 0.001) changes in red blood cell levels (11.6% increase), hemoglobin concentration (12% increase), and hematocrit levels (11.6% increase) compared to controls in euthanasia. These results suggest that in vivo administration of GDF traps with truncated ActRIIB extracellular domains can stimulate the proliferation of erythrocyte progenitors as part of their overall effect of increasing red blood cell levels.

[0312] Example 14. GDF Trap with Truncated ActRIIB Extracellular Domain Counteracts Chemotherapy-Induced Anemia in Mice

[0313] Applicants studied the effects of ActRIIB(L79D25-131)-hFc on erythropoietic parameters in a paclitaxel-based chemotherapy-induced mouse model of anemia, which inhibits cell division by blocking microtubule polymerization. Male C57BL / 6 mice (8 weeks old) were assigned to 1 of 4 treatments:

[0314] 1) Paclitaxel (25 mg / kg, intraperitoneal)

[0315] 2) ActRIIB(L79D 25-131)-hFc (10 mg / kg, intraperitoneal)

[0316] 3) Paclitaxel + ActRIIB(L79D 25-131)-hFc

[0317] 4) Vector (TBS).

[0318] Paclitaxel was administered on day 0, while ActRIIB(L79D 25-131)-hFc or vehicle was administered on days 0 and 3. Blood samples were collected from separate groups on days 1, 3, and 5 for CBC analysis, and the results for treatment groups 1-3 (described above) are expressed as the percentage difference from the vehicle at a given time point. The loss due to paclitaxel toxicity was a problem only in the paclitaxel group on day 3 (where n = 1), otherwise n = 3-5 / treatment / time point. Paclitaxel alone reduced hemoglobin concentration by almost 13% on day 5 compared to vehicle, while the addition of ActRIIB(L79D 25-131)-hFc prevented this paclitaxel-induced decline (Figure 11). Similar effects were seen for hematocrit and RBC levels. In the absence of paclitaxel, ActRIIB(L79D 25-131)-hFc increased hemoglobin concentration by 10% (0.02 mmol / l) on days 3 and 5 compared to vehicle. Fig.11 ). Thus, GDF traps carrying a truncated ActRIIB extracellular domain can increase erythrocyte levels sufficient to offset chemotherapy-induced anemia.

[0319] Example 15. GDF Trap with Truncated ActRIIB Extracellular Domain Reverses Nephrectomy-Induced Anemia in Mice

[0320] The applicant studied the effect of ActRIIB (L79D 25-131) -hFc on anemia in a nephrectomized mouse model of chronic renal disease. Male C57BL / 6 mice (11 weeks old) underwent sham surgery or unilateral nephrectomy to reduce the ability to produce erythropoietin. The mice were allowed to recover after 1 week of surgery and then treated twice a week with ActRIIB (L79D 25-131) -hFc (10 mg / kg, intraperitoneal; n = 15 / condition) or vehicle (TBS; n = 15 / condition) for 4 weeks in total. Blood samples were collected before the start of dosing and after 4 weeks of treatment. Although the nephrectomized mice treated with the vehicle showed a significant decrease in the number of red blood cells after a 4-week treatment period, treatment with ActRIIB (L79D 25-131) -hFc not only prevented the decrease, but also increased the level of red blood cells above the baseline by 17% (P < 0.001) ( Fig.12 ), despite reduced renal erythropoietin production capacity. In nephrectomized mice, ActRIIB(L79D 25-131)-hFc also produced a significant increase in hemoglobin concentration and hematocrit levels from baseline, and significantly stimulated each of these erythropoietic parameters under nephrectomy conditions to approximately the same extent as under sham conditions (Figure 13). Thus, GDF traps with truncated ActRIIB extracellular domains can increase red blood cell levels sufficient to reverse anemia in a chronic kidney disease model.

[0321] Example 16. GDF Trap with Truncated ActRIIB Extracellular Domain Improves Recovery from Blood Loss-Induced Anemia in Rats

[0322] Applicants studied the effects of ActRIIB(L79D 25-131)-hFc on erythropoietic parameters in a model of acute blood loss-induced anemia in rats (acute post-hemorrhagic anemia). Male Sprague-Dawley rats (approximately 300 g) received a chronic jugular catheter at the vendor (Harlan). On day -1, 20% of the total blood volume was withdrawn from each rat through the catheter over a 5-minute period under isoflurane anesthesia. The amount of blood removed was based on the value of the total blood volume calculated according to the following correlation, which was derived from Lee and colleagues (J Nucl Med 25:72-76, 1985) for rats weighing more than 120 g:

[0323] Total blood volume (ml) = 0.062 × body weight (g) + 0.0012.

[0324] An equal volume of phosphate buffered saline was replaced via the catheter at the time of blood draw. Rats were treated with ActRIIB(L79D 25-131)-hFc (10 mg / kg, subcutaneous; n = 5) or vehicle (TBS; n = 5) on days 0 and 3. Blood samples for CBC analysis were removed via the catheter on days -1 (baseline), 0, 2, 4, and 6.

[0325] In response to 20% blood loss in control rats, red blood cell levels dropped by nearly 15% by day 0. These levels remained significantly below baseline on days 2 and 4 and did not fully recover by day 6 ( Fig.14 ). Although rats treated with ActRIIB(L79D 25-131)-hFc showed nearly identical reductions in erythrocyte levels after 20% blood loss, these rats then demonstrated full recovery of these levels by day 2, followed by further increases on days 4 and 6, representing highly significant improvements relative to control levels at the corresponding time points ( Fig.14 ). Similar results were obtained for hemoglobin concentration. These findings suggest that GDF traps with truncated ActRIIB ectodomains can produce faster recovery of red blood cell levels from anemia caused by acute hemorrhage.

