AU2015202035B2 - Use of GDF traps to increase red blood cell levels - Google Patents

Use of GDF traps to increase red blood cell levels Download PDF

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AU2015202035B2
AU2015202035B2 AU2015202035A AU2015202035A AU2015202035B2 AU 2015202035 B2 AU2015202035 B2 AU 2015202035B2 AU 2015202035 A AU2015202035 A AU 2015202035A AU 2015202035 A AU2015202035 A AU 2015202035A AU 2015202035 B2 AU2015202035 B2 AU 2015202035B2
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amino acid
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acid sequence
polypeptide
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AU2015202035A1 (en
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Ravindra Kumar
Robert Scott Pearsall
Jasbir Seehra
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Acceleron Pharma Inc
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Acceleron Pharma Inc
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Abstract

In certain aspects, the present invention provides compositions and methods for increasing red blood cell and/or hemoglobin levels in vertebrates, including rodents and primates, and particularly in humans.

Description

USE OF GDF TRAPS TO INCREASE RED BLOOD CELL LEVELS
RELATED APPLICATIONS
The present application is a divisional application of Australian Application No. 2009282441, which is incorporated in its entirety herein by reference.
This application claims the benefit of U.S. Provisional Application Serial No. 61/189,094, filed August 14, 2008. All the teachings of the above-referenced application are incorperated herein by reference.
BACKGROUND OF THE INVENTION
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
The mature red blood cell, or erythrocyte, is responsible for oxygen transport in the circulatory systems of vertebrates. Red blood cells carry high concentrations of hemoglobin, a protein that binds oxygen in the lungs at relatively high partial pressure of oxygen (pCE) and delivers oxygen to areas of the body with a relatively low pCE·
Mature red blood cells are produced from pluripotent hematopoietic stem cells in a process termed erythropoiesis. Postnatal erythropoiesis occurs primarily in the bone marrow and in the red pulp of the spleen. The coordinated action of various signaling pathways control the balance of cell proliferation, differentiation, survival and death. Under normal conditions, red blood cells are produced at a rate that maintains a constant red cell mass in the body, and production may increase or decrease in response to various stimuli, including increased or decreased oxygen tension or tissue demand. The process of erythropoiesis begins with the formation of lineage committed precursor cells and proceeds through a series of distinct precursor cell types. The final stages of erythropoiesis occur as reticulocytes are released into the bloodstream and lose their mitochondria and ribosomes while assuming the morphology of mature red blood cell. An elevated level of reticulocytes, or an elevated reticulocyte:erythrocyte ratio, in the blood is indicative of increased red blood cell production rates.
Erythropoietin (Epo) is widely recognized as the most significant positive regulator of erythropoiesis in post-natal 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, elevated Epo levels promote red blood cell formation by stimulating the generation of erythroid progenitors in the bone marrow and spleen.
Various forms of recombinant Epo are used by physicians to increase red blood cell levels in a variety of clinical settings, and particularly for the treatment of anemia. Anemia is a broadly-defined condition characterized by lower than normal levels of hemoglobin or red blood cells in the blood. In some instances, anemia is caused by a primary disorder in the production or survival of red blood cells. More commonly, anemia is secondary to diseases of other systems (Weatherall & Provan (2000) Lancet 355, 1169-1175). Anemia may result from a reduced rate of production or increased rate of destruction of red blood cells or by loss of red blood cells due to bleeding. Anemia may result from a variety of disorders that include, for example, chronic renal failure, chemotherapy treatment, myelodysplastic syndrome, rheumatoid arthritis, and bone marrow transplantation.
Treatment with Epo typically causes a rise in hemoglobins by about 1-3 g/dL in healthy humans over a period of weeks. When administered to anemic individuals, this treatment regimen often provides substantial increases in hemoglobin and red blood cell levels and leads to improvements in quality of life and prolonged survival. Epo is not uniformly effective, and many individuals are refractory to even high doses (Horl et al. (2000) Nephrol Dial Transplant 15, 43-50). Over 50% of patients with cancer have an inadequate response to Epo, approximately 10% with end-stage renal disease are hyporesponsive (Glaspy et al. (1997) J Clin Oncol 15, 1218-1234; Demetri et al. (1998) J Clin Oncol 16, 3412-3425), and less than 10% with myelodysplastic syndrome respond favorably (Estey (2003) Curr Opin Hematol 10, 60-67). Several factors, including inflammation, iron and vitamin deficiency, inadequate dialysis, aluminum toxicity, and hyperparathyroidism may predict a poor therapeutic response, the molecular mechanisms of resistance to Epo are as yet unclear.
In one embodiment, the present disclosure relates to alternative compositions and methods for increasing red blood cell levels in patients.
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
SUMMARY OF THE INVENTION
According to a first aspect, the present invention provides a method for treating a hemoglobinopathy in a patient in need thereof, the method comprising administering to the patient an effective amount of a polypeptide comprising an amino acid sequence that is at least 95% identical to the sequence of amino acids 29-109 of SEQ ID NO: 1, wherein the polypeptide comprises an acidic amino acid at the position corresponding to position 79 of SEQ ID NO:1, and wherein the polypeptide is capable of inhibiting signaling by myostatin and/or GDF11 in a cell-based assay
According to a second aspect, the present invention provides use of a polypeptide comprising an amino acid sequence that is at least 95% identical to the sequence of amino acids 29-109 of SEQ ID NO: 1, wherein the polypeptide comprises an acidic amino acid at the position corresponding to position 79 of SEQ ID NO:1, and wherein the polypeptide is capable of inhibiting signaling by myostatin and/or GDF11 in a cell-based assay, for the manufacture of a medicament for treating a hemoglobinopathy.
Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.
In part, the disclosure demonstrates that GDF Traps may be used to increase red blood cell and hemoglobin levels. Variant ActRIIB polypeptides having a significantly decreased affinity for activin (e.g., activin A and/or activin B) relative to other ActRIIB ligands, such as GDF11 and/or myostatin, are referred to as GDF Traps. ActRIIB variants described herein are GDF Traps unless otherwise stated. In particular, the disclosure demonstrates, that a GDF Trap which is a soluble form of ActRUB polypeptide having an acidic residue at position 79 of S'EQ ID NO; \» -when administered »? Wvo, increases red blood cell levels in the blood. Therefore, in certain embodiments, the disclosure provides methods for using GDF Traps to increase red blood cell and hemoglobin levels in patients and to treat disorders 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, incorporated by reference herein* GDF Traps can be used to increase muscle mass and decrease tat mass.
In certain aspects* the present disclosure provides GDF Traps that are variant AciROB polypeptides, including ActRUB polypeptides having amino- and carfeoxy-termmai truncations and sequence alterations. Optionally, GDF Traps of the invention may be designed to preferentially antagonize one or more 1 igands of ActROB receptors* such as GDPS (also called myostaimg GDF.l 1, Nodal, and BMP? (also called OP-1), Examples of GDF Traps include a set of variants derived from ActRUB that have greatly diminished affinity for aetivin. These variants exhibit desirable effects on red blood cells while reducing 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: the position .corresponding to position 79 of SEQ ID NO.1, In certain embodiments, the GDF Trap polypeptide comprises an amino acid sequence that comprises, consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 7, 26, 28, 29,. 32, 37 or 38* and polypeptides that are at least 80%, 85%, 90%, 95%, 97%, 98%:, or 99% identical to any of the foregoing.
In certain aspects, the· disclosure provides pharmaceutical preparations comprising a GDF Trap that hinds to an ActRlfB ligand such as GDFS, GDF! I, act?vin (e.g., actlvin B), BMP? or nodal, and a pharmaceutically acceptable carrier. Optionally, the GDF Trap binds to an ActRUB ligand with a Kd less than 10 micromolar, less than 1 micromolar, less than 100 nanomolar, less than 10 nanomolar, or less than I nanomolar. Optionally, the GDF Trap inhibits ActRUB signaling, such as intracellular signal transduction events triggered by an AetRllB ligand. A GDF Trap for use in such a preparation may be any of those disclosed herein, including, for example, GDF Traps having an amino acid sequence selected from SEQ ID NOs: 2, 3, 7,11, 26, 28, 29, 32, 37,38:or 40, or GDF Traps having an amino acid sequence that is at least .80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selected Mm SEQ ID NOs: 2, 3, 7, 11., 26, 28., 29, 32, 37, 38 or 40, or GDF Traps 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 NQs: 2,3, ?, 1 I. 26, 28,29, 32, 37, 38 or 40 wherein the position corresponding to L79 in SEQ ID NO: 1 is an acidic amino acid. A preferred GDP Trap for use in such a preparation consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 2(3. A GDF Trap may include a functional fragment of a natural 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 a sequence of SEQ ID NO: 2, lacking the C-terminal Ϊ, 2, 3, 4, 5 or 10 to 15 amino acids and lacking I, 2, 3. 4 or 5 amino acids at the N-terminus. A preferred polypeptide will comprise a truncation relative to SEQ ID NO: 2 or 40 of between 2 and 5 amino acids at the N-terminus and no more than 3 amino acids at the C-terminus. A GDF Trap may include one or more alterations in the amino acid sequence of an ActRIIB polypeptide (e.g., in the ligand-binding domain) relative to a naturally occurring ActRIIB polypeptide. The alteration in the amino acid sequence may, for example, alter glycosylation of the polypeptide when produced in a mammalian, insect or other eukaryotic cell or alter proteolytic cleavage of the polypeptide relative to the naturally occurring ActRIIB polypeptide. A GDF Trap maybe a fusion protein that has, as one domain, an ActRIIB polypeptide {e.g,, a ligand-binding domain of an ActRIIB with one or more sequence variations) and one or more additional domains that provide a desirable property, such as improved pharmacokinetics, easier purification, targeting to particular tissues, etc. For example, a domain of a fusion protein may enhance one or more of in vivo stability, in vivo half life, uptake/adm inistradon, tissue localization or distribution, formation of protein complexes, multimerization of the fusion protein, and/or purification. GDF Trap fusion proteins may Include an immunoglobulin Fc domain (wild-type or mutant) or a serum albumin. In certain embodiments, a GDF Trap fusion comprises a relatively unstructured linker positioned between the Fc domain and the extracellular ActRIIB domain. This unstructured linker may correspond to the roughly 15 amino acid unstructured region at the C-tenninal end of the extracellular domain of ActRIIB (the “tail”), or it may be an artificial sequence of between 3 and 5, 15. 20, 30, 50 or more amino adds that are relatively free of secondary structure. A linker may be rich in glycine and proline residues and may, for example, contain repeating sequences of threonine/serine and glycines (e.g., TG4 (SEQ ID NO: 13) or SG* (SEQ ID NO: 14) singlets or repeats) or a series of three glycines. A fusion protein may include a purification subsequence, such as an epitope tag, a FLAG tag, a poiyhistkfine sequence, and a GST fusion. In certain embodiments, a GDF Trap fusion comprises.a leader sequence. The leader sequence may be a native AeiRUB leader sequence or a heterologous leader sequence. In certain embodiments., the leader sequence Is a Tissue Plasminogen Activator (TEA) leader sequence, in an embodiment, a GDF Trap 'fusion protein comprises an amino acid sequence as set: forth in the formula A-B-G. The B portion is an N- and €-terminally truncated AeiRUB polypeptide consisting of the amino acid sequence corresponding to amino acids 25-13! of SEQ ID NO: 2 or 40. The A and C portions may be independently zero, one or more than one amino adds, and both A and € portions are heterologous to B. The A and/or C portions may he attached to the B portion via a linker sequence.
Optionally, a GDF Trap includes a variant ActRliB polypeptide having one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGykued amino acid, a famesvlated amino acid, an acetyl ated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid, moiety, and: an amino acid conjugated to an organic dertvalizing agent. A pharmaceutical preparation may also include one or .more additional: compounds such as a. compound that is used to treat an AetRUB-associated disorder. Preferably, a pharmaceutical preparation Is substantially pyrogen, free, fn general, it is preferable that a GDF Trap be expressed m a mammalian cell line that mediates suitably natural glyoosylation of the GDF Trap so as to diminish the likelihood of an unfavorable immune response in a patient. Human and CHO cell lines have been used successfully, and it is expected that other common mammalian expression vectors will be useful. in certain aspects, the disclosure provides packaged pharmaceuticals 'comprising a pharmaceutical preparation described· herein and labeled for use in increasing red blood cell levels in a human.
In certain aspects, the disclosure provides GDF Traps which are soluble ActRliB polypeptides comprising an altered ligand-binding (e.g., G0F8-bihding) domain. GDF Traps with altered ligand-binding domains may comprise, for example, one or more mutations at amino acid residues such as E37, E39, R40, K5Ss R5b, Y60, AM, K74, W7S, L79, DS'O, F&2 and. FI01 of human ActRliB (numbering is relative to.'SEQ ID NO: !}. Optionally, the . altered ligand-binding domain can have increased selectivity fora ligand such as GDFS/GDFU relative to a wild-type i igand-binding. domain of an ActRliB receptor. To illustrate, these mutations are demonstrated herein to increase the selectivity of the altered ligand-binding domain for GDF1! (and therefore, presumably, GDFS) over activin; K74Y, K74F, K74I,. L79D, .L79.E, and 0801, The following mutations have the reverse effect, increasing the ratio of activin binding over GDFII: D54A, KS5A,. 1,..79 A and F82.A. The overall (GDFI I and activin) bidding activity can be increased by inclusion of the “tail” region or, presumably, an unstructured linker region, and also by use of a K74A mutation. Other mutations that caused an overall decrease in ligand binding affinity, include: R40A, E37A, R56A, W?8AS D80K, D80R, D80A, D80G, D80F, D80M artd D80N. Mutations may he combined to achieve desired effects. For example, many of the mutations that affect the ratio of GDFI I:Activin binding have an overall negative effect on ligand binding, and therefore, these may be combined with mutations that, generally increase ligand binding to produce an improved binding protein with ligand selectivity, in an exemplary embodiment, a GDF Trap is an ActRIIB polypeptide comprising an L?9D or L79E mutation, optionally in combination with additional amino acid substitutions, additions or deletions.
Optionally, a GDF Trap comprising an altered ligand-binding domain has a ratio of IQ for activin binding to Kfj for GDF8 binding that is at least 2, 5, 10, or even 100 fold greater relative to the ratio for the wild-type ligand-binding domain. Optionally, the GDF Trap comprising an altered ligand-binding domain has a ratio of'lCsa for inhibiting activin to IC50 for inhibiting GDF8/GDF11 that is at least. 2, 5» 10, or even 100 fold greater relative to the wild-type ActRIIB ligand-binding domain. Optionally, the GDF Trap comprising an altered ligand-binding domain inhibits GDF8/GBP! 1 with an ICso at least 2, 5, 10, or even 100 times less than the lC$o for inhibiting activin. These GDF Traps can be fusion proteins that include an immunoglobulin Fc domain (either wild-type or mutant). In certain eases, the subject soluble GDF Traps are antagonists (inhibitors) of GDF8 and/or GDFI I.
Other GDF Traps are contemplated, such as the. following. A -GDF Trap fusion protein comprising a portion derived from the ActRIIB sequence of SEQ ID NO: .1 or 39 and a second polypeptide portion, wherein the portion derived from ActRIIB corresponds to a sequence beginning at any of amino acids. 21 -29 of SEQ ID NO: I or 39 (optionally beginning at 22-25 of SEQ ID NO: 1 or 39) and ending at any of amino acids 109-134 of SEQ ID NO: 1 or 39, and wherein the GDF Trap fusion protein inhibits signaling by activin, myostatic and/or GDFI 1 in.a cell-based assay. The GDF Trap fusion prolfcm above, wherein the portion derived from' ActRIIB corresponds to a sequence beginning at any of amino acids 20-29 of SEQ ID NO: I or 39 (optionally beginning at 22-25 of SEQ ID NO: I .or 39) and ending at any of amino acids 109-133 of SEQ ID NO: I or 39, The GDF Trap fusion protein above, wherein the portion derived from Act ROB corresponds to a sequence, beginning at any of amino acids 20-24 of SEQ ID NO: 1 or 39 (optionally beginning at 22-25 of SEQ ID NO: I or 39) and ending at any of amino acids 109-133 of SEQ ID NO: 1 or 39., The GDF Trap fusion protein above, wherein the portion derived from AotR'IIB corresponds to a sequence beginning at any of amino acids 21-24 of SEQ ID NO: I or 39 and ending at any oi amino acids 109-134 of SEQ ID NO: l or 39. The GDF Trap fusion protein above, wherein the portion derived from Act RUB. corresponds to a sequence beginning at any of amino acids 2.024 of SEQ ID NO; I or 39 and ending at any of amino acids 11 8-1 33 of SBQ ID NO: I or 39. The GDF Trap fusion protein above, wherein the portion derived from ActEIIB corresponds- io a sequence 'beginning at any of amino acids 21 -2:4 of SEQ ID NO; 1 or 39 and ending at any of amino acids 118-134 of SEQ ID NO; 1 or 39. The GDF Trap fusion protein above, wherein the portion derived from ActRlIB corresponds to a sequence beginning at any of amino acids 20-24 of SEQ ID NO: 1 or 39 and ending at any of amine adds 128-133 of SEQ ID NO: 1 or 39. The GDF Trap fusion protein-above, wherein the portion derived froth ActRlIB corresponds- to a sequence beginning at any of amino acids 2024 of SEQ ID NO; 1 or 39 ami ending at any of amino acids 128-133 of SEQ ID NO: 1 or 39. The GDP Trap, fusion protein above, wherein the portion derived from ActRlIB corresponds to a sequence beginning at any of amino acids 21-2.9 of SEQ ID NO: i or .39 and ending at any of amino adds· I 18-134 of SEQ ID NO: I or 39. The GDF Trap fusion protein above, wherein the portion derived from AciRflB corresponds to a sequence beginning at any of amino acids 20-29 of SEQ ID NO: I. or 39 and ending at any of amino •acids 118-133 of SEQ ID NO: 1 or 39. The GDF Trap fusion protein above, wherein the portion derived from ActRlIB corresponds to a sequence beginning at any of amino acids 2129 of SEQ ID 'NO: 1 or 39 and ending at any of amino acids 128-134 of SEQ ID NO: 1 or 39. The GDF Trap fusion protein above, wherein the portion derived from ActRlIB corresponds to a sequence-beginning at any of amino acids 20-29 of SEQ ID NO: I and ending at any of amino acids 128-133 of SEQ ID NO: 1. or 39, Surprisingly, constructs beginning at 22-25 of SEQ ID NO:- I or 39 have activity levels greater than proteins having the fid! extracellular domain of human ActRlIB. in a preferred embodiment, the GDF Trap fusion protein comprises, consists essentially of, or consists of, an amino acid sequencebeginning at amino acid position 25. of SEQ ID NO: I or .39 and ending at amino acid position 13! of SEQ-ID NO: 1 or 39, in another preferred embodiments, the GDF Trap polypeptide consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 7, 26» 28» 29, 32, 37 or 38, Any of the above GDF Trap fission proteins may be produced as a homodimer. Any of the above GDF Trap fusion proteins may have a heterologous portion that comprises 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 the 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: I,
Other GDF Trap proteins are contemplated, such as the. following. A GDF Trap protein comprising an amino acid sequence that is at leas! 80% identical 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: I is an R or K, and wherein the GDF Trap protein inhibits signaling by activin,. myosiafin and/or GDF 11 in a cell-based assay. The GDF Trap protein above, wherein at least one alteration with respect to the sequence of SEQ ID NO: I or 39 is positioned outside of the ligand binding pocket. The GDF Trap protein above, wherein at least one alteration with respect to the sequence of SEQ ID NO: I or 39 is a conservative alteration positioned within the ligand binding pocket. The GDF Trap protein above, wherein at least one alteration with respect to the sequence of SEQ ID NO: 1 or 39 is an alteration at one or more positions selected from the group consisting of K74, R40, Q53, K55, F82 and L79, The GDF Trap protein above, wherein the protein comprises at least one N-X-S/T sequence at a position other than an endogenous N-X-S/T sequence of ActRHB, and at a position outside of the ligand binding pocket.