[0326] Example 17. GDF Traps with Truncated ActRIIB Extracellular Domain Increase Red Blood Cell Levels in Nonhuman Primates

[0327] The ability of two GDF traps, ActRIIB(L79D 20-134)-hFc and ActRIIB(L79D 25-131)-hFc, to stimulate erythrocyte production was evaluated in cynomolgus monkeys. Monkeys were treated subcutaneously with GDF traps (10 mg / kg; n = 4 males / 4 females) or vehicle (n = 2 males / 2 females) on days 1 and 8. Blood samples were collected on days 1 (pre-treatment baseline), 3, 8, 15, 29, and 44, and analyzed for erythrocyte levels ( Fig.15 ), hematocrit ( Fig.16 ), hemoglobin level ( Fig.17 ) and reticulocyte levels ( Fig.18 ). Vehicle-treated monkeys showed reduced levels of red blood cells, hematocrit, and hemoglobin at all post-treatment time points, an expected effect of repeated blood sampling. In contrast, treatment with ActRIIB(L79D 20-134)-hFc or ActRIIB(L79D 25-131)-hFc increased these parameters to the first post-treatment time point (Day 3) and maintained them at substantially elevated levels for the duration of the study ( Figure 15-17 Importantly, reticulocyte levels were substantially increased in monkeys treated with ActRIIB(L79D 20-134)-hFc or ActRIIB(L79D 25-131)-hFc compared to vehicle at days 8, 15, and 29 ( Fig.18 ). This result suggests that GDF Trap treatment increases erythroid precursor production, leading to increased erythroid levels.

[0328] Together, these data suggest that truncated GDF traps as well as full-length variants can serve as selective antagonists and potentially relevant ligands for GDF11 to increase erythroid formation in vivo.

[0329] Example 18. GDF Trap Derived from ActRIIB5

[0330] Others have reported an alternative soluble form of ActRIIB (designated ActRIIB5) in which exon 4, including the ActRIIB transmembrane domain, has been replaced by a different C-terminal sequence (WO2007 / 053775).

[0331] The sequence of native human ActRIIB5 without its leader sequence is as follows:

[0332]

[0333] A leucine-to-aspartic acid substitution or other acidic substitution can be made at the native position 79 (underlined and highlighted in grey) to construct a variant ActRIIB5 (L79D) having the following sequence:

[0334]

[0335] This variant can be linked to human Fc using a TGGG linker to generate a human ActRIIB5(L79D)-hFc fusion protein with the following sequence:

[0336]

[0337] This construct can be expressed in CHO cells.

[0338] Example 19. Effects of combined treatment with EPO and GDF traps with truncated ActRIIB extracellular domain in mice

[0339] EPO induces erythropoiesis by increasing the proliferation of erythroid precursors, and GDF traps can potentially affect erythropoiesis in a way that complements or enhances the effects of EPO. Therefore, the applicant studied the effects of combined treatment with EPO and ActRIIB (L79D 25-131) -hFc on erythropoietic parameters. Male C57BL / 6 mice (9 weeks old) were given a single intraperitoneal injection of recombinant human EPO alone (Epoetin α, 1800 units / kg), ActRIIB (L79D 25-131) -hFc alone (10 mg / kg), EPO and ActRIIB (L79D 25-131) -hFc, or vehicle (Tris-buffered saline). Mice were euthanized 72 h after dosing to collect blood, spleen and femur.

[0340] Spleens and femurs were processed to obtain erythroid progenitor cells for flow cytometric analysis. After removal, spleens were minced in Iscove's modified Dulbecco's medium containing 5% fetal bovine serum and mechanically dissociated by pushing the plunger from a sterile 1-mL syringe through a 70-µm cell strainer. Femurs were cleaned of any residual muscle or connective tissue and the ends were trimmed to allow collection of bone marrow by flushing the remaining diaphysis with Iscove's modified Dulbecco's medium containing 5% fetal bovine serum through a 21-gauge needle attached to a 3-mL syringe. The cell suspension was centrifuged (2000 rpm, 10 min) and the cell pellet was resuspended in PBS containing 5% fetal bovine serum. Cells from each tissue (10 6) were incubated with anti-mouse IgG to block nonspecific binding, then incubated with fluorescently labeled antibodies against mouse cell surface markers CD71 (transferrin receptor) and Ter119 (antigen bound to cell surface glycophorin A), washed, and analyzed by flow cytometry. Dead cells in the sample were excluded from the analysis by counterstaining with propidium iodide. Erythroid differentiation in the spleen or bone marrow was assessed by the degree of CD71 labeling that decreased during differentiation and the degree of Ter119 labeling that increased during terminal erythroid differentiation starting in the pre-erythroblast stage (Socolovsky et al., 2001, Blood 98:3261-3273; Ying et al., 2006, Blood 108:123-133). Therefore, flow cytometry was used to determine the pre-erythroblast (CD71 高 Ter119 低 ), basophils (CD71 高 Ter119 高 ), polychromatic + positively stained erythroblasts (CD71 中 Ter119 高 ) and late positively stained erythroblasts + reticulocytes (CD71 低 Ter119 高 ) as described.

[0341] Combined treatment with EPO and ActRIIB(L79D 25-131)-hFc resulted in a surprisingly dramatic increase in red blood cells. In the 72-h time frame of this experiment, neither EPO nor ActRIIB(L79D 25-131)-hFc alone significantly increased hematocrit compared to vehicle, whereas combined treatment with both agents resulted in an almost 25% increase in hematocrit that was unexpectedly synergistic, i.e., greater than the sum of their individual effects ( Fig.19 This type of synergistic effect is usually considered to demonstrate that the individual agents act through different cellular mechanisms. Similar results have also been reported for hemoglobin concentration ( Fig. 20 ) and red blood cell concentration ( Fig.21 ) were observed, each of which was also synergistically increased by the combined treatment.

[0342] Analysis of erythroid precursor levels revealed a more complex pattern. In mice, the spleen is thought to be the primary organ responsible for induced (“stress”) erythropoiesis. Flow cytometric analysis of spleen tissue at 72 h showed that EPO significantly altered the erythropoietic precursor profile compared with vehicle, increasing the number of basophilic erythroblasts by more than 170% at the expense of late precursors (late positively stained erythroblasts + reticulocytes), which decreased by more than one-third (Figure 22). Importantly, the combination treatment significantly increased basophilic erythroblasts compared with vehicle, but to a lesser extent than EPO alone, while supporting undiminished maturation of late precursors (Figure 23). Fig. 22 ). Therefore, combined treatment with EPO and ActRIIB (L79D 25-131) -hFc increases erythropoiesis through balanced enhancement of precursor proliferation and maturation. In contrast to the spleen, the profile of precursor cells in the bone marrow after combined treatment was not slightly different from that after EPO alone. Based on the spleen precursor profile, the applicant predicts that combined treatment will cause increased reticulocyte levels and will be accompanied by a sustained increase in mature erythrocyte levels if the experiment is extended for more than 72 h.

[0343] Taken together, these findings suggest that GDF traps with truncated ActR IIB extracellular domains can be administered in combination with EPO to synergistically increase erythrocyte formation in vivo. Through complementary but undefined mechanisms of action, GDF traps can mitigate the strong proliferative effects of EPO receptor activators alone and still allow target levels of erythrocytes to be achieved with lower doses of EPO receptor activators, thereby avoiding potential side effects or other problems associated with higher levels of EPO receptor activation.

[0344] Incorporated by Reference

[0345] All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0346] Although specific embodiments of the subject matter have been discussed, the above description is intended to be illustrative only and not limiting. Many variations of the invention will be apparent to those skilled in the art upon reading this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims and all their equivalents, as well as the specification and various variations. <110> Aceleron Pharmaceuticals <120> GDF trap combined with erythropoietin receptor activator to increase red blood cell levels <130> PHPH-040-102 <140> 12 / 856,420 <141> 2010-08-13 <150> PCT / US09 / 004659 <151> 2009-08-13 <150> 12 / 583,177 <151> 2009-08-13 <150> 61 / 305,901 <151> 2010-02-18 <160> 44 <170> PatentIn version 3.5 <210> 1 <211> 512 <212> PRT <213> Homo sapiens <400> 1 Met Thr Ala Pro Trp Val Ala Leu Ala Leu Leu Trp Gly Ser Leu Trp 1 5 10 15 Pro Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg 50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu Ser Leu Ile Val Leu Leu Ala Phe Trp Met Tyr 145 150 155 160 Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Ile His Glu Asp Pro 165 170 175 Gly Pro Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu Gln Leu 180 185 190 Leu Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln 195 200 205 Leu Met Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys 210 215 220 Gln Ser Trp Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly Met Lys 225 230 235 240 His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser Asn 245 250 255 Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe His Asp Lys Gly Ser 260 265 270 Leu Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr Trp Asn Glu Leu Cys 275 280 285 His Val Ala Glu Thr Met Ser Arg Gly Leu Ser Tyr Leu His Glu Asp 290 295 300 Val Pro Trp Cys Arg Gly Glu Gly His Lys Pro Ser Ile Ala His Arg 305 310 315 320 Asp Phe Lys Ser Lys Asn Val Leu Leu Lys Ser Asp Leu Thr Ala Val 325 330 335 Leu Ala Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro 340 345 350 Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu 355 360 365 Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile 370 375 380 Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Val Ser Arg Cys 385 390 395 400 Lys Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu 405 410 415 Glu Ile Gly Gln His Pro Ser Leu Glu Glu Leu Gln Glu Val Val Val 420 425 430 His Lys Lys Met Arg Pro Thr Ile Lys Asp His Trp Leu Lys His Pro 435 440 445 Gly Leu Ala Gln Leu Cys Val Thr Ile Glu Glu Cys Trp Asp His Asp 450 455 460 Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Val Ser Leu 465 470 475 480 Ile Arg Arg Ser Val Asn Gly Thr Thr Ser Asp Cys Leu Val Ser Leu 485 490 495 Val Thr Ser Val Thr Asn Val Asp Leu Pro Pro Lys Glu Ser Ser Ile 500 505 510 <210> 2 <211> 115 <212> PRT <213> Homo sapiens <400> 2 Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn 1 5 10 15 Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly 20 25 30 Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser 35 40 45 Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn 50 55 60 Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val 65 70 75 80 Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90 95 Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr 100 105 110 Ala Pro Thr 115 <210> 3 <211> 100 <212> PRT <213> Homo sapiens <400> 3 Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn 1 5 10 15 Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly 20 25 30 Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser 35 40 45 Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn 50 55 60 Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val 65 70 75 80 Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90 95 Leu Pro Glu Ala 100 <210> 4 <211> 1539 <212> DNA <213> Homo sapiens <400> 4 atgacggcgc cctgggtggc cctcgccctc ctctggggat cgctgtggcc cggctctggg 60 cgtggggagg ctgagacacg ggagtgcatc tactacaacg ccaactggga gctggagcgc 120 accaaccaga gcggcctgga gcgctgcgaa ggcgagcagg acaagcggct gcactgctac 180 gcctcctggg ccaacagctc tggcaccatc gagctcgtga agaagggctg ctggctagat 240 gacttcaact gctacgatag gcaggagtgt gtggccactg aggagaaccc ccaggtgtac 300 ttctgctgct gtgaaggcaa cttctgcaac gagcgcttca ctcatttgcc agaggctggg 360 ggcccggaag tcacgtacga gccacccccg acagccccca ccctgctcac ggtgctggcc 420 tactcactgc tgcccatcgg gggcctttcc ctcatcgtcc tgctggcctt ttggatgtac 480 cggcatcgca agccccccta cggtcatgtg gacatccatg aggaccctgg gcctccacca 540 ccatcccctc tggtgggcct gaagccactg cagctgctgg agatcaaggc tcgggggcgc 600 tttggctgtg tctggaaggc ccagctcatg aatgactttg tagctgtcaa gatcttccca 660 ctccaggaca agcagtcgtg gcagagtgaa cgggagatct tcagcacacc tggcatgaag 720 cacgagaacc tgctacagtt cattgctgcc gagaagcgag gctccaacct cgaagtagag 780 ctgtggctca tcacggcctt ccatgacaag ggctccctca cggattacct caaggggaac 840 atcatcacat ggaacgaact gtgtcatgta gcagagacga tgtcacgagg cctctcatac 900 ctgcatgagg atgtgccctg gtgccgtggc gagggccaca agccgtctat tgcccacagg 960 gactttaaaa gtaagaatgt attgctgaag agcgacctca cagccgtgct ggctgacttt 1020 ggcttggctg ttcgatttga gccagggaaa cctccagggg acacccacgg acaggtaggc 1080 acgagacggt acatggctcc tgaggtgctc gagggagcca tcaacttcca gagagatgcc 1140 ttcctgcgca ttgacatgta tgccatgggg ttggtgctgt gggagcttgt gtctcgctgc 1200 aaggctgcag acggacccgt ggatgagtac atgctgccct ttgaggaaga gattggccag 1260 cacccttcgt tggaggagct gcaggaggtg gtggtgcaca agaagatgag gcccaccatt 1320 aaagatcact ggttgaaaca cccgggcctg gcccagcttt gtgtgaccat cgaggagtgc 1380 tgggaccatg atgcagaggc tcgcttgtcc gcgggctgtg tggaggagcg ggtgtccctg 1440 attcggaggt cggtcaacgg cactacctcg gactgtctcg tttccctggt gacctctgtc 1500 accaatgtgg acctgccccc taaagagtca agcatctaa 1539 <210> 5 <211> 345 <212> DNA <213> Homo sapiens <400> 5 gggcgtgggg aggctgagac acgggagtgc atctactaca acgccaactg ggagctggag 60 cgcaccaacc agagcggcct ggagcgctgc gaaggcgagc aggacaagcg gctgcactgc 120 tacgcctcct gggccaacag ctctggcacc atcgagctcg tgaagaaggg ctgctggcta 180 gatgacttca actgctacga taggcaggag tgtgtggcca ctgaggagaa cccccaggtg 240 tacttctgct gctgtgaagg caacttctgc aacgagcgct tcactcattt gccagaggct 300 gggggcccgg aagtcacgta cgagccaccc ccgacagccc ccacc 345 <210> 6 <211> 225 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <220> <221> MOD_RES <222> (43)..(43) <223> Asp or Ala <220> <221> MOD_RES <222> (100)..(100) <223> Lys or Ala <220> <221> MOD_RES <222> (212)..