Other GDF Traps are contemplated, such as the following. A GDF Trap protein oomprismg an amino add sequence that is at least 80% identical to the sequence of amino acids 29-109 of SEQ I D NO; 1 or 39. and wherein the protein comprises at least one N-X-S/T sequence at a position other than an endogenous N-X-S/T sequence of ActRHB, and at a position outside of the ligand binding pocket. The GDF Trap above, wherein the GDF Trap .protein comprises an N at the position corresponding to position 24 of SEQ ID NO: 1 or 39 and an S or T at the position corresponding to position 26 of SEQ ID NO: ! or 39» and wherein the.GDF Trap inhibits signaling by activin, myosiafin and/or GDF 11 in a cell-based assay. The GDF Trap above, wherein the GDP Trap protein comprises an R or K. at the position corresponding to position 64 of S EQ ID NO; I or 39. The GDF Trap above, wherein the ActRI'18 protein comprises a D or E at the position corresponding to position 79 of SEQ ID NO; 1 or 39 and wherein the GDF Trap inhibits signaling by activin, myostatin and/or GOP! I in a cell-based assay. The GDP Trap above, wherein at least one alteration with respect to the sequence of SEQ ID NO: I or 39 is a conservative alteration positioned within the ligand binding pocket. The GDF Trap above, wherein at least one alteration with .respect to the sequence of SEQ ID NO: I or 39 is an alteration at one or more positions selected from the group consisting of K?4, R40, Q53, K.55, F82 and L?9. The GDF Trap above, wherein the protein is a fusion protein further comprising a heterologous portion. Any of the above GDP Trap fusion proteins may be produced as a homodimer. Any of the above GDF Trap fusion proteins may have.a heterologous portion that comprises a constant region from an IgG heavy chain, such as an Fc domain. in certain aspects, the disclosure provides nucleic acids encoding a GDP Trap polypeptide. An isolated polynucleotide may comprise a. coding sequence for a soluble GDP Trap polypeptide, such as described above. For example, an isolated nucleic-acid may include a sequence coding for a GDF Trap comprising an extracellular domain (e.g,, ligandbinding domain) of an ActRllB polypeptide having one. or more sequence variations and a sequence that would code for part or all of the trammembrane domain and/or-the cytoplasmic domain of an ActRllB polypeptide, but for a stop codon positioned within the transraembrane •domain or the cytoplasmic domain, or positioned between the extracellular domain and the transmembrane domain or cytoplasmic domain. For example, an isolated polynucleotide coding for a GDF Trap may comprise a· foil-length ActRllB -polynucleotide sequence such as SEQ ID NO: 4 having one or more variations, or a partially truncated version, said isolated polynucleotide further comprising a transcription termination codon at least six hundred nucleotides before the 3'Merminus or otherwise positioned such that translation of the polynucleotide gives rise to an extracellular domain optionally fused to a truncated portion of a foil-length ActRllB. Nucleic acids disclosed herein may be operably linked to a promoter for expression, and the disclosure provides cells transformed with such recombinant polynucleotides. Preferably the cel! is a mammalian eel! such as a OH0 cell
In certain aspects, the disclosure provides methods for making a GDF Trap polypeptide. Such a method may include expressing any of the nucleic acids (e.g. , SEQ ID NO; .5., 25, 27, 30 or 31) disclosed herein in a suitable cell, such as a Chinese hamster ovary (CHO) cell. Such a method.may comprise: a) culturing:a ceil under conditions suitable for expression of the GDF Trap polypeptide, wherein said cell is transformed with a GDF Trap expression construct; and b) recovering:the GDF Trap polypeptide so expressed. GDF Trap polypeptides may be recovered as crude, partially purified or highly purified fractions using any of the well known techniques for obtaining protein from cell cultures.
In certain aspects, a GDF Trap polypeptide' disclosed herein may be used in a method for promoting red blood cell production or increasing red blood cell levels, .in a subject. In certain embodiments, the disclosure provides methods tor treating a disorder associated with low red blood cell counts or low hemoglobin levels (e.g., an anemia), or to promote red blood cell production, in patients in need thereof. A method may comprise administering to a subject in need thereof an effective amount of a GDF Trap .polypeptide. In certain aspects, the disclosure provides· uses of GDF Trap polypeptides for making a medicament for the treatment of a· disorder or condition, as described herein.
In certain aspects, the disclosure provides methods for administering a GDF Trap polypeptide to a patient, 1.« part, the disclosure demonstrates that GDF Trap polypeptides can boused to increase red blood cell and hemoglobin levels, GDF Trap polypeptides may also be used tor treating or preventing other therapeutic uses such as promoting orosde growth. I.n certain instances, when administering a GDF Trap polypeptide for promoting muscle growth, it maybe desirable to monitor the effects on red blood cells during administration of the GDF Trap polypeptide, or to determine or adjust the dosing of the GDF Trap polypeptide, in order to reduce undesired effects on red blood cells. For example, increases in red blood cell levels, hemoglobin levels, or hematocrit levels may cause increases in blood pressure.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure I shows an alignment of the extracellular domains of human ActRSlA (SEQ 10 NO: 15) and human ActRllB (SEQ ID NO: 2) with the residues that are deduced herein, based on composite analysis of multiple ActRllB and ActRlIA crystal structures to directly contact ligand (the ligand binding pocket) indicated with boxes.
Figure 2 shows a multiple sequence alignment of various vertebrate ActRllB proteins and human ActRHA (SEQ 10 NOs: 16-23),
Figure 3 shows the full amino acid sequence for the GDF Trap ActR!iB(L?9D 20-l34)-hFc (SEQ ID NO; 11), including theTPA leader sequence (double underlined), ActRllB extracellular domain (residues 20-134 in SEQ ID NO: 1; underlined), and hPe..domain. The aspartate substituted at position ?9 in the native sequence is double underlined and highlighted, as is the glycine revealed by sequencing to be the bi-terminal residue in the mature fusion protein. figure 4 shows a nucleotide sequence encoding Aet'RI1B(L79D 20-134}~hf e, SEQ ID NO: 25 corresponds to the sense strand, and SEQ ID NO: 33 corresponds to the antisense strand. The TP A leader (nucleotides 1 -66) is double underlined,, and the ActRllB extracellular domain .(nucleotides 76-420) is underlined.
Figure 5 shows the full amino acid sequence for the truncated GDF Trap ActR118(L79D' 25-135 }-hFc (SEQ I D NO: 26), including the TPA leader (double underlined), truncated AetRlfB extracellular domain (residues 25-131 in SEQ ID NO: I; underlined), and hFc domain. The aspartate substituted at position 79 in the native sequence is-double underlined and highlighted, as is the glutamate revealed by .sequencing to be the N-terrainai residue in the mature fusion protein.
Figure 6 shows a nucleotide sequence encoding AetR118(L79D '25-131)-hfc. SEQ ID NO: 27 corresponds to the sense, strand, and SEQ ID NO: 34 corresponds to the antisense strand, The TPA leader {nucleotides 1-66) is double underlined, and the truncated ActRllB extracellular domain (nucleotides 76-396) is underlined. The'ammo acid sequence for the ActRllB extracellular domain (residues 25-131 in SEQ 10 NO: I) is also shown.
Figure 7 shows the amino acid sequence for the truncated GDF Trap ActRllB(L?9D 25-131 )-hFc without a leader (SEQ ID NO: 28). The truncated ActRllB extracellular domain (residues 25-131 in SEQ ID NO: 1) is underlined. The aspartate substituted at position 79 in the native sequence is double underlined and highlighted, as is the glutamate revealed by sequencing to be the N-tennhial residue in the mature fusion protein.
Figure 8 shows the amino acid sequence for the truncated GDF Trap ActRUB(L79D 25-131) without the leader, hFc domain, and linker (SEQ ID NO; 29), The aspartate substituted at position. 79 in the native sequence is underlined and highlighted, as is the glutamate revealed by sequencing to be the N-ternsinal residue in the mature fusion protein.
Figure 9 shows an alternative nucleotide .sequence encoding Ae?R!lB(L79D 25-131}-hFc. SEQ ID NO: 30 corresponds to the sense strand, and SEQ IP NO: 35 corresponds to the antisense strand. The TEA leader (nucleotides 1-66) is'double underlined, the truncated ActRllB extracellular domain (nucleotides 76-396) is underlined, and substitutions in the wildtype nucleotide sequence of the extracellular domain are double underlined and highlighted {compare with SEQ ID NO; 27» Figure 6). The amino acid sequence for the AetRIIB extracellular domain (residues 25-131 in SEQ ID NO: 1} is also shown.
Figure 10 shows nucleotides 7.6-3% (SEQ ID NO: 31} of the alternative nucleotide sequence shown in Figure 9 (SEQ ID NO: 30). The same nucleotide substitutions indicated in Figure 9 are also underlined and highlighted here. SEQ ID NO: 31 encodes only the truncated ActR UB extracellular domain, (corresponding to residues 25-131 in 'SEQ ID NO; I) with a L79D substitution. e.g.* AefRl!B(L?9P 25-131).
Figure 11 shows the effect of AetRIIB(L79.D 25-13 l)-hFc on hemoglobin concentration in a mouse model of chemotherapy-induced anemia. Data are means ± SEM, **, P < 0.01 vs,, paclitaxel at the same time point. This GDF Trap offset the anemia induced by gaditaxel treatment
Figure 12 shows the effect of ActRIIB(L?9D 25-131.)-hFc on red blood cell {RBC) levels in a unilaterally nephreetomized (NEPHX) mouse model of chronic kidney disease. Data are means * SEM ***, F < 0.001 vs. baseline. This GDF Trap reversed the nephrectomy-induced anemia observed in· control mice.
Figure 13 shows the effect of ActRIIB(L79D 25-131 )~hFe on red blood cell (RBC), hemoglobin (FIGS), and hematocrit (HCTJ levels in a unilaterally nephrectomized (NEPHX) mouse model of'chronic kidney disease.· Data are mean changes from baseline over 4 weeks (± SEM). *, P < 0.05; **» P < 0,0!; ***, P < 0.00! vs. NEPHX controls. This GDF Trap prevented the nephrectomy-associated decline in these erythrocytic parameters, increasing each by a magnitude similar to that In kidney-intact (sham) mice .
Figure 14 shows the effect of ActRIIB(L79D 25-131 }-ItFc on ted blood cell (RBC) levels in. a rat model of anemia induced by acute blood loss. Blood removal occurred on Day -I. with dosing on Days.0 and 3. Data, are means ± SEM. *'*, P < 0.01: *'**, P <· 0.001 vs, vehicle at same time point. This GDF Trap improved the rate and extent of recovery from .blood-loss-induced anemia.
Figure 1S shows the effect of treatment with ActRllB( L79D 20-134)-hFc (gray) or AciRllB(L79D 25-131) hFc (black) on the absolute change in red blood cell concentration from baseline in evnomolgus monkey. YEH vehicle. Data are means ± SEM. n ~ 4-8 per group.
Figure 16 shows the effect of treatment with AciRIIB(L?9D 20-134)-hFc (gray) or AciR!!B(L79D 25“ 131 }-hFc (black) on the· absolute change in hematocrit from baseline in eynornolgus monkey. VEH ~ vehicle. Data are means + SEM. n ~ 4~B per group.
Figure 1 ? shows the effect of treatment with AetR31BCL79D 20-!34)-hFc (gray) or AetRitB{L79D 25-131)-faFe (black) on the absolute change in hemoglobin concentration from baseline in eynornolgus monkey. VEH " vehicle. Data are means ± SEM, n ~ 4-8 per group.
Figure 18 shows the effect of treatment with AciRIIB(L?9D 2(M34)-hFc (gray) or AciRHB(L79D 25-l31)-hFc (black) on the absolute change in circulating reticulocyte concentration from baseline in eynornolgus -monkey. VEH :::: vehicle. Data ate means ± SEM. n ~ 4-8 per group. DETAILED DESCRIPTION OF THE INVENTION 1. Overview
The transforming growth factor-beta (TGF-beta.s supcrfamily contains a. variety of growth factors that share common sequence dements and structural motifs. These proteins arc known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates, Members of the.superfamily perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, ehondrogenesis, eardiogenesls, hematopoiesis, neurogenesis, and epithelial cell differentiation. The family is divided into two general branches; the BME/GDF and the TGF-beia/Acdvin/BMPI 0 branches, whose members have diverse, often complementary effects. By 'manipulating the activity of a member of the TGF-heta family, it is often possible to cause significant physiological changes in an organism. For example, the Piedmontese arid Belgian Blue cattle breeds carry a loss-of-function mutation in the GDP8 (also called myostafin) gene that causes a marked increase in muscle mass, Grobet et a!,, Nat Genet. 1997, ! 7(1):71 -4. Furthermore, in humans, inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength. Schnelke et a!,, N Engl I Med 2004,350:2682-8., TGP-β signals are mediated by Eeteromerie complexes of type 1 and type II serine/tfe'eonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon Hgand stimulation (Massague, 2000, Nat, Rev, Mol, Cell Biol, 1:169-178). These type I and type 11 receptors are transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threoniae specificity. Type I receptors are essential for signaling. Type II receptors are required for binding ligands and for expression of Type I receptors. Type I and II activin receptors form a stable complex after ligand binding, resulting in phosphorylation of Type 1 receptors by Type 11 receptors.
Two related Type II receptors (ActRli), ActRIIA and ActRllB, have been Identified as the Type II receptors for aetivins (Mathews and Vale, 1991, Cell 65:973*982; Attisanoet a!,, 1992, Cell 68: 97-408). Besides aetivins, ActRIIA and ActRllB-can biochemically interact with several other TOF-p family proteins, including BMP?, Nodal, GDF8, and GDF31 (Y-amashita et ah, 1995, j. Cell Biol. 130:21/-226; Lee and McPherron. 2001, Proc. Nath Acad. Scl. 98:9306-931 i; Yen and Whitman, 2001, Mol. Cell 7:949-957; Oh et ah, 2002, Genes .Dev. 16:2749-54), AUC4 Is the primary type 1 receptor for aetivins, particularly for activin A, and ALfi-7 may serve as a receptor for aetivins as well, particularly for activin B, In certain embodiments, the present invention relates to antagonizing a ligand of ActRllB receptors (also referred· to as an ActRllB ligand) with a subject GDP Trap polypeptide. Exemplary ligands· of ActRllB receptors include some TGF-β family members, such as activin, Nodal, GDF8, GDPI I, and BMP?.
Aetivins are dimeric polypeptide growth factors that belong to the TGF-feeta super&amp;mily:. There are three principal activin forms (A, B, and AB) that are homo/heterodimers of two closely related β subunits'(βχβ;ν β#Β, and PaPb, respectively), lire human genome also encodes an activin C and an activin E, which are primarily expressed in the liver, and heterodimerie forms containing pc or βε are also known. In the TGF-beta superfemily, activms are unique and muhifiioetiona! factors that can stimulate hormone production In ovarian and placental cells, support neuronal cell survival, -influence cell-cycle progress positively or negatively depending on cell type, and induce mesodermal differentiation at least in amphibian embryos (DePaolo el ah, 1991, Prop See Ep. Biol Med. 198:500-512; Dyson et ah, 1997, Curr Biol. 7:81-84; Woodruff, 1998-, Biochem Pharmacol. 55:953-963). Moreover, erythroid differentiation factor (EDF) isolated from the stimulated human monocytic leukemic cells was found to be identical to activin A (Mura-ta et ah, 1988, PNAS, 85:2434). It has been suggested that activin A promotes erythropoies-s in the bone marrow, in several tissues·, activin signaling is antagonized by its related heferodimer, inhibin. For example, during the release of follicle-stimulating hormone (FSH) from the pituitary, activin promotes FSH secretion and synthesis, while inhibin prevents FSH secretion and synthesis. Other proteins that may regulate activin bioactivity and/or bind to activin include foilistatm (FS), folhxtatm related protein (FSRP) and ob-macrogiohulin.
Nodal proteins have functions in mesoderm and endoderm induction and formation, as well as subsequent organization of axial structures such as heart and stomach in early ernhryogenesis, it has been demonstrated that dorsal tissue in a developing Vertebrate embryo contributes predominantly to the axial structures of the notochord and pre-chorda! 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 known as Smad proteins. Recent studies support the idea that AetRHA and ActRIIB serve as type 11 receptors for Nodal (Saknma et ah, Clares Cells. 2002, 7:401-12). It is suggested that Nodal ligands interact with their co-factors (e.g., cripto) to activate activin type I and type II receptors, which phosphorylaie Smad2. Nodal proteins are implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification. Experimental evidence has demonstrated that Nodal signaling activates pAR3~L«x, a Inciferase reporter previously shown to respond specifically to activin and TGF-beta. However, Nodal is unable to induce pT!x2-Lux, a reporter specifically responsive to bone morphogenetic proteins. Recent results provide direct biochemical evidence that Nodal signaling is mediated by both acttvin-TOF-beta pathway Smads. Smad2 and Smad3, Further evidence has shown that the extracellular cripto protein is required for Nodal signaling, making it distinct from activin or TGF-beta signaling.
Growth and Differentiation Factor-8 (GDF8) is also known .as myostatin. GDF8 is a negative regulator of skeletal muscle mass. GDPS is. highly expressed In the developing and adult skeletal muscle. The GDFS null mutation in transgenic mice is characterized by a marked hypertrophy and hyperplasia of the skeletal muscle (MePherron et ah, Nature, 1997, 387:83-90). Similar increases in skeletal muscle mass are evident 'in naturally occurring mutations ofG.DF8 in cattle (Ashmore et aL, 1974, Growth, 38:501-307; Swatland and Kieffer, h An up. Sci., 1994, 38:752-757; Mcfherron and Lee, Proe. Natl, Acad. Set, USA, 1997, 94:12457-12461; and tiamhadur et a!,. Genome Res„ 1997, 7:910-915) and, strikingly, in humans (Schuelke et ai,, N Engl J Med 2004:350:2682-8), Studies have also shown that muscle wasting associated with H?V~infection in humans is accompanied by increases in OD-F8 protein expression (Gorrealez-Cadavid et a!., PNAS, 1998, 95:14938-43). In addition, GDF8 can modulate the production of mosci.e-specifte enzymes (e.g., creatine kinase) and modulate myoblast cell proliferation (WO 00/43781). The ODF8 propeptide can noncovalentiy- bind to the mature GDF8 domain dimer, inactivating its biological activity {Miyazone et al. (15)88) i. Biol. Chem., 2:63; 6407-6415; Wakefield :et al. (15)88) I, Biol. Cbem.j 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3: 35-43). Other proteins which bind to GD!;8 nr structurally related proteins and inhibit their biological activity include fbllistatm, anti potentially, follistatin-related proteins-(Gamer et ah (15)99) Dev. Biol. , 208; 222-232).