(212) <223> Asn or Ala <400> 6 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 1 5 10 15 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 20 25 30 Arg Thr Pro Glu Val Thr Cys Val Val Val Xaa Val Ser His Glu Asp 35 40 45 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 50 55 60 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 65 70 75 80 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 85 90 95 Tyr Lys Cys Xaa Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu Lys 100 105 110 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 115 120 125 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 130 135 140 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 145 150 155 160 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 165 170 175 Asp Ser Asp Gly Pro Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 180 185 190 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 195 200 205 Ala Leu His Xaa His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210 215 220 Lys 225 <210> 7 <211> 343 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 7 Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn 1 5 10 15 Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly 20 25 30 Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser 35 40 45 Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Asp Asp Asp Phe Asn 50 55 60 Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val 65 70 75 80 Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90 95 Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr 100 105 110 Ala Pro Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys Pro Ala Pro 115 120 125 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 130 135 140 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 145 150 155 160 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 165 170 175 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 180 185 190 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 195 200 205 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 210 215 220 Pro Val Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 225 230 235 240 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 245 250 255 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 260 265 270 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 275 280 285 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 290 295 300 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 305 310 315 320 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 325 330 335 Leu Ser Leu Ser Pro Gly Lys 340 <210> 8 <211> twenty one <212> PRT <213> Italian Bee (Apis mellifera) <400> 8 Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile 1 5 10 15 Ser Tyr Ile Tyr Ala 20 <210> 9 <211> twenty two <212> PRT <213> unknown <220> <223> Unknown description: Tissue plasminogen activator <400> 9 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro 20 <210> 10 <211> 19 <212> PRT <213> unknown <220> <223> Unknown description: Natural peptide <400> 10 Met Thr Ala Pro Trp Val Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys 1 5 10 15 Ala Gly Ser <210> 11 <211> 368 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 11 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Gly Ala Ser Gly Arg Gly Glu Ala Glu Thr 20 25 30 Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn 35 40 45 Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His 50 55 60 Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys 65 70 75 80 Lys Gly Cys Trp Asp Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys 85 90 95 Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly 100 105 110 Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Ala Gly Gly Pro 115 120 125 Glu Val Thr Tyr Glu Pro Pro Pro Thr Ala Pro Thr Gly Gly Gly Thr 130 135 140 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 145 150 155 160 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 165 170 175 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 180 185 190 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 195 200 205 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 210 215 220 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 225 230 235 240 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu Lys Thr 245 250 255 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 260 265 270 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 275 280 285 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 290 295 300 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 305 310 315 320 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 325 330 335 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 340 345 350 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 365 <210> 12 <211> 1107 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic polynucleotide <400> 12 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgcccggcg cctctgggcg tggggaggct gagacacggg agtgcatcta ctacaacgcc 120 aactgggagc tggagcgcac caaccagagc ggcctggagc gctgcgaagg cgagcaggac 180 aagcggctgc actgctacgc ctcctggcgc aacagctctg gcaccatcga gctcgtgaag 240 aagggctgct gggacgatga cttcaactgc tacgataggc aggagtgtgt ggccactgag 300 gagaaccccc aggtgtactt ctgctgctgt gaaggcaact tctgcaacga gcgcttcact 360 catttgccag aggctgggg cccggaagtc acgtacgagc caccccgac agcccccacc 420 ggtggtggaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 480 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 540 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 600 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 660 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 720 aagtgcaagg tctccaacaa agccctccca gtccccatcg agaaaaccat ctccaaagcc 780 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 840 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 900 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 960 tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1020 gggacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1080 agcctctccc tgtctccggg taaatga 1107 <210> 13 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 13 Thr Gly Gly Gly Gly 1 5 <210> 14 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 14 Ser Gly Gly Gly Gly 1 5 <210> 15 <211> 116 <212> PRT <213> Homo sapiens <400> 15 Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn 1 5 10 15 Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly 20 25 30 Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser 35 40 45 Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn 50 55 60 Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp Ser Pro Glu Val 65 70 75 80 Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr 85 90 95 Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro 100 105 110 Lys Pro Pro Thr 115 <210> 16 <211> 150 <212> PRT <213> Rattus sp. <400> 16 Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys 1 5 10 15 Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Pro 50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu Pro Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu Ser 145 150 <210> 17 <211> 150 <212> PRT <213> Pig species (Sus sp.) <400> 17 Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys 1 5 10 15 Val Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg 50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu Ser 145 150 <210> 18 <211> 150 <212> PRT <213> Mus sp. <400> 18 Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys 1 5 10 15 Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg 50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu Pro Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu Ser 145 150 <210> 19 <211> 150 <212> PRT <213> Homo sapiens <400> 19 Met Thr Ala Pro Trp Val Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys 1 5 10 15 Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg 50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu Ser 145 150 <210> 20 <211> 150 <212> PRT <213> Bos sp. <400> 20 Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys 1 5 10 15 Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly Glu Arg Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg 50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Val Gly Gly Leu Ser 145 150 <210> 21 <211> 150 <212> PRT <213> Xenopus sp. <400> 21 Met Gly Ala Ser Val Ala Leu Thr Phe Leu Leu Leu Leu Ala Thr Phe 1 5 10 15 Arg Ala Gly Ser Gly His Asp Glu Val Glu Thr Arg Glu Cys Ile Tyr 20 25 30 Tyr Asn Ala Asn Trp Glu Leu Glu Lys Thr Asn Gln Ser Gly Val Glu 35 40 45 Arg Leu Val Glu Gly Lys Lys Asp Lys Arg Leu His Cys Tyr Ala Ser 50 55 60 Trp Arg Asn Asn Ser Gly Phe Ile Glu Leu Val Lys Lys Gly Cys Trp 65 70 75 80 Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Ile Ala Lys Glu 85 90 95 Glu Asn Pro Gln Val Phe Phe Cys Cys Cys Glu Gly Asn Tyr Cys Asn 100 105 110 Lys Lys Phe Thr His Leu Pro Glu Val Glu Thr Phe Asp Pro Lys Pro 115 120 125 Gln Pro Ser Ala Ser Val Leu Asn Ile Leu Ile Tyr Ser Leu Leu Pro 130 135 140 Ile Val Gly Leu Ser Met 145 150 <210> 22 <211> 150 <212> PRT <213> Homo sapiens <400> 22 Met Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys 1 5 10 15 Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe 20 25 30 Phe Asn Ala Asn Trp Glu Lys Asp Arg Thr Asn Gln Thr Gly Val Glu 35 40 45 Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp 50 55 60 Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu 65 70 75 80 Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp 85 90 95 Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu 100 105 110 Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn 115 120 125 Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu 130 135 140 Val Pro Leu Met Leu Ile 145 150 <210> twenty three <211> 154 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic consensus polypeptide <220> <221> MOD_RES <222> (8) <223> Thr or Ala <220> <221> MOD_RES <222> (121)..(121) <223> Pro, Ala, Val or Met <400> twenty three Met Thr Ala Pro Trp Ala Ala Xaa Leu Ala Leu Leu Trp Gly Ser Leu 1 5 10 15 Cys Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr 20 25 30 Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu 35 40 45 Arg Leu Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser 50 55 60 Trp Arg Asn Ser Ser Gly Thr Leu Glu Leu Val Lys Lys Gly Cys Trp 65 70 75 80 Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu 85 90 95 Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn 100 105 110 Glu Arg Phe Thr His Leu Pro Glu Xaa Gly Gly Pro Glu Val Thr Tyr 115 120 125 Glu Pro Lys Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr 130 135 140 Ser Leu Leu Pro Ile Gly Gly Leu Ser Met 145 150 <210> twenty four <211> 6 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic 6xHis tag <400> twenty four His His His His His 1 5 <210> 25 <211> 1107 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic polynucleotides <400> 25 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgccccggcg cctctgggcg tgggggaggct gagacacggg agtgcatcta ctacaacgcc 120 aactgggagc tggagcgcac caaccagagc ggcctggagc gctgcgaagg cgagcaggac 180 aagcggctgc actgctacgc ctcctggcgc aacagctctg gcaccatcga gctcgtgaag 240 aagggctgct gggatgatga cttcaactgc tacgataggc aggagtgtgt ggccactgag 300 gagaaccccc aggtgtactt ctgctgctgt gaaggcaact tctgcaacga gcgcttcact 360 catttgccag aggctgggg cccggaagtc acgtacgagc caccccgac agcccccacc 420 ggtggtggaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 480 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 540 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 600 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 660 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 720 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 780 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 840 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 900 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgctccc cgtgctggac 960 tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1020 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1080 agcctctccc tgtccccggg taaatga 1107 <210> 26 <211> 360 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 26 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Gly Ala Ala Glu Thr Arg Glu Cys Ile Tyr 20 25 30 Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu 35 40 45 Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp 50 55 60 Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Asp 65 70 75 80 Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu 85 90 95 Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu 100 105 110 Arg Phe Thr His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu 115 120 125 Pro Pro Pro Thr Gly Gly Gly Thr His Thr Cys Pro Pro Cys Pro Ala 130 135 140 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 145 150 155 160 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 165 170 175 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 180 185 190 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 195 200 205 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 210 215 220 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 225 230 235 240 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 245 250 255 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 260 265 270 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 275 280 285 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 290 295 300 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 305 310 315 320 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 325 330 335 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 340 345 350 Ser Leu Ser Leu Ser Pro Gly Lys 355 360 <210> 27 <211> 1083 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polynucleotide <220> <221> CDS <222> (76)..(396) <400> 27 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgcccggcg ccgctgagac acgggagtgc atctactaca acgccaactg g 111 Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp 1 5 10 gag ctg gag cgc acc aac cag agc ggc ctg gag cgc tgc gaa ggc gag 159 Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu 15 20 25 cag gac aag cgg ctg cac tgc tac gcc tcc tgg cgc aac agc tct ggc 207 Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly 30 35 40 acc atc gag ctc gtg aag aag ggc tgc tgg gac gat gac ttc aac tgc 255 Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Asp Asp Asp Phe Asn Cys 45 50 55 60 tac gat agg cag gag tgt gtg gcc act gag gag aac ccc cag gtg tac 303 Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr 65 70 75 ttc tgc tgc tgt gaa ggc aac ttc tgc aac gag cgc ttc act cat ttg 351 Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu 80 85 90 cca gag gct ggg ggc ccg gaa gtc acg tac gag cca ccc ccg aca 396 Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr 95 100 105 ggtggtggaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 456 gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 516 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 576 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 636 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 696 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 756 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 816 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 876 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 936 tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 996 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1056 agcctctccc tgtccccggg taaatga 1083 <210> 28 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 28 Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg 1 5 10 15 Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg 20 25 30 Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu 35 40 45 Val Lys Lys Gly Cys Trp Asp Asp Asp Phe Asn Cys Tyr Asp Arg Gln 50 55 60 Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys 65 70 75 80 Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Ala Gly 85 90 95 Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr Gly Gly Gly Thr His 100 105 110 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 145 150 155 160 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 225 230 235 240 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 245 250 255 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280 285 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 290 295 300 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 305 310 315 320 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 335 <210> 29 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 29 Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg 1 5 10 15 Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg 20 25 30 Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu 35 40 45 Val Lys Lys Gly Cys Trp Asp Asp Asp Phe Asn Cys Tyr Asp Arg Gln 50 55 60 Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys 65 70 75 80 Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Ala Gly 85 90 95 Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr 100 105 <210> 30 <211> 1083 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic polynucleotides <220> <221> CDS <222> (76)..