Growth and Differentiation Factor-11 (GDP 11), also known as BMP 11, is a secreted protein (McPhomm of. ah, 1999, Gat Genet. 22; 260-264). GDFI1 is expressed in the tail bud, limb bud, maxillary and mandibular arches, and dorsal root ganglia during mouse development (Nakashima et ah, 1999, Mech. Dev. 80; 185-189). GDFI 1 plays· a unique role in patterning both mesodermal and neural tissues (Gamer et al, 1999, Dev Biol, 208:22232). GDFI I was shown to be a negative regulator of chondrogenesis and myogenesis in developing chick Hmb (Garner et ah, 2001, Dev Biol. 229:407-20).. The expression of GDFI 1 in muscle also suggests its role in regulating muscle growth in a similar way to GDF8. hi addition, the expression of GDFI 1 in brain suggests that GDFI I may also possess activities that relate to the function of the nervous system. Interestingly, GDFI 1 was found to inhibit neurogenesis in the olfactory epithelium (Wu et a!., 2003, Neuron. 37:197-207). Hence, GDFI 1 may have in vitro and in vivo applications-in the treatment of diseases such as muscle diseases and neurodegenerafive diseases (e.g>, amyotrophic lateral sclerosis).
Bone morphogenetic protein (BMP?), also called osteogenic protein-1 (OP-1), is well known to induce cartilage and bone formation, in addition, BMP? regulates a wide array of physiological processes. For example, BMP7 may be the osteoinductive factor responsible: for the phenomenon of epithelial osteogenesis. It is also found that BMP? plays a role in calcium regulation and bone homeostasis. Like aetivin, BMP? binds toType 11 receptors. AGRBA and AetRIlB, However, BMP? and aetivin recruit distinct Type ! receptors into heteromenc receptor complexes. The major .BMP? Type 1 receptor observed was ALK2, while aetivin bound exclusively to AL.K4 (AciRIIB). BMP? and aetivin elicited distinct biological responses and activated different Smad pathways (Macias-Silva et al, 1998, J 'Biol Chem. 273:25628-36).
As demonstrated herein, a GDP Trap polypeptide, which is a variant ActRifB polypeptide (ActROB), is more effective at increasing red blood cell levels in vivo as compared to a wild-type soluble ActRIIB polypeptide and has-beneficial effects in a variety of models for anemias, it should be noted that hematopoiesis'is a complex process, regulated by a variety of factors, including erythropoietin, G-CSF and iron homeostasis. The terms “increase red blood cell levels’* and “promote red blood cell formation” refer to clinically observable metrics, such as hematocrit, red blood cell counts and hemoglobin measurements, and are intended to he neutral as to the mechanism by which such changes occur.
In addition to stimulating red blood cell levels, OOP Trap polypeptides are useful for a variety of therapeutic applications, including, for example, promoting muscle growth (see POT Publication Nos. WO 2006/012627 and WO 2008/097541, which are hereby incorporated by reference in their entirety). In certain instances, when administering a GDF Trap polypeptide for the purpose of increasing muscle, it may he desirable to reduce or minimize effects on. red blood cells. By monitoring various hematologic parameters in patients being treated with, or who are candidates for treatment with, a GDF Trap polypeptide, appropriate dosing (including amounts· and frequency of administration) may be determined based on an -individual patient’s needs, baseline hematologic parameters, and purpose for treatment. Furthermore, therapeutic progress and effects on one or more hematologic parameters over time, may be useful in managing patients being dosed with a GDF Trap polypeptide by facilitating patient care, determining appropriate maintenance dosing (both amounts and frequency), etc,
The terms used in this specification generally have their ordinary meanings in the art., within the context of this invention and in the specific context where each term is used. Certain terms are discussed below or elsewherein the specification, to provide additional guidance to the practitioner in describing the compositions and. methods of the invention and how to make and use them. The scope or meaning of any use of a term will he: apparent from the specific context in which the term is used. “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent..{%), preferably within 10%, and more preferably within 5% of a given value-or range of values.
Alternatively, and particularly in biological systems, the terms '"about” and “approximately” may mean values that are within an order of magnitude, preferably within 5fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
The methods of the invention may include steps of comparing sequences to each other, including wild-type sequence to one or more· mutants (sequence variants). Such comparisons typically comprise alignments of polymer sequences, e.g.., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, f ASTA and MEG ALIGN, to name a few). The skilled artisan can readily appreciate that, in such alignments, where a mutation contains a residue- insertion or deletion, the sequence alignment will introduce a “gap” (typically represented by a dash, or “A”) in the polymer sequence not containing the inserted or deleted residue. “Homologous,” in all its grammatical forms and spelling variations, refers to the relationship between .two proteins that possess.a “common evolutionary origin.” including proteins from super-families 'in the same species of organism, as wel l as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent-identity or by the presence of specific residues or motifs and conserved positions.
The term- “sequence similarity,” in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or aml.no.acid sequences that, mayor may not share a common evolutionary origin.
However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin. 2. GDF Trap Polypeptides
In certain aspects, the invention relates to OOF Trap polypeptides:, e.g., soluble variant ActRIlB polypeptides, including, for example, -fragments,;functional variants, and .modified forms of ActRHB polypeptides. In certain embodiments, the GDf Trap polypeptides have at least one similar or same biological activity as a corresponding wild-type ActRHB polypeptide. For example, a GDF Trap polypeptide of the invention may bind to and inhibit the function of an ActRHB ligand (e.g,, aetivin A, aetivin AB, aetivin B, Nodal, GDF8, GDFII or BMR7). Optionally, a GDF Trap polypeptide increases'red blood ceil levels. Examples of GDF Trap polypeptides include, human ActRHB precursor polypeptides (SEQ ID NO; I of 39) having one of more sequence variations, and soluble human ActRHB polypeptides (e.g., SEQ ID NOs: 2, 3, 7, 11, 26, 2.S, 29, 32,37, 38,46 and 41). having one or more sequence variations. A GDF Trap refers to an ActR HB polypeptide having a decreased affinity for aetivin relative to other ActRHB ligands, including tor example GDF11 and/or myosiatim
As used herein, the term “ActRHB” refers to a family of aetivin receptor type lib (ActRHB) proteins from any species and variants derived from such ActRHB proteins by mutagenesis or other modification. Reference to ActRHB herein is understood to be a reference to any one of the currently identified forms. Members of the ActRHB family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmem'brane domain, and a cytoplasmic domain with predicted serine/flueonine kinase activity. Amino acid sequences of human ActEIIA soluble extracellular domain (provided for comparison) and ActRHB soluble extracellular domain are illustrated in Figure I.
The term “ActRHB polypeptide” includes polypeptides comprising any naturally occurring, polypeptide of an ActRHB family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. See, for example, WO 2006/012627. For example, ActRHB polypeptides include polypeptides derived from the sequence of any known ActRHB having a sequence at least about 80% identical to the sequence of an ActRHB polypeptide, and optionally at least 85%, 90%, 95%, 97%, 99% or greater identity. For example,, an ActRHB polypeptide may bind to and inhibit the function of an ActR HB protein and/or aetivin. An ActRHB polypeptide which is a GDF Trap may be selected for activity in promoting red blood cell formation in vivo. Examples of ActRHB polypeptides include human ActRHB precursor polypeptide (SEQ ID NO: 1 and 39) and soluble human ActRHB polypeptides (e.g,, SEQ ID NO: 2, 3, 7, 11,26, 28, 29, 32,37, 38,40-and 41), Numbering of amino acids for all ActRHB-related polypeptides described herein is based on the numbering for SEQ ID NO:I, unless specifically designated otherwise.
The human Act RUB precursor protein sequence is as follows: MT^PWVAL&amp;LLWG$LWPGSG^SEAETRECIYSmamELERT|feSgLERC E GEQDKRL ECYA S WRjfe S GTIE LVKKGCMLD D Ft?C Y D RQE CVAT E EM PQ VYrCCCESNFCHESFrHLREAGGPEVTYEPPPTAETLLTVLAYSLLPIG' GLSLIVLLAFfiMYEHEKPPYGHVDIHEOPGPPPPSPLVGLKPLQLLElK ARGPrGCVWKAQLLIEDFVAVKXPPLQDKQSWQSEREIFSTPGMKBBflLL· QFIAAEKRGSNLEVELWLXTAFrlDKGSLTDYLKGNIXTWELCHVAETH SRGLS YLHEDV PWC FGEGHK PSIAHRDFKSKN V ELKS DLTAVIADFGLA VREE PGKP PG DTHGQVGTRRYMAPEVLEGAIHFQRDA FI, RIDMYAMGLV LWELVSECKAADGPVDEYHL'PFEEEIGQHPSLEELQSVVVHKKMRFTIK DHtCLKH PGLAQLC VT1EECWDH DAE ARLSAGCVEE.RVSL 1 RRS VEGTTS DCLVSLVTSVTNVDI.PPKESSX (SEQ ID NO; 1}
The signal peptide-is single- underlined; the extracellular domain is in bold and the potential N-Imked glycosyl-ation sites are in boxes, A form with an alanine at position 64 is also reported in the literature, as follows: EGEQDKRLHCYASWAgsSGTXELVKKGCMLODFMCYBSQECVATEEHPQ VYFCCCEGHFCMERFTHXFEAGGPEVTYEPFPTAPTL:LT VL A Y- S L h PIG GLS LIVLL A FtVM Y R H RK P P Y G Η. V DI HE 0 PG P F P P S P LVG L K PLQL L EIK ARGRFGCVWKAQIWNDFVAVKX EPLQDKQSliQSERE X FSTPGMKHENLL QFIAAEKRGSNLE VELWL X T AFH DKGSLTDYLKGNI IT&amp;IM ELCH VAETH SRGLSYLHFDVP-WCRGEGHKFSIAHRDFKSKNVLLKSDLTAVLADFGI-A V.RFE PGK PPG OTEGQyGTRRYMAPE.VLEGAIH.FQRDAFLRI DMYAMGLV LWELVSRCKAAOGPVDEYMLPFEEE.IGQHPS.LEELQEVVV.HKKHRPTXK DnSLKHPGLAQLCVTIEECWDHDAEAELSAGCVEEBVSLXPkSVGGETS OCLVSLV.TSVT-NVDLPPKESSX (SEQ 10 HO: 39)
The 'human ActRHB soluble (extracellular), processed polypeptide sequence is as follows: GRGE A ET R EC IY Y R AM W-ELER TF Q S G L E RC EG FQ OREL H C Y A S G RM S S G TI EL VKKGCVfLDDFN C Y DRQEC V AT FEN PQVY FCCC EGG FCNBR FT H LP BAGG PBYTYSPPPTAPT <SHQ ID NO; 2)
The alternative form with an A64 is as follows: GRGEAETRECJ YYNANWELERTiNQS GLERCE6EQ DKRLHC YASWMiSSG T1EL7KEGCWL DDFlSiC YDRQBCVATEEhi PQVY PCCCEGF FCMBRFTHLP E1AGG PBV v Y B E P ΡΊ (PPQ H> NO: 40)
In.some conditions, the protein may he produced with an "SGR.. A sequence at the N~ terminus. The C-terminal ‘Tail” of the extracellular domain is underlined. The sequence with the “tail’5 deleted (a ΔΙ5 sequence) is as follows;
GRGEASTRECIYYNANWELERTNQSGLERCEGEQDKRX.HCYhSWRNSSG
TiELVKKGCWLDDFPCYDKOECVATBERPQVYFCCCEGHFChERFTHLP BA (SEQ ID NO: 3)
The alternative form with an A64 is as follows: GRGEAETRBCJYYBAMRELERTRQSGXfoECEGFQDKRlfoCYASWAFSSG TlELVKKGCMLDDFRCYORQECVATEEhPQVYFGCCEGNFCRERFTHLP F;A (SEQ ID NO: 4!)
In some conditions, the protein maybe produced with an “SGR..." sequence at the N--terminus. The nucleic acid sequence encoding a human ActRHB precursor protein is as follows: (nucleotides 5-1543 of Geobank entry NMJJOI106)( the sequence as shown provides an alanine at position 64, and may be modified to provide an arginine instead)
AY GACGGC vjCGGY O ....A'.: GGCX, C i\,G:..\,C: G i.Y. fGC.U.A;/ > ] (.. (..0, YG'i GwG CCGGCTCTGGGCGTGGGGAGGCTGAGACACGGGAGTGCATCTAeTACAA CGCCAACTGGGAGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTGC '.GCAG/\v.:C.·/Vi..··:./\·..ί%c0:-1..GoC Yvr. At.· I.GC'.I j-X.·1.. iT.GrGG’GCGAAv·A GCTCTGGCACCATCGAGCTGGTGAAGAAGGGCTGCTGGCTAGATGACTT CAACTGCTACGATAGGCAGGAGTGTGTGGCCACTGAGGAGAACCCCCAG GTGTACTTCTGCTGCTGTGAAGGCAACTTGTGCAACGAGCGCTTCACTC atttgcgagaggctgggggcccggaagtcacgtaggagccacccccgac
A.GCCCCCACCCTGCT€ACGGTG€TGGGCTACfCAC?GCTGCCCATCGGG
GGCCTTTCCCTCATCGTCCTGCTGGCCTTTTGGATGTACCGGCATCGCA
AGCCCCCCTACGGTCATGTGGACATCCATGAGGACeCTGGGCCTCCACC
ACCATCCGCTCTGGTGGGCCTGAAGCCACTGCAGCTGCTGGAGATCAAG
GCTCGGGGGCGCTTTGGCTGTGTCTGGAAGGCCCAGCTCATGAATGACT
TTGTAGCTGTCAAGATCTTCCCACTCCAGGACAAGCAGTCGTGGCAGAG
TGAACGGGAGATCTTCAGCACACCTGGCATGAAGCACGAGAACCTGCTA
CAGTTCATTGCTGCGGAGAAGCGAGGCTCCAACGTCGAAGTAGAGCTGT
GGCTCATCACGGCCTTCCATGACAAGGGCTGCCTCACGGATTAGCTCAA ggggaacatcaicacatggaacgaactgtgtcatgtagcagagacgatg
TCAG:GAGGCCTCTCATA€CTGCATGAGGATGTGCCCTGGTGCCGTGGCG
AGGGCCACAAGCCGTCTATTGCCCACAGGGACTTTAAAAGTAAGAATGT
ATTGCTGAAGAGCGACCTCACAGCCGTGCTGGCTGACTTTGGCTTGGCT gttcgatttgagcoagggaaacctccaggggacacccacggacaggtag
GCACGAGACGGTACATGGGTCCTGAGGTGCTCGAGGGAGCCATCAACTT
GCAGAGAGATGCCTTCCTGCGCATTGACATGTATGCCATGGGGTTGGTG
CTGTGGGAGCTTGTGTCTCGCTGCAAGGCfGCAGACGGACCCGTGGATG agtacatgctciccctttgaggaagagattggcgagcacccttcgttgga GGAGCTGCAGGAGGTGGTGGTGCACAAGAAGATGAGGCCCACCATTAAA GATCACTGGTTGAAftCACCCGGGCCTGGCCCAGCTTTGTGTGACCAfCG AGGAGfGCTGGGACCATGATGCAGAGGCTCGCTTGTCCGCGGGCTGTGT GGAGGAGCGGGTGTCCCTGATTCGGAGGTCGGTCAACGGCACTACCTCG GACTGTCTCGTTTCCCTGGTGACCTCTGTCACGAATGTGGACCTGCCCC CTAAAGAGTGAAGCATCTAA {SEQ ID NO: 4}
The nucleic add sequence encoding a human ActRJIA soluble (exiracelluiar) poiypephde is as follows (the sequence as shown fSinovki.es an alanine a? position: (hi, and may be modified to provide an arginine instead}: GGGCGTGGGGAGGCTGAGACACGGGAGTGCATGTAGTACAACGCCAACT GGGAGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTGCGAAGGCGA v.::v AG GAS /;A(.K,Gb'. .1 Go.?v·.... Γ·.·.·;... e At. GGG1 GGG-... o AACAoA i oYoGL·
ACCATCGAGCTCGTGAAGAAGGGCTGCTGGCTAGATGAGTTCAACTGCT AGGATAGGCAGGAGTGTGTGGCCAGTGAGGAGAACCCGCAGGTGTACTT
CTGCTGCTGTGAAGGCAACTTCTGCAACGAGCCCTTCACTCATTTGCCA
GAGGCTGGGGGCCCGGA&amp;GTCACGTACGAGCCACCCCCGACAGCCCCCA CC {SEQ ID NO: 5}
In a specific embodiment,, the invention relates to GDF Trap polypeptides which are variant forms of soluble ActRIIB polypeptides. As described herein, the term “soluble ActRIIB polypeptide’* generally refers to polypeptides 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 as well as any variants thereof (including mutants, fragments and peptidomimetie. forms) that retain a useful activity. For example, the extracellular domain of an ActRIIB protein binds to a ligand and is generally sol uble. Examples of soluble ActRIIB polypeptides include ActRIIB soluble polypeptides (e.g., SEQ ID NOs: 32, 3. ?, 11,26, 2b, 29, 32, '17, 3b, 40 and 41). Other examples of soluble ActRIIB polypeptides comprise a signal sequence in addition to the extracellular domain of an ActRIIB protein, see Example 1. The signal sequence can be a nati ve signal sequence of an ActRIIB, or a signal sequence from another protein, such as a tissue plasminogen activator (TPA) signal sequence or a honey bee melitiin (HBM) signal sequence.
The disclosure identifies functionally active portions and variants of ActRIIB.
Applicants have ascertained that an Fc fusion protein having the sequence disclosed by iiiideo et al. (Blood, 1994 Apr 15;S3(8):2I 63-70), which has an Alanine at the position corresponding to amino acid 64 of SEQ ID HO: I (A64), has a relatively low affinity for activin. and GDF~11. By contrast, the same Fc fusion protein with an Arginine at position 64 (R.64) bas an affinity for aetivm and 6DF-11 in the low nanomolar to high picomolar range. Therefore, a sequence with an R64 is used as the wild-type reference sequence for human ActRIIB In this disclosure.
Attisano et al. (Cell. 1992 Ian 10;6S( 1):97-108) showed that a-deletion of the proline knot at the C-terminus of the extracellular domain of ActRIIB reduced the ..affinity of the receptor for activin. An ActRIlB-Fc fusion protein containing amino acids 20-119 of SEQ IB NO: 1, “A.ctRIIB(20-1Ί 9)~Fc*\ has reduced binding to GDP-11 and activin relative to an ActR 1 18(20-134)-FC, which.includes the proline knot region and the complete juxtamembraoe domain. However, an ActRiIB(20-I 29)~Fc protein retains similar but somewhat reduced activity relative to the wild type, even though the proline knot region Is disrupted. Thus, ActROB 'extracellular domains that stop at amino acid !34, 133, 132, 131, 130 and 129 are all expected to be active, hut constructs stopping at 134 or 133 may be most active. Similarly, mutations at any of residues 129-134 are not expected to· alter ligand binding affinity by large margins, in support of this, .mutations of P1.29 and P130 do not substantially decrease ligand binding. Therefore, a GDP Trap polypeptide -which is an ActRlIB-Fc fusion protein may end as early as amino acid 109 (the final cysteine), however, forms ending at or between 109 and 119 are expected to have reduced ligand binding. Amino acid. 119 is poorly conserved and so is readily altered or truncated. Forms ending at S 28 or later retain ligand binding activity. Forms ending at or between 119 and 127 will have an intermediate binding ability. Any of these forms may be desirable to use, depending on the clinical or experimental .setting.
At the N-teftninus of AelRilB, it is expected that a protein beginning at amino acid 29 or before will retain ligand binding activity. Amino acid IN represents the initial cysteine.
An alanine to asparagine mutation at position 24 introduces an Mdinked glycosyiatiort sequence without substantially affecting ligand binding. This confirms that mutations in the region between the signal cleavage peptide and the cysteine cross-linked region, corresponding to amino acids 20-29 are well tolerated. In particular, constructs beginning at position 20, 21,22., 23 and 24 will retain activity, and constructs beginning at positions 25, 20,..27, 28 and 29 are also expected to retain activity. Data-shown in the Examples demonstrates that, surprisingly, a construct beginning at 22,23, 24 or 25 will have the most activity.