(396) <400> 30 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgcccggcg ccgccgaac ccgcgaatgt attack atgctattg g 111 Glu Thr Arg Glu Cys Ile Tyr Asn Only Asn Trp 1 5 10 gaa ctc gaa cgg acg aac caa tcc ggg ctc gaa cgg tgt gag ggg gaa 159 Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu 15 20 25 cag gat aaa cgc ctc cat tgc tat gcg tcg tgg agg aac tcc tcc ggg 207 Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly 30 35 40 acg att gaa ctg gtc aag aaa ggg tgc tgg gac gac gat ttc aat tgt 255 Thr Ile Glu Val Lys Gly Cys Trp Asp Asp Asp Phe Asn Cys 45 50 55 60 tat gac cgc cag gaa tgt gtc gcg acc gaa gag at ccg cag gtc tat 303 Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr 65 70 75 ttc tgt tgt tgc gag ggg aat ttc tgt aat gaa cgg ttt acc cac ctc 351 Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu 80 85 90 ccc gaa gcc ggc ggg ccc gag gtg acc tat gaa ccc ccg ccc acc 396 Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr 95 100 105 ggtggtggaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 456 gtcttcctct tccccccaaa acccaaggac acctcatga tctcccggac ccctgaggtc 516 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 576 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 636 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 696 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 756 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccgggga ggagatgacc 816 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 876 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 936 tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 996 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1056 agcctctccc tgtccccggg taaatga 1083 <210> 31 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic polynucleotide <400> 31 gaaacccgcg aatgtattta ttacaatgct aattgggaac tcgaacggac gaaccaatcc 60 gggctcgaac ggtgtgaggg ggaacaggat aaacgcctcc attgctatgc gtcgtggagg 120 aactcctccg ggacgattga actggtcaag aaagggtgct gggacgacga tttcaattgt 180 tatgaccgcc aggaatgtgt cgcgaccgaa gagaatccgc aggtctattt ctgttgttgc 240 gaggggaatt tctgtaatga acggtttacc cacctccccg aagccggcgg gcccgaggtg 300 acctatgaac ccccgcccac c 321 <210> 32 <211> 115 <212> PRT <213> Homo sapiens <400> 32 Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn 1 5 10 15 Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly 20 25 30 Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser 35 40 45 Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Asp Asp Asp Phe Asn 50 55 60 Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val 65 70 75 80 Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90 95 Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr 100 105 110 Ala Pro Thr 115 <210> 33 <211> 1107 <212> DNA <213> Synthetic sequence <220> <223> Synthetic sequence description: Synthetic polynucleotide <400> 33 tacctacgtt acttctctcc cgagacgaca cacgacgacg acacacctcg tcagaagcaa 60 agcgggccgc ggagacccgc acccctccga ctctgtgccc tcacgtagat gatgttgcgg 120 ttgaccctcg acctcgcgtg gttggtctcg ccggacctcg cgacgcttcc gctcgtcctg 180 ttcgccgacg tgacgatgcg gaggaccgcg ttgtcgagac cgtggtagct cgagcacttc 240 ttcccgacga ccctactact gaagttgacg atgctatccg tcctcacaca ccggtgactc 300 ctcttggggg tccacatgaa gacgacgaca cttccgttga agacgttgct cgcgaagtga 360 gtaaacggtc tccgaccccc gggccttcag tgcatgctcg gtgggggctg tcgggggtgg 420 ccaccacctt gagtgtgtac gggtggcacg ggtcgtggac ttgaggaccc ccctggcagt 480 cagaaggaga aggggggttt tgggttcctg tgggagtact agagggcctg gggactccag 540 tgtacgcacc accacctgca ctcggtgctt ctgggactcc agttcaagtt gaccatgcac 600 ctgccgcacc tccacgtatt acggttctgt ttcggcgccc tcctcgtcat gttgtcgtgc 660 atggcacacc agtcgcagga gtggcaggac gtggtcctga ccgacttacc gttcctcatg 720 ttcacgttcc agaggttgtt tcgggagggt cgggggtagc tcttttggta gaggtttcgg 780 tttcccgtcg gggctcttgg tgtccacatg tgggacgggg gtagggccct cctctactgg 840 ttcttggtcc agtcggactg gacggaccag tttccgaaga tagggtcgct gtagcggcac 900 ctcaccctct cgttacccgt cggcctcttg ttgatgttct ggtgcggagg gcacgacctg 960 aggctgccga ggaagaagga gatatcgttc gagtggcacc tgttctcgtc caccgtcgtc 1020 cccttgcaga agagtacgag gcactacgta ctccgagacg tgttggtgat gtgcgtcttc 1080 tcggagaggg acaggggccc atttact 1107 <210> 34 <211> 1083 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polynucleotide <400> 34 tacctacgtt acttctctcc cgagacgaca cacgacgacg acacacctcg tcagaagcaa 60 agcgggccgc ggcgactctg tgccctcacg tagatgatgt tgcggttgac cctcgacctc 120 gcgtggttgg tctcgccgga cctcgcgacg cttccgctcg tcctgttcgc cgacgtgacg 180 atgcggagga ccgcgttgtc gagaccgtgg tagctcgagc acttcttccc gacgaccctg 240 ctactgaagt tgacgatgct atccgtcctc acacaccggt gactcctctt gggggtccac 300 atgaagacga cgacacttcc gttgaagacg ttgctcgcga agtgagtaaa cggtctccga 360 cccccgggcc ttcagtgcat gctcggtggg ggctgtccac caccttgagt gtgtacgggt 420 ggcacgggtc gtggacttga ggacccccct ggcagtcaga aggagaaggg gggttttggg 480 ttcctgtggg agtactagag ggcctgggga ctccagtgta cgcaccacca cctgcactcg 540 gtgcttctgg gactccagtt caagttgacc atgcacctgc cgcacctcca cgtattacgg 600 ttctgtttcg gcgccctcct cgtcatgttg tcgtgcatgg cacaccagtc gcaggagtgg 660 caggacgtgg tcctgaccga cttaccgttc ctcatgttca cgttccagag gttgtttcgg 720 gagggtcggg ggtagctctt ttggtagagg tttcggtttc ccgtcggggc tcttggtgtc 780 cacatgtggg acgggggtag ggccctcctc tactggttct tggtccagtc ggactggacg 840 gaccagtttc cgaagatagg gtcgctgtag cggcacctca ccctctcgtt acccgtcggc 900 ctcttgttga tgttctggtg cggagggcac gacctgaggc tgccgaggaa gaaggagata 960 tcgttcgagt ggcacctgtt ctcgtccacc gtcgtcccct tgcagaagag tacgaggcac 1020 tacgtactcc gagacgtgtt ggtgatgtgc gtcttctcgg agagggacag gggcccattt 1080 act 1083 <210> 35 <211> 1083 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic polynucleotide <400> 35 tacctacgtt acttctctcc cgagacgaca cacgacgacg acacacctcg tcagaagcaa 60 agcgggccgc ggcggctttg ggcgcttaca taaataatgt tacgattaac ccttgagctt 120 gcctgcttgg ttaggcccga gcttgccaca ctcccccttg tcctatttgc ggaggtaacg 180 atacgcagca cctccttgag gaggccctgc taacttgacc agttctttcc cacgaccctg 240 ctgctaaagt taacaatact ggcggtcctt acacagcgct ggcttctctt aggcgtccag 300 ataaagacaa caacgctccc cttaaagaca ttacttgcca aatgggtgga ggggcttcgg 360 ccgcccgggc tccactggat acttgggggc gggtggccac caccttgagt gtgtacgggt 420 ggcacgggtc gtggacttga ggacccccct ggcagtcaga aggagaaggg gggttttggg 480 ttcctgtggg agtactagag ggcctgggga ctccagtgta cgcaccacca cctgcactcg 540 gtgcttctgg gactccagtt caagttgacc atgcacctgc cgcacctcca cgtattacgg 600 ttctgtttcg gcgccctcct cgtcatgttg tcgtgcatgg cacaccagtc gcaggagtgg 660 caggacgtgg tcctgaccga cttaccgttc ctcatgttca cgttccagag gttgtttcgg 720 gagggtcggg ggtagctctt ttggtagagg tttcggtttc ccgtcggggc tcttggtgtc 780 cacatgtggg acgggggtag ggccctcctc tactggttct tggtccagtc ggactggacg 840 gaccagtttc cgaagatagg gtcgctgtag cggcacctca ccctctcgtt acccgtcggc 900 ctcttgttga tgttctggtg cggagggcac gacctgaggc tgccgaggaa gaaggagata 960 tcgttcgagt ggcacctgtt ctcgtccacc gtcgtcccct tgcagaagag tacgaggcac 1020 tacgtactcc gagacgtgtt ggtgatgtgc gtcttctcgg agagggacag gggcccattt 1080 act 1083 <210> 36 <211> 141 <212> PRT <213> Homo sapiens <400> 36 Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn 1 5 10 15 Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly 20 25 30 Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser 35 40 45 Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn 50 55 60 Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val 65 70 75 80 Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90 95 Leu Pro Glu Ala Gly Gly Pro Glu Gly Pro Trp Ala Ser Thr Thr Ile 100 105 110 Pro Ser Gly Gly Pro Glu Ala Thr Ala Ala Ala Gly Asp Gln Gly Ser 115 120 125 Gly Ala Leu Trp Leu Cys Leu Glu Gly Pro Ala His Glu 130 135 140 <210> 37 <211> 141 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 37 Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn 1 5 10 15 Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly 20 25 30 Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser 35 40 45 Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Asp Asp Asp Phe Asn 50 55 60 Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val 65 70 75 80 Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90 95 Leu Pro Glu Ala Gly Gly Pro Glu Gly Pro Trp Ala Ser Thr Thr Ile 100 105 110 Pro Ser Gly Gly Pro Glu Ala Thr Ala Ala Ala Gly Asp Gln