Taken together, an active portion of ActRIlB comprises amino acids 29-109 ofSEQ ID NO; 1, and GDF Trap constructs may, for example, comprise a portion of ActRIlB beginning at a residue corresponding to amino acids 20-29 of SEQ ID NO; I or 39 and ending at a position corresponding .to amino acids 109-134 of SEQ ID NO; I or 39. Other examples include 'constructs that begin at a position from 20-2.9 or 21-29 and end at a position from 119-134, 119-133, 129-134, or 129-03 ofSEQ ID NO; 1 or 39. Other examples include constructs that begin at a position from 20-24 (or 21.-24, or 22-25) and end at a position from 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 having at least 80%. 85%, 90%. 95% or 99% identity 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 25131 of SBQ ID NO: I or 39. In certain embodiments, the GDF Trap polypeptide comprises, consists essentially of, or consists of, a polypeptide having an amino add sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical 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 NO: 7,26,28, 29, 32, 37 or 38,
The disclosure includes the results of an analysis of composite ActRIIB structures, shown in Figure I, demonstrating that the ligand binding pocket is defined by residues Y31, N33, N3S, 1.38 through T4I, E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87, A.92, and E94 through F10I. At these positions, it is expected that conservative mutations will be tolerated, although a K74A mutation is well-tolerated, as are R40A, K55A, F82A and mutations at position L79. R40 is a K in Xenopos, indicating that basic amino acids at this position will he tolerated. Q53 is R in bovine ActRIIB and K in Xenopus ActRIIB,-and therefore amino acids including. R, K, Q, N and II will be tolerated at this position. Thus, a general formula for a GDF Trap protein is one that comprises amino adds 29-109 of SEQ ID NO: 1 or 39, but optionally beginning at a position ranging from 2024 or 22-25 and ending at a position ranging from 1294 34, and comprising no more than 1, 2, 5, 10 or 15 conservative amino acid changes in the ligand binding pocket, and aero, one or more non-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82 in the ligand binding pocket, 'Such a protein may retain greater than 80%, '90%, 95% or 99% sequence identity to the sequence of am ino acids 29-109 of SEQ ID NO: 1 or 39. Sites outside the. binding pocket, at which variability may be particularly well tolerated, include the amino and carboxy termini of the extracellular domain (as noted above), and positions 42-46 and 65-73. An asparagine to alanine alteration, at position 65 (N65 A) actually improves ligand binding in the A6'4 background, and is thus expected to have no detrimental effect on ligand binding in the R64 background. This change probably eliminates 'glyeosylation at N6S in the A64 background, thus demonstrating that a significant change in this region is likely to be tolerated. While an R64A change is poorly tolerated, R64K1 is well-tolerated, and thus another basic residue, such as H may be tolerated at position 64.
ActRIIB is well-conserved across nearly all vertebrates, with large stretches of the extracellular domain conserved completely. Many of the ligands that bind to ActRIIB are •also highly conserved. Accordingly, comparisons of AeiRIJB sequences from various vertebrate organisms provide insights into residues that may be altered. Therefore, an active, human ActRIIB variant polypeptide useful as a GDP Trap may include one or more amino acids at corresponding positions front the sequence of another vertebrate ActRIIB, or may include a residue that is similar to that in the human or other vertebrate sequence. The following examples illustrate this approach to defining an active ActRIIB variant. L46 is a valine in Xenopus ActRIIB, and so this position may he-altered, and optionally may be altered to another hydrophobic residue, such as V, I or F, or a non-polar residue such as A. E52 is a K in'Xenopus, indicating that this site may be tolerant of a wide variety of changes, including polar residues, such as E, D, K, R, B, S, T, P, G,· Y and probably A, T93 is a K in Xenopus, indicating that a wide structural variation is tolerated at this position, with polar residues favored, such as S, K, R, E, D, H, G, P, G and Y. FI 08 is a Y in Xenopus, and therefore Y or other hydrophobic group, such as 1, V or L should be tolerated. El 11 is K in Xenopus, indicating that charged, residues will be tolerated at this position, including D, R, K and B, as well as Q and N. Ill 12 is K in Xenopus, indicating that basic residues are·tolerated at this position, including R and H, A at position 119 is relatively poorly conserved, and appears as P in rodents and V in Xenopus, thus essentially any amino acid should be tolerated at this position.
The disclosure demonstrates that the addition of a further N-Iinked glycosyiation site (N-X-S/T) 'increases the serum half-life of an ActRIIB-Fc fusion protein, relative to the ActR!IS'(R64)~Fc form. By introducing an asparagine at position 24 (A24X construct), an NXT sequence is created that confers a longer half-life. Other ΝΧίΤ/S) sequences are found at 42-44 (NQS) and 65-67 (NSS). although the latter may not be efficiently glycosylated with the R at position 64, N-X-S/T sequences may be generally introduced at positions outside the ligand binding pocket defined in Figure I, Particularly suitable sites for the introduction of non-endogenous N-X-S/T sequences include amino acids 20-29, 20-24, 22-25, 109-134, 120134 or 129-134. N-X-S/T sequences mayUlso.be introduced into the linker between the ActRIIB sequence and the fe or other fusion component. Such a site may he:introduced with minimal effort by introducing.an N in the correct position with respect to a pre-existing $ or T, or by introducing an S or T at a position corresponding to a pre-existing N, Thus, desirable alterations that would create an N~!inked glycosyiation. site are: A24N, R64N, S67N (possibly combined with an N65A alteration), El06N, R112N, G! 20N, Η123N, F129N, A132N, III T2S and R1 Γ2Τ, Any S that is predicted to be glycosylated may be altered to a T without creating an immunogenic site, because of the protection afforded by the giycosylation. Likewise, any T that is predicted to be glycosylated may be altered to an S. Thus the alterations Sb?T and S44T are contemplated. Likewise, in an A24N variant, an S26T alteration may be used. Accordingly, a GDF Trap may be an ActRlIB variant having one or more additional, non-endogenous N-linked giycosylation consensus sequences.
Position L79 of ActRlIB may fee altered to confer altered activin - myostatic (GDF-11) binding properties, L79A or L79R reduces GDF-11 binding to a greater extent than activin binding, L79E or L?9D retains GDF-11 binding. Remarkably, the L79E and L79D variants have greatly reduced activin binding, ht vivo experiments indicate that these non-activin receptors retain significant ability to increase red blood cells but show decreased effects on other tissues. These data demonstrate the desirability and feasibility for obtaining polypeptides with reduced effects on activin. In exemplary embodiments, the methods described herein utilize a GDF Trap polypeptide which is a variant ActRlIB polypeptide comprising an acidic amino acid {e.g,, D or E) at the 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.
The variations described may be combined in various ways. Additionally, the results of the mutagenesis program described herein indicate that there are amino acid positions in ActRlIB that are often beneficial to conserve. These include position 64 (basic amino acid), position 80 (acidic or hydrophobic amino acid), position 78 (hydrophobic, and particularly tryptophan), position 37 (acidic, and particularly aspartic or glutamic acid), position 56 (basic amino acid), position 60 (hydrophobic amino acid, particularly phenylalanine or tyrosine). Thus, in each of the variants disclosed herein, the disclosure provides a framework of amino acids that may bo:conserved. Other positions that may be desirable to conserve areas follows; position 52 (acidic amino acid), position 55 (basic amino acid), position SI (acidic), 9S (polar or charged, particularly E, D, R or K.). in certain embodiments, isolated fragments of ActRlIB polypeptides can be obtained by screening polypeptides recombinamly produced from the corresponding fragment of the nucleic acid encoding an ActRlIB polypeptide (e.g.. SEQ ID NOs: 4 and S). In addition, fragments can be chemically synthesized using techniques known In the art such as .conventional MerrifteJd solid phase f-Moc or t-Boc chemistry. The fragments can be produced {recomhinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function, for example, as antagonists (inhibitors) or agonists (activators) of an ActRIIB protein or an ActRIIB ligand.
In certain embodiments, GDF Trap polypeptide is a,variant AetRI'IB polypeptide having an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NGs: 2: A ?,. 11,26, 28,29, 32,37, 38,40 or 41. In certain cases, the •GDF Trap has an amino add sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 11, 86, 28, 29, 32,37,38, 40 or 41. In certain emohdiments, the GDF Trap comprises, consists essentially of, or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs; 2, 3, 7, 11, 26, 28,29, 32, 37, 38,40 or 41 t wherein the position "corresponding to L79 of SEQ I D NO: I is an acidic amino acid (e.g., a D or E amino add.residue).
In certain embodiments, the present invention contemplates making functional variants by modifying the structure· of a GDF Trap polypeptide for such purposes as enhancing therapeutic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo), GDF Trap polypeptides can also be produced by amino acid substitution, deletion, or addition. For instance, it is reasonable to expect, that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement, of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements, are those that take place 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 results in a functional variant can be readily determined by assessing the ability of the GDF Trap polypeptide to produce.a response in cells relative to the unmodified GDF Trap polypeptide or a wild-type ActRIIB polypeptide, or to hind to one or more ligands, such as activin, GDF-1.1 or myostatic as compared to the unmodified GDF Trap polypeptide or a wild-type ActRIIB polypeptide.
In certain speci fic embodiments, the present invention contemplates making mutations in the extracellular domain (also referred to as ligand-binding domain) of an AetR I IB polypeptide such that the ActRIIB polypeptide lias altered ligand-binding activities: (e.g,, binding affinity or binding, specificity). In certain eases, such GDF Trap polypeptides have altered (elevated or reduced) binding affinity for a specific ligand, In other cases, the GDF Trap polypeptides have altered binding speci ficity for ActRIIB ligands.
For example, the disclosure provides GDF Trap polypeptides that preferentially bind to GDF8/GDF11 relative to acti'vinsi The disclosure further establishes the desirability of such polypeptides for reducing off-target effects,, although such selective variants may be less desirable for the treatment of severe diseases where very large gains in red blood cell levels may be needed for therapeutic effect and where some level of off-target effect is acceptable. For example, amino acid residues of the ActRIIB protein, such as IGF, K5S, Y60, K74, W7S, D80, and FI 01, are in the ligand-binding pocket and mediate binding tu ns ligands such as activin and GDFS. Thus, the present invention provides a GDF Trap comprising an altered ligand-binding domain (e.g., GDFB-binding domain) of-an ActRIIB receptor, which comprises one or more mutations at those amino acid residues. Optionally, the altered ligand-binding domain can have increased selectivity for a ligand such as GDFS relative to a wild-type ligand-binding domain, of an ActRIIB receptor. To illustrate, these mutations increase the selectivity of the altered ligand-binding domain, for GDFS over aeitvin. Optionally, the altered ligand-binding domain has a ratio of IQ for activin binding to IQ for GDFS binding that is at least 2., 5, 10, or even 100 fold greater relative to the ratio for the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has a ratio of ICso for inhibiting aeitvin to 1C so for inhibiting GDFS that is at least. 2,5, 10, or even 100 fold greater relative to the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain inhibits GDFS with an !€$« at least 2. 5, 10, or even 100 times less than the IC50 for inhibiting activin.
As a specific example, the positively-charged amino acid residue Asp (D8.0) of the ligand-binding domain of ActRII B can be mutated to a different amino acid residue to produce a GDF Trap polypeptide that preferentially binds to GDPS, but not activin. Preferably, the D80 residue is changed to an amino acid residue selected from the group consisting of: an uncharged amino acid residue, a negati ve amino acid residue, and a hydrophobic amino acid residue. As a further specific example, the .hydrophobic residue, L79, can be altered to the acidic amino acids aspartic acid or glutamic acid to greatly reduce activin binding while retainingGDFl 1 binding. As will be recognized by one of skill In the art, most of the described mutations, variants, or modifications may be made at the nucleic acid level or, in some eases, by post translational modification or chemical synthesis. Such techniques ate well known in the art, in certain embodiments* the present invention contemplates GDF Trap polypeptides having specific mutations in ActRlIB so as to alter the glycosylation of the ActRlIB-polypeptide. Exemplary glycosylation sites in GDF Trap polypeptides-are illustrated in Figure I (e.g,, the underlined NX(S/T) sites). Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as G-h'nked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X~threonine (where “X* is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes. The alteration may also he made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the wild-type ActRlIB polypeptide (for 0-1 inked glycosylation sites). A variety of amino acid substitutions or deletions at One or both of the first or third amino acid positions of a glycosylation. recognition site (and/or amino acid deletion at the second position) results in nou-glycosylatinu at the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties on a GDF Trap polypeptide is by chemical or •enzymatic coupling of glycosides to the GDF Trap polypeptide. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulihydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. These methods are described in WO 87/05330 and in Aplin and Wriston 11981) CRC Crit Rev. Biochem,, pp. 259-306, incorporated by reference herein. Removal of one or more carbohydrate moieties present on a GDF Trap polypeptide maybe accompli ahed chemically and/or enzymatically. Chemical degiycosylation may involve, for example, exposure of the GDF Trap polypeptide to the compound trifluoromethanesultonic -acid, or an equivalent compound. This treatment results i n the cleavage of most or all sugars except the linking sugar (15-acetylglucosamine or N~ acetylgalactosamine). while leaving the amino acid sequence .intact. Chemical· degiycosylation is further described by Halamuddin et al. (1987) Arch. Biochem, Biophvs. 259:52 and by Edge et al. (1981} Anal Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on GDF Trap polypeptides can foe achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. (1987) Meth, Enzyme), 138:350, The sequence of a GDF Trap polypeptide may be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide. In general, GD.F Trap polypeptides for use in humans will be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are expected to be. useful as well.
This- disclosure further contemplates a method of generating variants, particularly sets of combinatorial variants of a GD.F Trap polypeptide, including, optionally, truncation variants; pools of combinatorial mutants are especially useful for identifying GDF Trap sequences. The purpose of screening such combinatorial libraries may be to generate, for example, GDF Trap polypeptide variants which have altered properties, such as'altered pharmacokinetics, or altered ligand binding. A variety of screening assays are provided below, and such assays, may be used to evaluate variants. For example, a GD.F Trap polypeptide variant may be screened for the ability to bind'to an ActRIIB polypeptide, to prevent binding of an ActRUB ligand to an ActRIIB polypeptide or to interfere with signaling caused by an ActRIIB ligand.
The activity of a GDP Trap polypeptide or its variants may also be tested in a cell· based or in vivo assay. For example, the effect of a GDI·' Trap polypeptide variant on the expression of genes involved in hematopoiesis may be assessed. This may, as needed, be performed in the presence of one or more recombinant ActRIIB ligand proteins (e.g., activin), and cells maybe transfected so as to produce a GDF Trap polypeptide and/or variants thereof, and optionally, an ActRIIB ligand. Likewise, a GDF Trap polypeptide may be administered to a mouse or other animal, and one or more blood measurements, such as an RBC.count, hemoglobin levels, hematocrit levels., iron stores, or reticulocyte count may be assessed using art recognized methods.
Combinaforially-derived variants can be generated which have a selective potency relative to a reference GDF Trap polypeptide. Such variant proteins, when expressed from recombinant DMA constructs, can be used in gene therapy protocols. Likewise, mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding unmodified GDF Trap polypeptide, For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other processes which result in destruction of, or otherwise Inactivation of an unmodified GDF Trap polypeptide. Such variants, and the genes which encode them, can he utilized to alter GDF Trap polypeptide levels by modulating the half-life of the GDP Trap polypeptides. For instance, a short half-life can give rise to more transient biological effects and, when part of an inducible expression system, can allow tighter control of recombinant GDF Trap polypeptide levels within the cell in an Fcfusion protein, mutations may be made in the linker (if any > and/or the Pc portion to alter the half-life of the protein.
In certain embodiments, the GDP Trap polypeptides of the invention may further comprise post-translational modifications in addition to any that are naturally present in the AciRIIB polypeptides. Such modifications include, but are not limited to, acetylation, earboxylation, glycosylafion, phosphorylation, lipidation, and acylation. As a .result, GDF Trap polypeptides may contain non-amino add elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates, Effects of such non-amino acid elements on the functiouuittv of a GDF Trap polypeptide may be tested as described herein for other GDF Trap polypeptide variants. When a GDF Trap polypeptide is produced in cells by cleaving a nascent form of the GDF Trap, polypeptide, post-translational processing may also be important for correct folding and/or function of the protein. Different cells (such as O-iO, HeLa, MDCK, 293, WI38, N111-3T3 or HEK.293) have specific cellular machinery and characteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of the GDF Trap polypeptides.
In certain aspects, GDF Trap polypeptides include fusion proteins having at least a portion of an ActRBB polypeptide and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, poiyhisiidme, Giu-Giu,glutathione S •transferase (GST), thioredoxm. protein A, protein G, an immunoglobulin heavy chain constant region (e.g., an fe), maltose binding protein (M8P), or human serum albumin, A fusion domain may be selected so as to confer a desired property. For example, some fusion domains are. particularly useful for isolation of the fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt- corrugated resins are used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system .and the QIAexpress,M system (Qiagen) useful with (HIS§) fusion partners. As another example, a fusion domain maybe selected so as to facilitate detection of the GDF Trap polypeptides. Examples of such detection domains include the various fluorescent proteins (eg., GFP) as Wei! as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available. Well known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza vims haemagglutfoin (HA), and c-myc tags. In some cases, the fusion domains have a protease cleavage site, such as for factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the-fusion domain by subsequent chromatographic separation, in certain preferred embodiments, a GDF Trap polypeptide is fused with a domain that stabilizes the GDF Trap polypeptide in vivo (a “stabilizer” domain). By “stabilizing” is meant anything that increases serum half life, regardless of whether this is because of decreased destruction., decreased clearance by - the 'kidney, or other pharmacokinetic effect, fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, .fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may he selected include multimerizrog (e.g.„ dimerizing, tetramerizing) domains and functional domains (that confer an additional biologic.d function, such as further increasing red blood cell levels).
As a specific example, the present invention provides GDF Imp that is an ActRlIB-Fc fusion protein which comprises an extracellular (e.g,, ligand-binding) domain of ActRlIB polypeptide fused to an Fc domain. The sequence of an exemplary Fc domain is shown below (SEQ ID NO; 6).
THTCPFCPAPELLGG-PSVFLFP PKPKDTLMISRTPBVTCVVVD (A) VSH EDPEVK Y VDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEyKCK(A)VSNKALPVPIEKTISKAK GQPREPQVYTLPPSREEMTKNGVSLTCLVKGFYRSDIAVEWESNGQPENNYKTTPPVLDSDG PFFLYSKLTVDKSRWQQGEVESCSVMHEA'LHN (A 1 HYTQKSLSLSPGK*
Optionally, the Fc domain has one or more mutations at residues.-such as Asp-265, lysine 322, and Asn-434. In certain cases, the mutant Fe domain having one or more of these mutations (e.g., Asp-2G5 mutation) h. is reduced ability of binding to. the Fey receptor relative to a wi idtype Fc domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g., Asn-434 mutation) has increased ability of binding to the MHC class 1-related Fe-receptor (FORM) relative to a wildtype Fc domain.
It is understood that different elements of the fusion proteins may be arranged in any manner that is consistent with the desired, functionality. For example, a GDF Trap polypeptide may be placed €~ierniinai to a heterologous domain» or, alternatively, a heterologous domain maybe placed C-termisial to a GDP Trap polypeptide, The GDF Trap polypeptide domain and the heterologous- domain need not be adjacent in a fusion protein» -and additional domains or amino acid sequences may be included C~ or N-terminal to either domain or between the domains. in certain embodiments, a GDF Trap fusion protein comprises an amino acid sequence as set forth in the formula A-8-C. The B portion is an N- and C-terminaMy truncated ActRl IB polypept ide consisting of the amino add sequence corresponding to ammo acids 26-132 of SEQ ID NO: 26, The A and € portions may be independently zero, one or more than one amino acids, and both the A and C portions when present are heterologous to B. The A and/or C portions may he attached to the B portion via a linker sequence. Exemplary linkers are· include short polypeptide linkers such, as 2-10» 2-5., 2-4, 2-3 Glycine residues, such as, for example, a Gly-Gly-Gly linker. Other suitable linkers are described herein above. In certain embodiments, a GDF Trap fusion protein comprises an amino add sequence as set forth in the formula A-B--C, wherein A is a leader-sequence, B consists, of amino adds 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, formation of protein complexes, -and/or purification, in certain embodiments, a GDF Trap fusion protein comprises an amino add sequence as set forth in the formula A-B- C, wherein A is a 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,
In certain embodiments, the GDF Trap polypeptides of the.present invention contain one or more modifications that are capable of stabilizing the GDF Trap polypeptides. For example, such modifications enhance the in vfo-o half life of the GDF Trap polypeptides, enhance circulatory half life of the GDF Trap polypeptides or reducing proteolytic degradation of the GDF Trap polypeptides. Such stabilizing modifications include, but are not limited to, fusion proteins f including,, for-example, fusion proteins comprising-an GDF Trap polypeptide and a stabilizer domain), modifications of a glyeosylation site (including, for example, addition of a glyeosylation site to a GDF Trap polypeptide), and modifications of carbohydrate moiety (including, lor example, removal of carbohydrate, moieties from a GDF Trap polypeptide). In the case of fission proteins, a GDF Trap polypeptide is fused to a stabilizer domain such as an IgG molecule (e.g., an Fe domain). As used herein, the term ‘‘stabilizer domain’5 hot only refers to a fusion domain (e.g., Fc) as in the ease of fusion proteins, hut also includes nonproteinaceous modifications such as a carbohydrate moiety, or nonproieinaceous polymer, such as polyethylene glycol.
In certain embodiments,, the present invention makes available isolated and/or purified forms of the GDF Trap polypeptides, which are isolated from, or otherwise substantially free of,, other proteins.
In certain embodiments, GDF Trap polypeptides, {unmodified or modified) of the invention can be produced by a variety of art-known techniques. For example, such GDF Trap polypeptides can fee synthesized using standard protein chemistry techniques such as those described in Bodansky, M> Principles of Peptide Synthesis, Springer Verlag, Berlin (1.993) and Grant G. A, (ed.), Synthetic Peptides: A User’s Guide, W. H, Freeman and Company, New York ¢1992). In addition, automated peptide synthesizers arc commercially available (e.g,. Advanced ChemTech Model 396; Milligen/Biosearch 9600). Alternatively, the GDF Trap polypeptides, fragments or variants thereof may be recombinantly produced using various expression systems (e.g·., E* cob, Chinese Hamster Ovary (CHO) cells, COS cells, bacalovirus) as is well known in the art. In a further embodiment, the modified or unmodified GDF Trap polypeptides maybe produced fey digestion of .recombinantly produced full-length GDF Trap polypeptides by using, tor example, a protease, e.g,, trypsin, themiolysin, chymotrypsin, pepsin, or paired basic amino add converting enzyme (FACE). Computer analysis (using a commercially available software, e.g,, MacVecfor, Omega, PCGene, Molecular Simulation, Irsc j can be used to identify proteolytic cleavage sites. Alternatively, such GDF Trap polypeptides may be. produced from recombinantly produced full-length·GDF Trap polypeptides such as standard techniques-known in the art such as by chemical cleavage-(e.g., cyanogen bromide, hydroxylamine). 3. Nucleic Acids Encoding GDF Trap Polypeptides
In certain aspects, the invention provides isolated and/or recombinant nucleic acids: encoding any of the GDF Trap polypeptides disclosed herein. SEQ ID NOr 4 encodes a naturally occurring AetRIIB precursor polypeptide, while SEQ ID NO; 5 encodes a soluble Act RUB polypeptide, and SEQ ID NOs: 25,.27, 30 and 31 encode soluble GDF Traps. The subject nucleic acids may be single-stranded or double stranded. Such nucleic adds may be DNA or RNA molecules. These nucleic acids may be used, for example, so methods for making GDF Trap polypeptides or as direct therapeutic agents (dg., in a gene therapy approach).
In certain aspects, the subject nucleic acids encoding GDF Trap polypeptides are further understood to include nucleic acids that are variants ofSEQ ID NOs: 5,25, 2?, 30 and 31. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include coding sequences that differ from the nucleotide sequence of the coding sequence designated in SEQ ID NOs: 5, 25, 27, 30 and 31.
In certain embodiments, the Invention provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or .100% identical to SEQ ID 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 3!, and variants of SEQ ID NO; 5, 25, 27, 30 or 31. are also within the scope of this invention. In further embodiments, the nucleic acid sequences of the invention can be isol ated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library. in other embodiments, nucleic acids of the invention also Include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NO: 5, 25, 27, 30 or 31, complement sequence ofSEQ ID NO: 5, 25, 27, .30 or 31, or fragments thereof. As discussed above, one of ordinary skill in the art will understand readily- that appropriate stringency conditions which promote DNA hybridization can be varied, for example, one could perform the hybridization at 6.0 x sodium ehloride/sodium citrate ($$€) at about. 45 °C, followed by a wash of 2.0 x SSC <n $0 °C. For example, the salt concentration in the wash step can be selected from a low stringency of about 2,0 x SSC at 50 °Cto a high stringency of about 0.2 x SSC at 50 °C, In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 *€. to high stringency conditions at about 65 °C. Both temperature, and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed. In one embodiment, the invention provides nucleic acids which hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at mom temperature.
Isolated nucleic acids which ditter from the nucleic adds as set forth in SBQ ID NO: 5, 25, 27, 30 or 31 due io degeneracy in the-genetic code are also within the scope of the invention. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms lor histidine) may result in “silent” mutations which do not affect the amino acid sequence of the protein. In certain embodiments,, the GDP Trap polypeptide will be encoded by an alternative nucleotide sequence. Alternative nucleotide sequences are degenerate with respect to the native GDF Trap nucleic acid sequence but still encode for the same fusion protein. In certain embodiments, the GDP 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 that do lead to· changes in the amino acid sequences of the subject proteins will exist among mammalian cells. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.
In certain embodiments, the recombinant nucleic acids.of the invention may be operahly linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide.sequences will generally be appropriate to the host cell used for expression. Numerous types of appropriate -expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells. Typically, said one or more regulatory nucleotide sequences may include, but are not limited to. promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the ait are contemplated by the invention. The promoters may be either naturally occurring promoters. Or hybrid promoters that combine .dements of mure than one promoter. An expression construct may be present in a cell oil an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In.a preferred: embodiment, the expression vector contains a selectable marker gene· to allow the. selection of transformed host cells. Selectable marker genes are well known id the art and wit! vary with the host cell used.
In certain aspects of the invention, the subject nucleic acid ss provided in an expression vector comprising a nucleotide sequence encoding a GDP Trap polypeptide and operabiy linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of the GDF Trap polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gem Expression. 7'ecknohgy: Methods m'Enzymotagy, Academic Press, San Diego, CA {1990). For instance, any of a wide variety of expression control sequences that control the expression of a DMA sequence when operatively linked to it may he used in these vectors to express DMA sequences encoding a GDF Trap polypeptide. Such useful expression control sequences, include, for example, the early and late promoters of'SV'40, let promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the tip system, the TAC or TRC system, T? promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3 * phosphogiycefate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS, the promoters of the yeast ot~niaiing factors, the polyhedron promoter of the haculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the cho ice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also bo considered.
A recombinant nucleic acid of the invention can he produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinant GDF' Trap polypeptide incl ude plasmids and other vectors. For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derlved plasmids, pBTac-derived plasmids and pUC-derivsd plasmids: for expression in prokaryotic cells, such as £1 coiL
Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic 'transcript son units- that are expressed in eukaryotic cells. The pcDNAt/arnp,, peDNAl/neo., pRc/CMV, p$V2gpt, pSV'lneo, pSY2-dhif, pTk2, pRSVneo,. pIVISG, pS VT?, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacteria! plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma vims (BPV-1), or Epstein-Barrvirus (pHEBo, pREP-derived and p205) can be used for transient expressi on of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems cm be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and hi transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Eriisch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant polypeptides by the use of a baculovirus expression system , Examples of such baculovirus expression systems Include pVL-derived vectors (such as pVL'13'92, pVLI393 and pVL.941), pAcUW-derived vectors (such as pAcUWI), and pBlueBac-derived vectors (such as the 8-gai containing pBlueBac HI).
In a preferred embodiment, a vector will be designed for production of the subject GDF Trap polypeptides in CHO cells, such as a Pcmv-Scripi vector (Stratagene, La Jolla, Calif), pcDNA4 vectors (Invitrogen, Carlsbad, Calif) and p€!~neo vectors (Promega, Madison, Wise.). As will be apparent, the subject gene constructs can be used to cause expression of the subject GDF Trap polypeptides in cells propagated in culture, e g.. to produce proteins, including fusion proteins or variant proteins, for purification.
This invention also pertains to a host cell transfected with a recombinant gene including a coding sequence (e.g., SEQ I D NO: 4. 5,25,22, 30 or 31) for one or more of the subject GDP Trap polypeptides. The host cel! may be any prokaryotic or eukaryotic eel!, For example, a GDF Trap polypeptide of the invention may be expressed in bacteria! cells Such as £. colL insect cells (e.g,, using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled In the art.
Accordingly, the present invention further pertains to methods of producing the subject GDF Trap polypeptides. For example, a host cell transfected with an expression vector encoding a GDF Trap polypeptide can be cultured under appropriate conditions to al low expression of the GDF Trap polypeptide to occur. The C.1D.F Trap pol ypept ide may be secreted and isolated from a mixture of cells and medium containing the GDF Trap polypeptide. Alternatively* the G DF Trap polypeptide may be retained cytoplasmically or in a membrane fraction and the'cells -harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts- Suitable media for cell culture are well known in the an. The subject GDF Trap polypeptides can be isolated from cell culture medium, host cells, or both,, using'techniques known in the art for purslying proteins, including' ion-exchange chromatography, gel filtration chromatography, «Itrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the GDF Trap polypeptides.. In a preferred embodiment, the GDF Trap polypeptide is a fusion protein containing.a domain which facilitates its purification.
In another embodiment, a fusion gene coding lor a purification leader sequence, such as a poi y-(His) /enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant GDF Trap pol ypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni"^ metal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified GDF Trap polypeptide fe.g,, see Hoehuli et a!.., (1987)«/. Chromatography 411;! 77; and Janknecht e? al,yPN'A$ USA 88:8972).
Techniques for making fusion genes are well known. Essentially, the joining of various. ONA fragments coding for different polypeptide sequences ss performed in accordance with conventional techniques, employing blunt.~en.ded or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation, in another embodiment, the fusion gene can be .synthesized by conventional techniques including automated ONA synthesizers. Alternatively, PGR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene -fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology* eds. Ausubei efak, John Wiley &amp; Sons: 1992). 4, Screening Assays
In certain aspects, the present invention relates to the use of the subject GDF Trap polypeptides (e.g,, soluble variant ActRIIB polypeptides) to identify compounds (agents) which are agonist or antagonists of Act RIIB polypeptides. Compounds identified through this screening can be tested to assess their ability to modulate red blood cell, hemoglobin and/or reticulocyte levels in vim or in vi/m. "These compounds can be tested, tor example, in animal models.
There are numerous approaches to screening for therapeutic agents for Increasing red blood cell or hemoglobin levels by targeting ActRIIB signaling. In certain embodiments^ high-throughput screening of compounds can be carried out to identify agents that perturb ActRIIB-mediated effects on a selected cell line, in certain embodiments, the assay is carried out to screen and identify compounds that specifically inhibit or reduce binding of an ActRIIB polypeptide to its binding partner, such as an ActRIIB ligand (e,g,, activin. Nodal, GDP8, CIDPI1 or BMP?}. Alternatively, the assay can be used to identify compounds that enhance binding of an ActRIIB polypeptide to its binding partner such as an ActRIIB ligand. In a further embodiment, the compounds can he identified by their ability lo interact with an ActRIIB polypeptide,: A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein will nevertheless be comprehended by one of ordinary skill in the. art. As described herein, the test compounds (agents) of the invention may be created by any combinatorial chemical method. Alternatively,, the subject compounds may be naturally occurring biomolecules synthesized w vivo or in vitro. Compounds (agents) to be tested for their ability to act as modulators of tissue growth can be produced, for example, by bacteria, yeast, plants or other organisms (e.g,, natural products), produced: chemically (e.g,, small molecules, including peptidomimeries}, Or produced: recombmantly. Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidoinimefics., sugars, hormones, and nucleic acid molecules. In a specific embodiment, the test agent is a small organic molecule having a molecular weight of less than about 2,000 Daltons.
The test compounds of the invention can be provided as single, discrete entities, or provided in libraries of greater complexity, such as made by combinatorial chemistry. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds* Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in 'initial screening steps. Optionally, the compounds may be optionally derivafized with other compounds and have derivatizing groups that, facilitate isolation of the compounds. Nonlimiting examples of derivatizing groups include biotin, fluorescein, digoxygemn, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S transferase (GST), photoactivahbie crosslinkers or any combinations thereof.
In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed.in ceil-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as “primary” screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity or hioavaiiabiiiiy of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect, of the drug on the molecular target as may be manifest m an alteration of binding affinity between an AeiRIIB pol ypeptide and its binding partner (e,g., an ActRIIB ligand).
Merely to illustrate, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified ActRIIB polypeptide which Is ordinarily capable of binding to an ActRIIB ligand, as appropriate for the'intention of the assay. To the mixture of the compound and ActRIIB· polypeptide is then added to a composition containing-an ActRIIB ligand. Detection and quantification of AetRIIB/ActRHB ligand complexes provides a means for determining the'compound's efficacy at inhibiting (or potentiating) complex formation between the ActRIIB polypeptide and its binding protein. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations ofl.be test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. For example, in a control assay, isolated and purified AeiRIIB ligand is added to a composition containing the ActRl.l B polypeptide, and the formation, of Ac.tR 11 B/AciRIIB ligand complex is quantitated, in the absence of the test compound. It will he understood that, in general» the order in which the reactants may he admixed can be varied, and can be admixed simultaneously. Moreover, in place of purified proteins, cellular extracts and lysates may be used to render a stutable-cell-free-assay system.
Complex formation between the ActRIIB polypeptide and its binding protein may be detected by a variety of techniques, for instance, modulation of the formation of complexes can be quantitated using, for example, delectably labeled proteins such as radiolabeled (e.g., ^P, '^S, HC or Ή), fluorescently labeled (e.g., Fi'TC), or enzymatically labeled ActRIIB polypeptide or its binding protein, by immunoassay, or by chromatographic detection.
In certain embodiments, the present invention contemplates the. use of fluorescence polarisation assays and fluorescence resonance energy transfer (FRET) assays in measuring, either directly or indirectly, the degree of interaction between an ActRIIB polypeptide and its binding, protein. Further, other modes of detection, such as those based on optical waveguides (PCT Publication WO 96/26432 and U.S. Pal. Ho. $,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodiments of the invention.
Moreover, the present invention contemplates the use of an interaction trap assay, also known as the “two hybrid assay.” for identifying agents that disrupt or potentiate '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 el al. (1993) i Biol Chem 268:12046-1:2054; Bartel et al. (1993) Biotechniques 14:920-924: and Iwabuchi et al. (1993) Oncogene 8:1693-1690), In a specific- embodiment, the present invention contemplates the use of reverse two hybrid systems to identify compounds (e,g., small molecules or peptides) that dissociate interactions between an ActRIIB polypeptide and its binding protein. See.for example, Vidal and Legfam, (1999) Nucleic Acids Res 27:919-29; Vida! and Legrain, (1999) "TrendsB.iotecimol 17:374-8!; and U.S. Fat. Nos. 5,525,490: 5,955,280; and 5,963,368.
In certain embodiments, the subject. compounds are identified by their ability to interact with an ActRIIB polypeptide. The interaction between the compound and the ActRIIB polypeptide may be covalent or non-eovalent, For example, such hueraetion can be identified at the proteln level using in vitro biochemical methods, including- photocrosslinking, radiolabeled· ligand binding, and affinity chromatography iJakoby WB et al., 1974, Methods in Enzymolog-y 46: 1). in certain cases, the compounds may be screened in a mechanism based assay, such as an assay to detect compounds which bind to an Act RUB polypeptide.. This may include a solid phase or fluid phase binding event. Alternatively, the gene encoding an Act RUB polypeptide can he transfected with a reporter system (e.g., p-galactosidase, lucl (erase, or green fluorescent protein) into a cell and screened against the library preferably by a high throughput screening or with individual members of the library, Other mechanism based binding assays may be used, for example, binding assays which detect changes, in free energy. Binding assays can be performed with the target fixed to a well, bead or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. The bound compounds may be detected usually using colorimetric or fluorescence or .surface pfasmon resonance. 5. Exemplary Therapeutic Uses in certain embodiments, the GDP Trap polypeptides of the present invention can be used to increase red blood cell levels in mammals such as rodents and primates, and particularly human patients. In certain embodiments, the present invention provides methods of treating or preventing anemia in an individual in need thereof by administering to the individual a therapeutically effective amount of a GDP Trap-polypeptide. These methods may be used for therapeutic and prophylactic treatments of mammals, and particularly humans.
As used herein, a therapeutic that ‘‘prevents*5 a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or -condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. The term “treating55 as used herein includes prophylaxis of the named condition or amelioration or elimination of the condition once it hm been established. In either case, prevention or treatment may be discerned in the diagnosis provided by a physician or other health care provider and the. Intended result of administration- of the therapeutic agent.
As shown herein, GDF Trap polypeptides may he used to increase red blood cell, hemoglobin or reticulocyte levels in healthy individuals, and such GDF Trap polypeptides may be used In selected patient populations. Examples of appropriate patient populations include those with undesirably low red blood cell or hemoglobin levels, such as patients having an anemia, and those that are at risk for developing undesirably low red blood cell or hemoglobin levels, such as those patients.that are about to undergo major surgery or other procedures that may result in substantial blood loss. 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 transfusions. GDF Trap polypeptides disclosed herein may be used to increase red blood cell levels in patients having an anemia. When observing hemoglobin levels in humans, a level of less than normal for the appropriate age and gender category may be indicative of anemia, although individual variations are taken into account. For example, a hemoglobin level of 12 g/dt is generally considered the lower limit of normal in the general adult population. Potential causes include blood-loss, .nutritional· deficits, medication reaction, various problems with the bone marrow and many diseases. More particularly, anemia has been associated with a variety of disorders that include, for example, chronic renal failure, myelodysplastic· syndrome, rheumatoid arthritis, bone marrow"transplantation. Anemia may also be associated with the following conditions: solid tumors (e.g. breast cancer, lung cancer, colon cancer); tumors of the lymphatic system {e.g. chronic lymphocyte leukemia, non-Hodgkins and Hodgkins lymphomas ), tumors of the hematopoietic system (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 arthritides, systemic lupus erythematosa (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; situations where transfusion of red blood cells is not possible due to patient slid- or auto-antibodies, and/or for religious reasons (e.g. some Jehovah's Witnesses); infections (e,g. malaria, osteomyelitis); hemoglobinopathies, including, for example, sickle cell disease, thalassemias; drug use or abuse, e.g. alcohol misuse; pediatric patients with anemia from any cause to avoid transfusion; and elderly patients or patients with underlying cardiopulmonary disease with anemia who cannot receive transfusions due to concerns about circulatory overload. GOF Trap polypeptides would be appropriate for treating anemias of .hypopreli.ferattve bone marrrow, which are typically associated with little change in red blood cell (RBC) morphology, Hypoprolifeirative anemias include: 1) anemia of chronic disease, 2:) anemia of kidney disease, and 3) anemia associated with hypometabolic states. In each of these types, endogenous.erythropoietin levels are inappropriately km tor the degree of anemia observed. Other hypoprolifeiative anemias include: 4) early· stage iron-deficient .anemia, and 5) anemia caused by damage to the bone marrow, In these types, endogenous erythropoietin levels are appropriately elevated tor the degree of anemia observed.