Gly Ser 115 120 125 Gly Ala Leu Trp Leu Cys Leu Glu Gly Pro Ala His Glu 130 135 140 <210> 38 <211> 370 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide sequence description: <400> 38 Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn 1 5 10 15 Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly 20 25 30 Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser 35 40 45 Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Asp Asp Asp Phe Asn 50 55 60 Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val 65 70 75 80 Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90 95 Leu Pro Glu Ala Gly Gly Pro Glu Gly Pro Trp Ala Ser Thr Thr Ile 100 105 110 Pro Ser Gly Gly Pro Glu Ala Thr Ala Ala Ala Gly Asp Gln Gly Ser 115 120 125 Gly Ala Leu Trp Leu Cys Leu Glu Gly Pro Ala His Glu Thr Gly Gly 130 135 140 Gly Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 145 150 155 160 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 165 170 175 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 180 185 190 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 195 200 205 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 210 215 220 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 225 230 235 240 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 245 250 255 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 260 265 270 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 275 280 285 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 290 295 300 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 305 310 315 320 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 325 330 335 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 340 345 350 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 355 360 365 Gly Lys 370 <210> 39 <211> 512 <212> PRT <213> Homo sapiens <400> 39 Met Thr Ala Pro Trp Val Ala Leu Ala Leu Leu Trp Gly Ser Leu Trp 1 5 10 15 Pro Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40 45 Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Ala 50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu Ala Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly Leu Ser Leu Ile Val Leu Leu Ala Phe Trp Met Tyr 145 150 155 160 Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Ile His Glu Asp Pro 165 170 175 Gly Pro Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu Gln Leu 180 185 190 Leu Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln 195 200 205 Leu Met Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys 210 215 220 Gln Ser Trp Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly Met Lys 225 230 235 240 His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser Asn 245 250 255 Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe His Asp Lys Gly Ser 260 265 270 Leu Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr Trp Asn Glu Leu Cys 275 280 285 His Val Ala Glu Thr Met Ser Arg Gly Leu Ser Tyr Leu His Glu Asp 290 295 300 Val Pro Trp Cys Arg Gly Glu Gly His Lys Pro Ser Ile Ala His Arg 305 310 315 320 Asp Phe Lys Ser Lys Asn Val Leu Leu Lys Ser Asp Leu Thr Ala Val 325 330 335 Leu Ala Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro 340 345 350 Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu 355 360 365 Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile 370 375 380 Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Val Ser Arg Cys 385 390 395 400 Lys Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu 405 410 415 Glu Ile Gly Gln His Pro Ser Leu Glu Glu Leu Gln Glu Val Val Val 420 425 430 His Lys Lys Met Arg Pro Thr Ile Lys Asp His Trp Leu Lys His Pro 435 440 445 Gly Leu Ala Gln Leu Cys Val Thr Ile Glu Glu Cys Trp Asp His Asp 450 455 460 Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Val Ser Leu 465 470 475 480 Ile Arg Arg Ser Val Asn Gly Thr Thr Ser Asp Cys Leu Val Ser Leu 485 490 495 Val Thr Ser Val Thr Asn Val Asp Leu Pro Pro Lys Glu Ser Ser Ile 500 505 510 <210> 40 <211> 115 <212> PRT <213> Homo sapiens <400> 40 Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn 1 5 10 15 Tryptophan Glutamic acid Leucine Glutamic acid Arginine Threonine Asparagine Glutamine Serine Glycine Leucine Glutamic acid Arginine Cysteine Glutamic acid Glycine 20 25 30 Glutamic acid Glutamine Aspartic acid Lysine Arginine Leucine Histidine Cysteine Tyrosine Alanine Serine Tryptophan Alanine Asparagine Serine Serine 35 40 45 Glycine Threonine Isoleucine Glutamic acid Leucine Valine Lysine Lysine Glycine Cysteine Tryptophan Leucine Aspartic acid Aspartic acid Phenylalanine Asparagine 50 55 60 Cysteine Tyrosine Aspartic acid Arginine Glutamine Glutamic acid Cysteine Valine Alanine Threonine Glutamic acid Glutamic acid Asparagine Proline Glutamine Valine 65 70 75 80 Tyrosine Phenylalanine Cysteine Cysteine Cysteine Glutamic acid Glycine Asparagine Phenylalanine Cysteine Asparagine Glutamic acid Arginine Phenylalanine Threonine Histidine 85 90 95 Leucine Proline Glutamic acid Alanine Glycine Glycine Proline Glutamic acid Valine Threonine Tyrosine Glutamic acid Proline Proline Proline Threonine 100 105 110 Alanine Proline Threonine 115 <210> 41 <211> 100 <212> PRT <213> Homo sapiens <400> 41 Glycine Arginine Glycine Glutamic acid Alanine Glutamic acid Threonine Arginine Glutamic acid Cysteine Isoleucine Tyrosine Tyrosine Asparagine Alanine Asparagine 1 5 10 15 Tryptophan Glutamic acid Leucine Glutamic acid Arginine Threonine Asparagine Glutamine Serine Glycine Leucine Glutamic acid Arginine Cysteine Glutamic acid Glycine 20 25 30 Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Ala Asn Ser Ser 35 40 45 Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn 50 55 60 Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val 65 70 75 80 Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His 85 90 95 Leu Pro Glu Ala 100 <210> 42 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 42 Thr Gly Gly Gly 1 <210> 43 <211> 368 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 43 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Gly Ala Ser Gly Arg Gly Glu Ala Glu Thr 20 25 30 Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn 35 40 45 Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His 50 55 60 Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys 65 70 75 80 Lys Gly Cys Trp Asp Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys 85 90 95 Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly 100 105 110 Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Ala Gly Gly Pro 115 120 125 Glu Val Thr Tyr Glu Pro Pro Pro Thr Ala Pro Thr Gly Gly Gly Thr 130 135 140 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 145 150 155 160 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 165 170 175 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 180 185 190 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 195 200 205 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 210 215 220 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 225 230 235 240 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 245 250 255 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 260 265 270 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 275 280 285 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 290 295 300 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 305 310 315 320 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 325 330 335 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 340 345 350 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 365 <210> 44 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 44 Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg 1 5 10 15 Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg 20 25 30 Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu 35 40 45 Val Lys Lys Gly Cys Trp Asp Asp Asp Phe Asn Cys Tyr Asp Arg Gln 50 55 60 Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys 65 70 75 80 Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Ala Gly 85 90 95 Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr 100 105