The most common type is anemia of chronic disease, which encompasses inflammation, infection, tissue injury, and conditions such as cancer, and is distinguished by both low erythropoietin levels and an inadequate response to.erythropoietin in the hone marrow {Adamson, 2008, Harrison’s Principles of Internal Medicine, 17th ed.; McGraw Hill, New York, pp 628-634). Many factors can contribute to cancer-related anemia. Some are associated with the disease process itself and the generation of railamaiory cytokines such as interleukin-1, interferon-gamma, and tumor necrosis factor (Bron et al, 2001, Semin Oncol 28(Suppi 8): t -6), Among its effects, inflammation induces the key iron-regulatory peptide heneidm, thereby inhibiting iron export from macrophages and generally limiting Iron availability for erythropoiesis (Ganz, 2007, J Am Soc Nephrol 18:394-400). Blood loss through various routes can also contribute to. cancer-related, anemia. The prevalence of anemia due to cancer progression varies with cancer type, ranging from 5% in prostate cancer up to 90% in multiple myeloma. Cancer-related anemia has profound consequences for patients, including fatigue and reduced quali ty of life, reduced treatment efficacy, and i ncreased mo rial hy,
Chronic kidney disease is associated with hypoproliterative anemia that varies, in .severity with the degree ofrenal impairment; Such anemia is primarily due to inadequate production of erythropoietin and reduced Survival of red blood cel ls. Chronic kidney disease usually proceeds gradually over a period of years or decades to end-stage (Stage-5) disease, at which point .dialysis: or kidney transplantation is required for patient survival. Anemia often develops early in this process and worsens as disease: progresses. The clinical consequences of anemia of kidney disease, are well-documented and include 'development of left ventricular hypertrophy, impaired cognitive function, reduced quality of life, and altered immune function (Levin et al., 1999, Am i Kidney Dis 27:347-354; Nissenson, 1962, Am i
Kidney Dis 20{Supp! 1):21-24; Revicki el al, 1995, Am J Kidney Dis 25:548-554; Gaiter et aL 1994, Kidney hit 45:224-231). As demonstrated by the Applicants in a mouse model of chronic kidney disease {see Example below), a GDF Trap polypeptide can be used to treat anemia of kidney disease.
Many conditions resulting in a hypometabofie rate can produce a mild-to-moderate hypoproliferative anemia. Among such conditions are endocrine deficiency states. For example, anemia can occur in Addison’s disease, hypothyroidism, hyperparathyroidism, or males who are castrated or treated with estrogen. Mild-to-moderate anemia can also occur with reduced dietary intake of protein, a condition particularly prevalent in the elderly. Finally, anemia can develop in patients with chronic liver disease arising from nearly any cause (Adamson. 2008, Harrison’s Principles of Internal Medicine, 17th ed.; McGraw Hill, New York, pp 628-634),
Anemia resulting from acute blood loss of sufficient volume, such as from trauma or postpartum hemorrhage, is known as acute post-hemorrhagic anemia. Acute blood loss initially causes hypovolemia without anemia since there is proportional depletion ofRBCs along with other blood constituents. However, hypovolemia will rapidly trigger physiologic mechanisms that shift fluid from the extravascular to the vascular compartment, which results in hemodilution and anemia. If chronic, blood loss gradually depletes body iron stores and eventually leads to iron deficiency. As demonstrated by the Applicants in a mouse model (see Example below), a GDF Trap polypeptide can be used to speed recovery from anemia of acute blood loss.
Iron-deficiency anemia is the final stage in a graded progression of increasing iron deficiency which includes negative iron balance and iron-deficient crytbropoiesis as intermediate stages. Iron deficiency can result from increased iron demand, decreased iron intake, or increased iron loss, as exemplified in conditions such as pregnancy, inadequate diet, intestinal malabsorption, acute or chronic inflammation, and acute or chronic blood loss. With mild-to-moderate anemia of this type, the hone marrow remains hypopfohferati ve, and R8C morphology is largely normal; however, even mild anemia can result in some microcytic hypochromic RBCs, and the transition to severe iron-deficient anemia is accompanied by &amp;>j?erpro1tferation of the bone marrow and increasingly prevalent microcytic and hypochromic RBCs (Adamson, 2008, Harrison’s Principles of Internal Medicine, i 7th ed.; McGraw Hill, New York, pp 628-634), Appropriate therapy tor iron-deficiency anemia depends on its cause and severity, with ora! iron preparations, parenteral iron formulations, and RBC transfusion as major conventional options. An GDP Trap polypeptide could be used to treat chronic iron-deficiency anemias alone or in combination with conventional therapeutic approaches, particularly to treat anemias of multi factorial origin.
Hypoproliferative anemias can result from primary dysfunction or failure of the bone marrow, instead of dysfunction -secondary to inflammation, infection, or cancer progression. Prominent examples would be myelosuppresslon caused by cancer chemotherapeutic drugs or cancer radiation therapy. A broad review of clinical trials found that mild anemia can occur In 100% of patients after chemotherapy, while more severe anem ia can occur in up to 80% of such patients (Groopman et ah, 1999,1 Natl Cancer Inst. 91:1616-1634). Myelosuppressive drugs include: I ) alkylating agents such as nitrogen mustards (e.g., melphalan} and nitrosoureas (e.g., streptoxocm); 2} antimetaholites such as folic acid antagonists (e.g., methotrexate), purine analogs (e.g., thioguanine), and pyrimidine analogs (e.g., gemcitabme); 3) cytotoxic antibotbs such as anthracyclines (e.g,. doxorubicin); 4) kinase inhibitors (e.g,, gefitinih); 5) mitotic inhibitors such as taxanes (e.g,, paclitaxel) and vinca alkaloids (e.g., vinorelbine); 6) monoclonal antibodies (e.g., rltuxlmab); and ?) topoisomerase inhibitors (e,g.} topotecan and etoposide). As demonstrated in a mouse model of chemotherapy-induced anemia (see Example below), a GDP Trap polypeptide can be used to treat anemia caused by chemotherapeutic agents and/or radiation therapy. GDF Trap polypeptides would also be appropriate for treating anemias of disordered RBC maturation, which are characterised in part by undersized (mierocydc), oversized (macrocytic), misshapen, or abnormally colored (hypochromic) RBCs.
Patients may be treated with a dosing regimen intended to restore the patient to a target hemoglobin level, usually between about 10 g/dl and.about 12,5 g/dl, and typically about 11 ,.0 g/dl (see also Jacobs et ah (2000) 'Nephrol Dial Transplant '15,15-19), although lower target levels may cause fewer cardiovascular side effects. Alternatively, hematocrit levels (percerstage of the volume of a blood sample occupied by the cells) can be used as a .measure for the condition of red blood cells. Hematocrit: levels for healthy individuals range from 41 to 51% for adult males arid from 35 to 45% for adult females. Target hematocrit levels are usually around 30-3395. Moreover, hemoglobin/hematocrit levels vary from person to person. Thus, optimally, the target hemoglobin/hemafoerlt level can be individualized for each patient.
The rapid effect on red blood cell levels of the GDF Trap polypeptides disclosed herein indicate that these agents act by a different -mechanism than Fpo. Accordingly, these antagonists maybe useful for increasing red blood cell and hemoglobin levels in patients that do not respond well to Bpo, For example, a GDF Trap polypeptide may be beneficial for a patient in which administration of a normal to increased (>300 llJ/kg/week) dose of Epo does not result in the increase of hemoglobin level up to the target level Patients with an inadequate- Epo response -are found for all types of anemia, but higher numbers of nonresponders have been observed particularly frequently in patients with cancers and patients with end-stage renal disease; An inadequate response to Epo can be either constitutive (i.e. observed upon the first treatment with Epo) or acquired (e,g. observed upon repeated treatment with Epo).
The GDF Trap polypeptides may also be used to treat patients that, are susceptible to adverse effects of Epo. The primary adverse effects of Epo are an excessive increase in the hematocrit or hemoglobin levels and polycythemia, Elevated hematocrit levels can lead to hypertension (more particularly aggravation of hypertension) and vascular thrombosis. Other adverse effects of Epo which have been reported, some of which related to hypertension,-are headaches, influenza-like syndrome, obstruction of shunts, myocardial infarctions and cerebral convulsions due to thrombosis, hypertensive encephalopathy, and red cell blood cell applasia {Strsglbarii, (1994) I, Clin Investlg ?2(suppi 6). S36-S43; Horl et ai. (2000) Nephrol Dial Transplant l'5(suppl 4), 51 -56; Defantv et al, (1997) Neurology 49, 686-689; Bunn (2002) N Engl J Med 346(7), 522-523), GDF traps may also boused in. combination with Epo and other agents that activate the erythropoietin pathway., in some instances, this may permit lower dosing of each drug in the combination. in certain embodiments, the present invention provider methods for managing, a patient that has been treated with, or is a candidate to be treated with,, a GDF Trap polypeptide by measuring one or more hematologic parameters in the patient. The hematologic parameters may be used to evaluate appropriate dosing for a patient who is a candidate to be treated with a GDF Trap polypeptide, to monitor the hematologic parameters during treatment with a GDF Trap polypeptide, to evaluate whether to adjust the dosage during treatment with a GDF Trap polypeptide, and/or to evaluate an appropriate maintenance dose of a GDF Trap polypeptide, if one or more of the hematologic parameters are outside the norma! level, dosing with a GDF Trap polypeptide maybe reduced, delayed or terminated.
Hematologic parameters that maybe measured in accordance with the methods provided herein include, for example, red blood cell levels,, blood pressure, iron stores, and other agents found in bodily fluids that correlate with increased red Wood cell levels, using art recognized methods. Such parameters may be determined using a blood sample from a patient. Increases in red blood'cell levels, hemoglobin levels, and/or hematocrit levels may cause increases in blood pressure.
In one embodiment, If one or more hematologic parameters are outside the normal range, or on the high side of normal, in a patient who is a candidate to he treated with a GDF Trap polypeptide then onset of administration of the GDF Trap polypeptide may be delayed until the hematologic parameters have returned to a normal or acceptable level either naturally or via therapeutic intervention, For example, if a candidate patient is hypertensive or prehypertensive* then the patient may be treated with a blood pressure lowering agent in order to reduce the patient’s blood pressure; Any blood pressure towering agent appropriate for the individual patient’s condition may be used including, for example, diuretics, adrenergic inhibitors (including alpha blockers and beta blockers), vasodilators, calcium channel blockers., angiotensin-convertlng enzyme (ACE) inhibitors, or angiotensin II receptor blockers. Blood pressure may alternatively he treated using a diet and exercise regimen. Similarly, if .a candidate patient has iron stores that are lower than normal, or on the low side of normal, then the patient may be treated with an appropriate regimen of diet and/or iron supplements until the patient’s Iron stores have returned to a normal or acceptable level. For patients having higher than normal red blood cell levels and/or hemoglobin levels, then administration of the GDF Trap polypeptide may be delayed until the levels have returned to a normal or acceptable level.
In certain embodiments, If one or more hematologic parameters are outside the normal range, or on the high side of normal, in a patient who is a candidate to be treated with a. GDF Trap: polypeptide then the onset of administration may be not be delayed. However, the dosage amount or frequency of dosing of the GDF Trap polypeptide may be set at an amount that would reduce the risk of an unacceptable increase in the hematologic parameters arising upon administration of the GDF Trap polypeptide. Alternatively, a therapeutic regimen may be developed for the patient that combines a GDF Trap polypeptide with a therapeutic agent that addresses the undesirable level of the hematologic parameter. For example, if the patient has elevated blood pressure, then a therapeutic regimen involving' administration of a GDF Trap polypeptide and a blood pressure lowering agent may he designed. For a patient having lower than desired iron stores, a therapeutic regimen of a GDF Trap polypeptide and iron supplementation maybe developed.
In one embodiment, baseline parameters) for one or more hematologic parameters may be established for a patient who is a candidate to be treated with a GDF Trap polypeptide and an appropriate dosing regimen establish for that patient based on the baseline valuers). Alternatively, established baseline parameters based on a patient's medical history could be used to inform an appropriate GDF Trap polypeptide dosing regimen for a patient. For example, if a healthy patient has an established baseline blood pressure readi ng that is above the defined normal range it may not be necessary to bring the patient's blood pressure into the range that is considered normal tor the general population prior to treatment with the GDF Trap polypeptide.. A patient’s baseline values for one or more hematologic parameters prior to treatment with a GDF Trap polypeptide may also be used as the relevant comparative values for monitoring any changes to the hematologic parameters during treatment with the GDF Trap polypeptide.
In certain embodiments, one or more hematologic parameters are measured in patients who are being treated with a GDF Trap polypeptide. The hematologic parameters· may be used to monitor the patient during treatment and permit adjustment or termination of the dosing with the GDF Trap polypeptide or additional dosing with another therapeutic agent. For example, if administration of a GDF Trap polypeptide results in an Increase in blood pressure, red blood cell level, or hemoglobin level, or a reduction in iron stores, then the dose of the GDF Trap polypeptide may be reduced in amount or frequency in order to decrease the effects ofthe· GDF Trap· polypeptide on the one or more hematologic parameters. If administration or a GDF Trap polypeptide results in a change In one or more hematologic parameters that is adverse to the patient, then the dosing ofthe GDF Trap polypeptide may be terminated either temporarily, until the hematologic parameier(s) return to an acceptable level, or permanently. Similarly, if one or more hematologic parameters are not brought within an acceptable range after reducing the dose or frequency of administration ofthe GDF Trap polypeptide then the dosing may be terminated. As an .alternative, or in addition to, reducing or terminating the dosing with the GDF Trap polypeptide, the patient may be dosed *itb an additional therapeutic agent that addresses the undesirable level in the hematologic parameters), such as, for example, &amp; blood pressure lowering agent or an iron supplement. For example, i f a patient being treated with a GDF Trap polypeptide has elevated blood pressure, then dosing with the GDF Trap polypeptide may continue at the same level and a blood pressure lowering agent is added to the treatment regimen, dosing with the GDF Trap polypeptide may he reduce (e.g., in amount and/or frequency) and a blood pressure lowering agent is added to the treatment regimen, or dosing with the GDF Trap polypeptide maybe-terminated and the patient may be treated with a blood pressure lowering agent
In certain embodiments, patients being treated with a GDF Trap polypeptide, or candidate patients to fee treated with a GDF Trap polypeptide, are patients in need of muscle growth, such as patients suffering from, or at risk of developing, a neuromuscular disorder or musculogenerative disorder. For example, patients or candidate patients may be suffering from, or at risk tor developing, Lou Gehrig’s disease (ALS), cancer anorexia-cachexia syndrome, muscular dystrophy, muscle atrophy, congestive obstructive pulmonary disease (and muscle wasting associated with COFD), muscle wasting syndrome, sarcopema, or cachexia. Muscular dystrophy refers to a group of degenerative muscle diseases characterized fey gradual weakening and deterioration of skeletal muscles and sometimes the heart, and respiratory muscles. Exemplary muscular dystrophies that can be treated with a regimen including the subject GDF Trap polypeptides include: Duehenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Bmery-Dreifuss Muscular Dystrophy (EDMD), Limb-Girdle Muscular Dystrophy (LGMD), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD) (also known as Landouxy-Dejerine), Myotonic Dystrophy (MMD) (also known as Steinerfs Disease), Oculopharyngeal Muscular Dystrophy (OPMD)j Distal Muscular Dystrophy (DO), Congenital Muscular Dystrophy (CMD). 6. Pharmaceutical Compositions
In certain embodiments, compounds (e.g., GDF Trap polypeptides) of the present invention are. formulated with a pharmaceutically·acceptable carrier. For example, a GDF Trap polypeptide can he -administered alone or as a component of a pharmaceutical fonnu'latton (therapeutic composition). The subject compounds may be formulated for administration in any convenient way for use in human or veterinary medium.·.
In-certain embodiments, the therapeutic method of the invention includes administering the composition sysiemically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-tree, physiologically-acceptable form. Therapeutically useful agents other than the CDF Trap polypeptides which may also optionally be included in the composition as described above,, may be administered 'simultaneously or sequentially with the subject compounds (e.g,, GDF Trap polypeptides) in the methods of the invention.
Typical k, compounds will he 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 isotome aqueous or nonaqueous solutions, dispersions, suspensions or-emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacieriostats, solutes which render the formulation isotonic with the blood of the ««ended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the-invention include water, ethanol, polyols {such as glycerol, propylene glycol,-polyethylene glycol* and the like), and suitable mixtures thereof,, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can he maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case-of dispersions, and by the use of surfactants,
Further, the composition may be encapsulated or injected m a form for delivery to a target tissue site (e.g., bone marrow), in certain embodiments, compositions of the present invention may include a matrix capable of delivering one or more therapeutic compounds (e.g., GDF Trap polypeptides) to a target, tissue site (e.g., bone marrow), providing a structure for the- developing tissue and optimally capable of being resorbed into the body. For example, the matrix may provide slow' release of the GDF Trap polypeptides. Such matrices may he. formed of materials, presently in use for other implanted medical applications.
The choice of matrix material is based on hfocornpatsbihty, biodegradabilUy, mechanical properties, cosmetic appearance and: interface properties. The particular application of the subject compositions will define the appropriate formulation. Potential matrices for the compositions .may be biodegradable and chemically defined calcium sulfate. tricalciumpho$'phate» hydroxyapatite, polylactic acid and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are ncmfofodegradable am! chemically defined, such as sintered hydroxyapatite, bioglass, aiuminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material,, such as polylactic acid and hydroxyapatite or collagen and tricalciomphosphate. The bioceramics may be altered in composition, such as in calcium-alummate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradabihty.
In certain embodiments, methods of the invention can, he administered for orally, e,.g,, in the form of capsules, cachets, pills, tablets, lozenges (using-a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or no«-aqueous· liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an Inert base, such as: gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient An agent may also be administered as a bolus, electuary or paste. la solid dosage forms for oral administration -(capsules, tablets, pills, dragees, powders, granules, and the like), one or more therapeutic compounds of the present invention maybe mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicaldum phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carhoxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone* sucrose, and/or acacia; (3) huinectants, such as .glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, aiginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators,· such as quaternary ammonium, compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay: (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like, "Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, mieroernulsioos, solutions, suspensions, syrups, and elixirs. In addition to the active: ingredient., the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils' (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters ofsorbitan, and mixtures thereof Besides inert diluents, the oral compositions-can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxyiated isosteary! alcohols, 'polyoxyethylene sorbitol, and sorbitan esters, rnicrocrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
The compositions of the: invention may also contain adjuvants, such as preservatives* wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben,. e-hlorobutanol, phenol sorbic add, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin..
It Is understood that the dosage regimen will be determined by the attending physician, considering various factors which modify the action of the subject compounds of the invention: (e;g., GDF Trap polypeptides). The various factors Include, hut are not limited to, the patienTs red -blood cell count, hemoglobin level or other diagnostic assessments, the desired target red blood cel!-count, the patient's age. sex, and diet, the severity of any disease that may he contributing to-a depressed red blood cell level, lime of administration, and other clinical factors. The addition of other known growth factors to the final composition may also affect the dosage. Progress can he monitored by periodic assessment of red blood cell and hemoglobin levels, as well as assessments, of reticulocyte- levels and other indicators of the hematopoietic process.
In certain embodiments, the present invention also provides gene therapy for the in vivo production of GDP Trap polypeptides. Such therapy would achieve its therapeutic effect by introduction of the GDF Trap polynucleotide sequences into cells or tissues having-the disorders as listed above. Delivery of GDF Trap polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Preferred for therapeutic delivery of GDP Trap polynucleotide sequences is the use of targeted liposomes.
Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or an R’NA virus such as a retrovirus. The retroviral vector may be a deri vative of a murine or avian· retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, hut are not limited to: Moloney murine leukemia vims (MoMuLV), Harvey murine sarcoma vims (HaMuSV), murine mammary inmor virus (MuMTV), and Rons Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and gems ated Retroviral vectors can he made targebspeeific by attaching, for example, a sugar,-a glycolipid, or a protein. Preferred targeting is- accomplished by using an antibody. Those of skill in the art will recognize that specific polynucleotide sequences can he inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the GDF Trap polynucleotide.
Alternatively, tissue-culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, po! and env, by conventional calcium phosphate transfection. These cells ate then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector Into the culture medium.
Another targeted delivery system for GDF Trap polynucleotides Is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, mmocapsules, microspheres, heads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes ore artificial membrane vesicles which are useful as delivery vehicles in vitm and in vim. RNA, DMA and intact virions can he encapsulated within the aqueous interior and be 'delivered to cells in a biologically active form {see e.g., Fraley, el si,/Trends Biochem. Sci., 6:77, 1981), Methods: for efficient gene transfer using a liposome vehicle, are known in the art, see e.g,, Mannino, et al,, Biotechniques, 6:682, 1988. The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. 'Examples of lipids-useful in liposome production include phosphatidyl compounds, such as phosphatidyl glycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, eerebrosides, and gangliosides. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphafidylcholine, and. distearoylphosphatidylchOline, The targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art,
EXEMPLIFICATION
The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments and embodiments of the present invention,, and are -not intended to limit the Invention,
Example I . Generation of a GI>F Trap,
Applicants constructed-a GDF Trap as follows. A polypeptide having a modified extracellular domain of ActR'OB with greatly reduced-aciivin A binding relative to GDf 11 and/or myostatic (as a consequence of a leueme-to-aspartate substitution at position 79 in SEQ ID NO; I) was fused to a human or mouse Fc domain with a minimal linker (three glycine amino acids) in between. The constructs arc referred to as ActlNIBf L.79D 20-134)-h-Fc and ActRlTB(L79D 2C)-134)-mFc, respectively. Alternative-forms with a glutamate rather than.an .aspartate at position 79 performed- similarly (L79E). Alternative forms with an alanine rather than a valine at- position 226 with respect to SEQ ID NO: 7, below were also generated and performed equivalently in-all respects tested. The aspartate at position '79 (relative to SEQ ID NO: I, or position 60 relative to SEQ ID NO: 7) is highlighted in gray below. The valine at position 226 relative to SEQ ID NO: 7 is also highlighted in gray below.
The GDF Trap Aetil!IB(L7‘->D 20-134)-hFc is shown below as purified· from CHO cel! lines (SEQ 10 NO: ?>,
GRGIH AETRECIYYN AN WBLERTNQSGLERCEG EQDKR LHCY ASWRNSSGTIELV KK
GCWDOOFNCYDRQECVATEENPQVyFCCCEGNFCNERFTHLPEAGGPEVTYEPPPT
AP1TK3GT|jTCPFCPAPEtj^GPSVFLFgFKPKDTLMISETPEyTCVVVDySHEDEEYK EMWYYDGVEVHSAKTKZREEQYl·^^
PjPIEKBSKMGQFREFQYrrWSKa^T^
Fllffillime EFLYSKi LSFGE
The ActRUB-derived portion of the GDF Trap has an amino acid sequence set forth below (SEQ ID NO: 32), and that portion could be used as a monomer or as a non~Fc fusion protein as a monomer, dimer or greater order complex .
GRGEAETRECIYYNANWELERTNQSGLERCEOEQDKRLHCYASWRNSSGTIE LVKKGCw||DDFNCYDRQBCVATEENPQVYFCCCEGNFGNERFTHLPEAGGPEVTYE PPPTAPT (SR) ID NO: 32)
The GDF Trap protein was expressed in CHO eel! lines. Three different leader sequences were considered: (i) Honey bee melittin (FIBML): MKFLYN VALVFMYV YfSYIYA (SEQ ID NO: 8} (it) Tissue Plasminogen Activator (TPA); MDAMfCRGLCCVLLLCGAVFVSP (SEQ ID NO: 9) (in) Native: MTAPWVALALLWCSLCAGS (SEQ ID NO: 10}..
The selected form employs the TPA leader and has the following unprocessed amino acid sequence:
MS A MKRGLGCVIIRG B A ETR EC 1Y YN A N WE l, ERTN QSG L F RC E geqdkrlhcyaswrnssgtielvkkgcwdddfhcydrqegvateenpqyyfccce GNFCN ERFTH LPEAGGPEVTYEPFPTAPTGGGTHTCPPCPAFELLGGFSVFLFPFRPRD llMtSRTP£yTCyVyiDYSFIEDPEVKFN,WYVDGVEYjlNAKTMPREEQYNSTYRVV$V mamsmi^MiscsMNKAU)yeE^^
VStTCLVKCFYPSpiAVEWESMGOPHNNYRT fP.PV LPSPGSFF LYSK LT VDKSR WOO GMYFSCSVMI^^ CSEQ IDNO: 11}
This polypeptide is encoded by the following nucleic acid sequence t'SEQ ID NO; 12):
A TGGATGCAAT GAAGAGAGGG CTCTGGTGTG TGC7GCTGCT GTG7GGAGGA G'fCTTCGTTT CGCCGGGCGC CTCTGGGCGT GGGGAGGCTG AG AC AC GG GA GTGCATCTAC TAGAACGC C A ACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGGC C.TGGCAAGGC GAG C A GG AC A AGCGOC TGCA CTGCTACGCC TCC7GGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGA AGGGCTGCTG GGACGATGAC ??OAACTGCT AC'jATAGGGA GGAGTGTGTG GCGACTGAGG AGAACCCCCA GGTGTACTTC TGGTGCTGTG AA·GGCAACTT CTGCAACGAG CGCTTCACTC AT'TTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTA.CGAGCC ACCCCCGACA GCCCCCACCG G7GGTGGAAC TCACACA'TGC CC.AGCGTGCG CAGCACCTGA ACTCCTGGGG GCACCGTCAG TCTTCCTCTT GCCGCGAAAA CCCAAGGACA CCCTCATOAT CTCCCGGAC.'C CCTGAG'GTCA CATGCGTGGT GGTGGACGTG AGCCXCGAAG ACCC?GAGGT CAAGTTCAAC TGGTACGTGG ACuGOGT GG A G<jTG CA'i AA'i' GCCaAGACAR AGCCgCgGGA. OWAGCAu λ AC AA.CGv.;:vACGT ACCGTGTGGT CAGCGTCCTC ACCGTGCTGC ACCAGGACTG GCTGAATGGC A.AGG?-\GTACA AGTGCAAGGT CTCCAACAAA GCCOTCCCAG TCCCC.ATCGA GA.AAACCATC TCCAAAGCCA AAGGGCAGCC C C GAGAAC 0 A CACGTGTACA CCCTGGCCCC ATC CC GG (5 AG CAGATGACCA AGAACCAGGT CAGCCTGACC TGGCTGGTCA' AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG AG i'GGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACT CCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT G7CTGCGGGT AAATGA
Purification could bo achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenyl sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification could be completed with viral filtration and buffer exchange. In an example of a purification scheme, the cell culture medium is passed over a protein A column, washed .in 150 mM Tris/NaCI (pH 8.0), then washed in 50 mM Tris/NaCI (pH 8.0) and eluted with 0.1 M glycine, pH 3.0. The low pH d'uate is kept at room temperature for 30 minutes as a viral clearance step. The eluate is then neutralized and passed over a Q sepharose ion exchange column and washed in 50 mM Tris pH 8.0, 50 mM NaCI, and eluted in 50 mM Tris pH .8.0,· with an NaCl concentration between 150 mM and 300 mM. The eluate is then changed into SO mM Tris pH 8.0, 1.1 M ammonium sulfate and passed over a phenyl sepharose column, washed, and eluted in 50 mM Tris pH 8.0. with ammonium sulfate between 150 and 300 mM. The eluate is dialyzed and filtered for use.
Additional GDF Traps (ActRIlB-Fc fusion proteinsmodified so as to reduce the ratio o:f aetivio A binding relative to royestaiie or GDF! I) are described in PCT/US:20O8/OO!5Ob and WO 2000/012627, incorporated by reference herein.
Example 2,, Bioassay for GDF-J1 and Aeflvin-mediated signaling.
An A-204 Reporter Gene Assay was used to evaluate the. effects of AetRIIB-Fe proteins-and GDF Traps on signaling by GDP-11 and Activin A, Cell line: Human Rhabdomyosarcoma (derived from muscle). Reporter vector: pGL3(CAGA)l 2 (Described in Dennier et a!, 199&amp;, EMBO Γ7; 3091-3100). The GAGA 12 motif is present in TGF-Beta responsive genes ( PAH gene), so this vector is of general use for factors signaling through Smad2 and .1.
Day l: Split A-204 cells into 48-well plate.
Day 2: A-204 cells transfected with iO.ug pGt3fCAGA)i2 or pGU(CAGA) 12(10 ug)v pRLCMV (1 ug) and fugene.
Day 3; Add factors (diluted into medium* 0.1 % BSA). Inhibitors need to he preincubated with Factors for 1 hr before, adding to cells. 6 hrs later,-cells rinsed with PBS. and lyse cells.
This is followed by a Luct(erase assay. In the absence of any inhibitors, Activin A showed 10 fold stimulation of reporter gene expression and an BD50 2 ng/mt GDP-11: 16 fold, stimulation, ED50: - 1.5 ng/ml.
AeiRlIB(20-134) is a .potent inhibitor of activin, GDF-8 and GDF-11 activity in this assay. Variants were tested in this assay as well.
Example 3« GDP-11 Inhibition by N-teonma! and C-terndnal Truncations
Variants of ActR 118(20-134)-hFc with truncations at the Ν-terminus and/or C-terminus were generated and tested for-activity as inhibitors of GDF-11 and activin. The activities are shown below fas measured in conditioned media): C-terminal. AetRHR-hFe Truneation-s:
As can be seen, truncations of three (ending with ,, ,PPT), six (ending with ,, .YEP) or mote amino acids at the C-terminus causes a threefold or greater decrease in the activity of the molecule. The truncation of the final 15 amino acids of the AetRIlB portion causes a greater loss of activity (see WD2006/G12627),
Amino terminal truncations were made in the background of an AetR1JB(20- 131 )-h.Fc protein. The activities am shown below (as measured in conditioned media); N-termlnal AetRMB-hFe Truncations:
Accordingly, truncations of two,· three or four amino acids from (heN-terminus lead to the production of a more active protein than the versions with a foil-length extracellular domain. Additional experiments show that a truncation of five amino acids, AetR 118(25-131 )-hFc has activity equivalent to the untamcated form, and additional deletions at the N~ terminus continue to degrade the activity of the protein. Therefore, optima! constructs will have a C-terminus ending between amino acid 133-134 of SEQ ID NO; 1 and an N~ terminus beginning at amino acids 22*24 of SEQ 10 NO; 1. An N denuinus corresponding to amino acids 2! or 25 will give activity that is similar to the Act.ROB(20-!.34)-hFc construct, These truncations may also he used in the Context of GDF Traps, such as an L79D or L79E variant.
Example 4, AclRilB-Fe Variants, Cetl-tmed Activity.
Activity of AetRIIB-Fc proteins and GDF Traps was tested so a cell based assay, as described above. Results are sumsoarked -in the table below. Some variants'were tested in different C-terminal truncation -constructs. As· discussed above, truncations of five or fifteen amino acids caused reduction in activity. The GDF Traps {L79D and L79E variants) showed substantial loss of activin binding while retaining almost wild-type inhibition of GDF-11.
Soluble ActRHB-Fc binding to GDF! I and Acttvln A:
•r Poor activity {roughly lx 1 O'* Kj) •r+ Moderate activity (roughly 1x1 £Γ Kt)
Good {.wild-type}.activity'.{roughly IxlD^'K*) f-i-t-f Greater than wild-type activity
Several variants have been assessed for serum half-life in rats, AetRIiB(2{}-134)~Fc has a serum half-life of approximately 70 hours, ActRHB(A24N 20-l34)-Fc has a serum half-life of approximately 100-.1 SO hours. The A24N variant has activity in the cell-based assay (above) and in vivo assays (below) that are .equivalent to the wild-type molecule. Coupled with the longer halRifc, this means that over time an A24N variant will give greater effect per unit of protein than the wild-type molecule. The A24N variant, and any of the other variants'tested above, may be combined with the GDP Trap molecules, such as the L?9D or L79E variants.
Example 5, Gf>E~!l and Actlvia A Binding.
Binding of certain ActRlIB-Fc proteins and GDF Traps to ligands was tested in a BiaCore assay.
The Ac?RUB~fe variants or wild-type protein were captured onto the system using an anti-KFc antibody. Ligands were injected and flowed over the captured receptor proteins. Results are summarized in the tables, below.
Ligand binding specificity IIB variants.
These data confirm the cell based assay data, demonstrating that the A24N variant retains ligand-binding activity that is similar to that of the ActRIIB{20- 134)-hFc molecule, and that the L79D or L79E molecule retains myostatin and G.DF1 1 binding hut. shows markedly decreased (non-quantifiable)'binding to Activin A.
Other variants have been generated and tested, as reported in W02006/01262? (incorporated herein by reference in its entirety) see e.g., pp. 59-60, using ligands coupled to the device and flowing receptor over the coupled ligands. Notably, K74Y, K74F, K741 (and presumably other hydrophobic substitutions at K?4,. such as K.74L), and D80I, cause a decrease in the ratio of A ctivin A binding to CSDP1I binding, relative to the wild-type K.74 molecule, A table of data with respect to these variants is reproduced below;
Soluble AetRlIS-Fe variants binding to GW'!! and Activin A (BlaCore assay)
* No observed binding
- < 1/SWT binding ' - ~ 1/2· WT binding •r WT ' •f-f < 2x increased binding +++ ~5x increased binding ++++ - IOx increased binding e- t-f'5'+ 40x increased binding
Example 6. AetRlIB~hFc Stimulates Eryrhrej>oie$i$ m No»>H«nta«. Primates
AetRl!Bf20~i34)4iFeOgGl) was administered oneea week for 1 month to male and female cynomolgus monkeys by subcutaneous injection. Forty-eight cynomolgus monkeys (24/sex) were assigned to one of four treatment groups (6 animais/sex/group) and were administered subcutaneous injections of either vehicle or ActRliB-hFc at 3,10, or 30 mg/kg once weekly for 4 weeks (total of 5 doses). Parameters evaluated included general clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis), ActtUIB-hFe caused statistically significant elevated mean absolute reticulocyte values by day 15 in treated, animals. By day 36, ActRHB-hFc caused several hematological changes, including elevated mean absolute reticulocyte and fed blood cell distribution width \ slues and lower mean corpuscular hemoglobin concentration. All treated groups and both sexes were affected. These effects are consistent with a positive effect of ActRHB-hFc on the release of immature reticulocytes from the bone marrow. Thi s effect was reversed after drug was washed out of the treated animals (by study day 56)., Accordingly, we conclude that ActRHB-hFc sti.mulates erythropoiesis,
Example 7, A€tRilB~mPe Promotes Aspects of Erythropoiesls In Mice by Stimulation of Splenic Erythropoietic Activities
In this,study the effects of the m -vivo .admi msirsttoh of ActR 118(20-134)-m be on the frequency of hematopoietic progenitors in hone marrow and spleen was analyzed One group οί€5?Β1/6 mice was injected with PBS as a control and a second group of mice administered two doses of AesRIIB-mFc at 10 mg/kg and both groups sacrificed after 8 days. Peripheral blood was used to perform complete blood counts and femurs and spleens were used to perform in vitro do.nogenie assays to assess the .lymphoid, erythroid and myeloid progenitor eel! content in each organ. In the brief time frame of this study, no significant changes were seen in red blood cell, hemoglobin or white blood cell levels in treated mice. In the femurs there was no difference in the nucleated cell numbers or progenitor content between the. control and treated groups. In the spleens, the compound treated group experienced a statistically significant increase in the mature erythroid progenitor (CFli-E) colony number per dish, frequency and total progenitor number per spleen, in addition, and increase was seen in the number of myeloid (CFU-GM), immature erythroid (BFU-E) and total progenitor number per spleen.
Animals;
Sixteen C57BL/6 female mice 6-S weeks of age were used in the study. Eight mice were injected subcutaneously with test compound ActRIlB~mFc at days 1 and 3 at. a dose of 10 mg/kg and eight mice were injected subcutaneously with vehicle control, phosphate buffered saline (PBS},.at a volume of 100 pt per mouse. All mice were .sacrificed' 8 days after first injection in accordance with the relevant Animal Care Guidelines. Peripheral blood (PS) samples from individual animals were collected by cardiac puncture and used for complete blood counts and differential (CBOPill). Femurs and spleens were harvested from each mouse.
Tests performed: CBC/Diff Counts FB from each mouse was collected via cardiac puncture· and placed into the appropriate microtainer tubes. Samples were sent to CLV for analysis on a CcllDyn 3500 counter,
Clonogeuic Assays
Cionogenic. progenitors of the myeloid, erythroid and lymphoid lineages were assessed using the in viira meihylcellulose-based media systems described below.
Mature Erythroid Progenitors:
Clohogenie progenitors of the mature erythroid {CFli-E) lineages were cultured in MethoCuilTM 3334, a methylce1!u!ose~hased:medium containing recombinant human (rh) Erythropoietin (3 DimL),
Lymphoid Progenitors:
Clenogemc progenitors of the lymphoid <CFU*pre-B) lineage were cultured' in MethoCult# 3630, a methyl cellulose-based medium containingrh Interleukin 7 CIO ng/mt).
Myeloid and immature Erythroid Progenitors':
Glonogenic progenitors of the granulocyte-monocyte iCFU-GM)* erythroid (BPU-E) and multipotential (CFU-(SEMM) lineages were cultured In MethoCuItTM 3434, a methylceliuloscrbased medium containing recombinant murine (rm) Stem Cell -Factor (50 ng/mt), rh Interleukin 6 <10 ng/mL)s rm interleukin 3 (10 ng/mt) and rh Erythropoietin (3 0/mL).
Methods:
Mouse femurs and spleens were processed by standard protocols. Briefly, bone marrow was obtained' by flushing the femoral cavity with lscove’s Modified Duibeeco's Media containing 2% fetal bovine serum (1MDM 2% FBS) using a 21 gauge needle and 1 cc syringe. Spleen cells were obtained by crushing spleens through a 70 pM filter and rinsing the filter with 1MDM 2% FBS. Nucleated cell counts in 3% glacial acetic acid were then performed on the single cells suspensions using a Neubaoer counting chamber so that the total cells per organ could he calculated. To remove contaminating red blood cells, total spleen cells were then diluted with 3 times the volume, of ammonium chloride lysis buffer and incubated on ice 10 minutes. The cells were then washed and resuspended in IMDM 2% FBS and a second cell count were performed to determine the cell concentration of cell:', after lysis.