Claims

1. Use of an ActRII fusion protein in the preparation of a medicament for treating myelodysplastic syndrome in a patient in need thereof, comprising administering to the patient an effective amount of an ActRII fusion protein, the fusion protein comprising: (i) an ActRII protein consisting of the amino acid sequence of SEQ ID NO: 29; (ii) immunoglobulin Fc domain; and (iii) a linker domain located between the ActRII protein and the immunoglobulin Fc domain, the linker domain comprising an amino acid sequence selected from the group consisting of GGG, SEQ ID NO: 13, or SEQ ID NO: 14; wherein the position corresponding to position 79 of SEQ ID NO: 1 in the ActRII fusion protein is aspartic acid, and Compared to an ActRII fusion protein in which position 79 of SEQ ID NO: 1 is leucine, the ActRII fusion protein exhibits loss of binding to activin A and activin B, but retains binding to GDF11.

2. The method of claim 1, wherein the immunoglobulin Fc domain is derived from an IgG heavy chain.

3. The method of claim 2, wherein the fusion protein forms a homodimer.

4. The use of claim 3, wherein the fusion protein is administered by subcutaneous injection.

5. The use of claim 4, wherein the fusion protein is administered to a patient at a dose of up to 30 mg / kg.

6. The use of claim 5, wherein the fusion protein is administered to a patient at a dose of up to 10 mg / kg.

7. The use of claim 6, wherein the fusion protein is administered to a patient at a dose of up to 3 mg / kg.

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