Cell stocks -were made and added to each methylcellulose-based media formulation to obtain the optimal plating concentrations for each tissue in each media formulation. Bone marrow cells were plated at 1x1 if cells per dish in MethoCuftTM 3334 to assess mature erythroid progenitors, 2x103 cells per dish in McthoGultTM 3630 .to assess lymphoid progen dors and 3xl04 cells per dish in MethoCuItTM 3434 to assess ...immature· erythroid and myeloid progenitors. Spleen cells were plated at 4.x 10' cells per dish in MethoCuItTM 3334 to assess mature erythroid progenitors,.4x10' cells per dish in MethoCuItTM 3630 to assess lymphoid progenitors and 2x1(2 cells per dish in MethoCuItTM 3434 to assess immature erythroid and myeloid progenitors. Cultures plated in triplicate dishes were Incubated at 370CS 5% C02 until colony enumeration and evaluation was performed by trained personnel.
Mature erythroid progenitors were cultured for 2 days, lymphoid progenitors were cultured for 7 days and mature erythroid and myeloid progenitors were cultured for 12 days.
Analysis:
The mean +/-1 'standard deviation was calculated for the triplicate cultures of the clonogemc assays and for the control and treatment groups for ail data sets.
Frequency of colony forming cells (CFC) in each tissue was calculated as follows:
Cells plated per dish
Mean CFC scored per dish
Total CFC per femur or spleen was calculated as follows:
Total CFC scored x nucleated cell count per femur·or spleen (following RBC lysis)
Number of nucleated cells cultured
Standard Wests were performed to assess if there was a differences in the mean number of cells or 'hematopoietic progeni tors between the PBS control mice and compound treated mice. Due to the potential subjectivity of colony enumeration, a p value of less than 0.01 is deemed significant. Mean values (+/- SD) for each group are shown in the tables below.
Tablet Hematologic Parameters
Table: CFC From Femur oust Spleen
* preliminary analysis indicates p - 0 0$
Treatment of mice wi th ActROE(20^134)-mFc, in the brief time frame of this study, did not result in significant increases in red blood cell or'hemoglobin content. However, the effect on progenitor cell content was notable. In the femurs there was no difference in the nucleated cell numbers or progenitor content between the control and treated groups. In the spleens., the compound treated group experienced a statistically significant increase in the nucleated, cell number before red blood cell lysis and in the mature erythrok! progenitor (CPU-E) colony number per dish, frequency and total progenitor number per spleen. In addition, an increase was seen in the number of myeloid (CPU--GM), immature erythrokl (BFU-E) and total progenitor number per spleen. Accordingly, it is expected that over a longer time course, .ActROBt 20-13.4)-mFc treatment may result in elevated red blood cell and hemogl obi n con tent,
Example 8: A GOP Trap Increases Med Blood Cell Levels m vivo
Twelve-week-old male C$?BL/6MTac mice were assigned to one of two treatment groups (N~1G). Mice were dosed with either vehicle or with a variant ActRHB polypeptide (“GDF Trap*) [AetRilB{L79D 20-134}~hPcj by subcutaneous injection (SC) at 10 mgr kg twice per week for 4 weeks. At study termination, whole blood was collected by cardiac puncture into BPTA containing tubes and analyzed for cel I distribution using an ,HM2 hematology analyzer (Abaxis. Inc),
Group Designation ....................................................................................................................................
Treatment with the GOF Trap did not have a statistically significant effect on the number of white blood cells i WBC) compared to the vehicle controls. Red blood ceil (REG) numbers were increased in the treated: group relative to the controls (see table below). Both the·hemoglobin content (HGB) and hematocrit (HCT) were also increased due to the additional red blood cells. The average width of the red blood cells (RDWc) was higher in the treated animals, indicating an increase in the pool of immature red blood cells. Therefore, treatment with the GOF Trap leads to increases in red blood cells, with no distinguishable effects on white blood cell populations.
Hematology Bespits..............................................................................................................
Example 9; A CDF Trap is Superior to AciRIlB-Fe for Increasing Red Blood Coll Levels in vim.
Nineteen-week-old male C57BL/6NTae mice were randomly assigned to one of three groups. Mice were dosed with vehicle (10 mfvl Iris Buffered Saline, TBS), wild-type AetRIi8{2(M 34)~mFe* or GD.F trap ActRIIB(L?9D 20~134)-hPc by subcutaneous injection twice per week for three weeks. Blood was collected cheek bleed at baseline and after three weeks of dosing and analyzed for cell distribution using a hematology analyzer (HM2, Abaxis, Inc.)
Treatment with AeiRIIB-Fe or the GDP trap did not have a signi ficant effect on white blood cell (WBC) numbers compared to vehicle controls. The red blood cell count (RBC), hematocrit (HCT), and hemoglobin levels were all elevated in GDF Trap treated mice compared to either the controls or the wild Type construct (sec table below). Therefore, in a direct comparison, the GDF trap promotes increases in red blood cells to a significantly greater extent than a wild-type ActRHB-Fc protein. In fact, in this experiment, the wild-type ActRIlB-Fc protein did not cause a statistically significant increase in red blood cells, suggesting that longer or higher dosing would be needed to reveal this effect.
Hematology Results after three weeks of dosing
Example 10. Generation of a GDF Trap with Truncated ActRIIB Extracellular Domain
As described In Example I» a GDF Trap referred to as ActRHB(L?9D 2G-134)~hFc was generated by N-terminal fusion of TP A leader to the ActRIIB extracellular domain (residues 20-134 in SEQ ID NO: I) containing a leucine-to-aspartate substitution (at residue 79 in SEQ ID NO; I) and C-terminal fusion of human Fc domain with minimal linker (three glycine residues) (Figure 3). A nucleotide sequence corresponding to this fusion protein is shown in Figure 4. A GDF Trap with truncated ActR IIB extracellular domain, referred to as AciRIfB(L79D 25-!31)«hFc, was generated by N-terminal fusion of TP A leader to truncated extracellular domain (residues 25-131 in SEQ ID NO: I) containing a leucine-to-aspartate substitution (at residue 79 in SEQ ID NO: 1) and C-terminal fusion of human Fc domain with minimal linker (three glycine residues) (Figure 5). A nucleotide sequence corresponding to this fusion protein is shown in Figure 6.
Example II. Selective Ligand Binding by GDF Trap with Double-Truncated ActRIIB Extraeeiiuar Domain
The affinity of GDF Traps and other ActRHB-hFc proteins for several ligands was evaluated in vitro with a Biaeore™ instrument. Results are summarized in the table below. ICd values were obtained by steady-state affinity fit due to the very rapid association and . dissociation of the complex, which prevented accurate determination of k„„ and kfify.
Ligand Selectivity of ActRf IB-bFc Variants:
The GDF Trap with a truncated extracellular domain, ActRIiB(L?9D 2'5-!3l)»hFc, equaled or surpassed the ligand selectivity displayed by the longer variant, ActRiIB(L79D 20--1 ViLhFc, with-pronounced loss of activin A and. actwin B binding and nearly foil retention of GDF! 1 binding compared to ActROB-hFc counterparts lacking the L79D substitution. Note that fruneadort alone (without L79D substitution) did not alter selectivity among the ligands displayed here [compare ActRllB(L79 25-l31)~hFc with ActRHB(L79 20“134}“hFc|,
Example 12. Generation of AcfRlIB{L79D 25-13 l)-hFc. with Alternative Nucleotide .
To generate AeiRi!S(L79D 25-.131)-hFe, the human Act RUB extracellular domain with an aspartate-substitution at native position 79 (SEQ ID NO: 1} and with N-terrainaJ and € Terminal truncations (residues 23-131 in SEQ ID NO: 1} was fused N-terminally with a TEA leader sequence instead of the nati ve ActRIIB leader and C-terrninaliy with a human Fc domain via a minimal linker (three glycine residues) (Figure $), One nucleotide sequence encoding this fusion protein is shown in Figure 6 (SEQ ID NO: 27), and an alternative nucleotide sequence encoding exactly the same fusion protein is shown in Figure 9 (SEQ ID NO: 30). This protein was expressed and purified using the methodology described in Example 1,
Example .13, GDF Trap with a Truncated ActRIIB Extracellular Domain Increases Proliferation of Erytlvroicl Progenitors in Mice
AetRlIB{L?9D 25-131 )-hf c was evaluated to determine its effect on proliferation of erythrotd progenitors. Male €578126 mice (8 weeks edd) were treated with ActRHB{L79D 25-131 )-hFc (10 mg/kg, s.c.; π « 6) or vehicle (TBS; n = 6) on Days I and 4, then euthanized on Day 8 for collection of spleens, tibias, lemurs, and blood. Cells of foe spleen md bone marrow were isolated, diluted in fscove's modified Dulbeceo’s medium continuing 5% fetal bovine serum, suspended in specialized methylcelluiose-based medium, and cultured for either 2 or 12 days ίο assess levels of clonogenic progenitors at the colony-forming unii-erythroid (CFU-E) and burst forming umi-eryihroid (BFU-E) stages,, respectively. Methylcellulose-based medium for BFU-E determination (MethoCult M3434, Stem Cell Technologies) included recombinant murine stem cell factor, mter!eukm~3, and interleukin-6, which were not present in methylcellulose medium for CFU-E determination (MethoCult ΚΊ3334, Stem Cell Technologies), while.both media contained erythropoietin, among other constituents. For both BFU-E and CFU -E, the number o f colonies were determined in duplicate culture plates derived from each tissue sample, and statistical analysis of the results was based on the number of mice per treatment group.
Spleen-derived cultures from mice treated with AetRI!B(L79D 25-131)-hFc had twice the number of CFU-E colonies as did corresponding cultures from control mice (P < 0,05), whereas the number of BFU-E colonies did not dit ier significantly with treatment in vivo.
The number-of CFU-E or BFU-E colonies from bone marrow cultures also did not. differ significantly with treatment. As expected, increased numbers of CFU-E colonies in spleen-derived cultures-wereaccompanied by highly significant (P < 0,001) changes in red blood cell level (11.6% increase), hemoglobin concentration {12% increase), and hematocrit level (11.6% increase) at euthanasia in mice treated with ActEllB(L79D 25-13 l)-hFc compared to controls. These results indicate that in vivo administration -of a (IDF Trap with truncated AetRlIB extracellular domain can stimulate proliferation of erythroid progenitors as part of its overall effect to increase red blood cell levels.
Example 14, GF ►F Trap with a Truncated ActRllB Extracellular Domain Offsets 'Chemotherapy-Induced Anemia in Mice
Applicants investigated the effect of ActR 1.1.8(1..790 25-!3t}-hPc on erythropoietic parameters in a mouse model of chemotherapy-induced anemia based on paelitaxel, which •inhibits·cell division by blocking microtubule polymerization·, Male C57BL/6 mice (8 weeks old) were assigned to One of four treatments: 1) paclitaxel (25 mg/kg, i.p.) 2) .AciR118(1.,79.0 25-13 l)-hFc (10 mg/kg:; i.p.) 3) paclitaxel + AeiRIlBf L?9D 25-131 )-hFc 4) vehicle (TBS). .Ifoclitaxel was administered on Day 0, while AciRlI 8{L79D 25-131 )-hFc or vehicle were administered on Days 0 and 3. Blood samples were collected for CBC analysis from separate cohorts, on Days 1, .1, and 5, and results for treatment .groups 1-3 (above) -were -expressed as percent' difference from vehicle at a give» time point. Attrition doe to paclitaxel toxicity was an issue in the paeliiaxei-only cohort on Day 3 (where n = 1); otherwise, n ~ 3-5 per treatment per time point.. Compared to vehicle, paclitaxel alone decreased hemoglobin concentration by nearly 13% at Day 5, whereas addition of ActRfIB(L79D 25-13l)-hFc prevented this paclitaxel-induced-dedine (Figure .11). Similar effects were observed for hematocrit and RBC levels·. In the absence of paclitaxel, AciRllB(L79D 25~131)--hFc increased hemoglobin concentration by 10% compared to vehicle on Days 3 and 5 (Figure II). Thus, a GDP Trap with truncated ActRIlB extracellular domain can increase levels of red blood ceils sufficiently to offset chemotherapy-induced anemia.
Example 15* GDFTrap with a Truncated ActRIlB Extracellular Domain Reverses Nephrectomy*Induced. Anemia in Mice
Applicants Investigated the effect of ActRI.iB(L79D 25-131}~hFc on anemia in a nephrectoraked mouse model of chronic kidney disease. Male C57BL/S mice (11 weeks old) underwent either a sham operation or a unilateral nephrectomy to reduce the capaeity for erythropoietin production. Mice were allowed a week for postsurgica! recovery and then treated twice-weekly with ActRIIB(L79D 25-131)-¾¾ (10 mg/kg, ip.; ns:: 15 per-condition) or vehicle (TBS; n ::: I S per condition) for a total of 4 weeks. Blood samples were collected before the onset of dosing and after 4 weeks of treatment. Whereas vehicle-treated nephrectomixed mice displayed a significant decline in red blood cell number oyer the 4-week treatment period, treatment, with ActRII8(L79D 25-! 3!}~hFe not only prevented the decline but increased ted blood cell levels 17% (:P < 0.001) above baseline (Figure 12), despite reduced renal capacity for erythropoietin production, in oephrectomlzed mice, ActRllB(L79D .25-131 }-hfe also generated significant increases from baseline in hemoglobin concentration and hematocrit level and, notably, stimulated each of these erythropoietic parameters to approximately the same extent under nephrectom?zed conditions as under sham-operated conditions (Figure 13), Thus, a G.D.F Trap with truncated ActRIlB extracellular domain can increase red blood cell levels 'sufficiently to reverse anemia in a model of chronic kidney disease.
Example '16, GBFTrap with a Truncated ActEHB Extracellular Domain I mproves Recovery from Anemia Induced by Blood Loss in Rats
Applicants investigated the effect of ActRHB{L79D 25-13l)-hFc on erythropoietic parameters in a rat model of anemia induced by acute blood loss (acute post-hemorrhagic anemia). Male Sprague-Dawley rats (approximately 300 g} received a chronic jugular catheter at the vendor (Harlan). On Day - L 20% of total blood volume was withdraw?! from each rat over a 5-mmute period via the catheter under isoflurane anesthesia. The volume of blood removed was based on a value· for total blood volume calculated according to the following relationship derived by Lee and co-workers (i Nucl Med 25:72-76, 1985) for rats •with body weight greater than 120 g:
Total blood volume (ml) « 0.062 x body weight (g) -f 0.0012
An equal volume of phosphate-buffered saline was replaced via the catheter at the time of blood removal. Rats were treated with AeiRIlB(.L?9D 25-13!)-hPc (10 mg/kg, sxv, n - 5) or vehicle (TBS; n:::: 5) on Days 0 and 3. Blood sample for CBC analysis were removed via the catheter on Days - I (baseline), 0, 2,4, and 6.
Control rats responded to 20% blood loss with a drop of nearly 15% in red-biood~cel ! levels by Day 0. These levels remained significantly lower than baseline on Days 2 and 4, and had not recovered fully by Day 6 (Figure 14). Although rats treated with ActRliB(L.79D 25-131)~hFe showed a nearly identical drop in red-blood-cell levels after 20% blood loss, these rats then displayed a complete recovery in such levels by Day 2, followed by further elevation on Days 4 and 6, which represents a highly significant improvement over control levels at the corresponding time points (Figure 14). Similar results were obtained for hemoglobin concentration. These findings demonstrate that a GDF Trap with truncated ActRIIB extracellular domain can produce-a faster recovery of red blood cell levels from anemia caused by acute hemorrhage.
Example 17. GDF Trap with Truncated ActfUlB;Extracell»ar Domain Increases Levels of Red Blond Cells in Non-tinman Primates
Two GDF Traps, AetRl 18(0790 20-l34)-hFc and ActRI?B(L?9D 25H3I)-hFe, were evaluated for their ability to stimulate red blood cell production in cynomolgas monkey. Monkeys were treated subcutaneously with GDF Trap (10.mg/kg; n ~ 4 males/4 females), or vehicle (n “ 2 males/2 females)'op Days I and 8. Blood samples .were collected on Days 1 {pretreatment baseline), 3, 8, ? 5, 29» and 44, and were analyzed for red blood cel! levels (Figure ] 5), hematocrit (Figure 16), hemoglobin levels (Figure 17), and reticulocyte levels (Figure 18). Vehicle-treated monkeys exhibited decreased 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 ActRllB(L79D 20-134)-hFc or ActRHB(L79D 2S-!31)-hFe increased these parameters by the first post-treatment time point (Day 3) and maintained them at substantially elevated levels for the duration of the shady (Figures 15-17), importantly, reticulocyte levels in monkeys treated with ActRIIB(L79D 20-134)-hFe or ActRllB(L79D 25-131 )~hFe were substantially increased at Days 8» 1-5,.and 29 compared to vehicle (Figure 18). This result demonstrates that GDP Trap treatment increased production of red blood cell precursors-, resulting in elevated red blood cell levels.
Taken together, these data demonstrate that truncated GDF Traps, as well as a full-length variants, can be used as selective antagonists of GDP 11 and potentially related ligands to increase red blood cell formation in vivo.
Example ML GDF Trap Derived from ActEtlBS
Others have reported an alternate, soluble form of AetRIIB (designated AciRlIBS), in which exon 4, including the AetRIIB transmembrane domain, has been replaced by a different C-terminai sequence (WG2007/05377S),
The sequence of native human ActRHBS without its leader is as follows: GRGEAETRECIYYRANBELERTEQSGITRCEGgQDKRldiCYASERbSSGTIELVK KGCWLDDFNC¥DPQECVATEEGPQVYFCCC EGE FCEERFTH LFEAGG PEGPW AST T X P S GG P EATAAAG DQG S G ALWIXL EG PA H £ (SEQ ID MO: 36)
An.leudneTp-aspartate substitution, or other acidic substitutions, may be performed at"native position 79 (underlined and highlighted) as described to construct the variant A.ctRil85(L79P), which has dm folio wing sequence: GRGEAETRECIYYHAEWELEETEDSGLEBGEGEQDKRLHCYASWRESSGTIELVK KGC7«DDDEECYDRQEC¥ATEEMEQVYFCCCEGEFCEERrTHLPEAGGFEGFWAST Τ,ϊP S GG P EA T A A.AG DQG S G A L WLC L EG P A Η E (SEQ ID NO; 37)
This variant may be connected to human Fc with a TGGG linker to generate a human ActRIIB5fL79D)-hFe fusion protein· with the following sequence: GRGSAETRECIYYNANhfSLERTNQSGLERCEGEQDKRLHCY^SWBNSSGTIELVK KGCWGDDFECYDRGECVATEENPgVYECCCEGKECGERETHLPEAGGPEGPbiAST TIP S G G PEATAAAG DQG 3 G ALW LC L EG PAH ETGGGT H TC P P CPA P EL LG G P 3 V FI. FPFKPKDTLMrSRTPEVTCyWDVSfi£OPEVKFNWYV.DGVEVHNAKTKPRE£QYN STYRVVSVLTVLEQDWLEGKEYECKVSEKALPAPIEKTISKAKGQPREPQPYTLP PSGsEEhffKMQySLTClPv KG ΓΥ PS DlAVE^FiSNGQPENEYKTTPPVLDSDGSF’FEY SKLTVDKGBYiQQGMVTSCSVPiHEALHNHYTQKSLSLSPGK (SEQ ID NO; 38}
This construct may be expressed in CHO cells.
INCORPORATION BY REFERENCE AH publications and patents mentioned, herein are hereby incorporated by reference in their entirely as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
While specific embodiments of the subject matter have been discussed, the above specification is illustrative-and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The-full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations..

Claims (42)

  1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
    1. A method for treating a hemoglobinopathy in a patient in need thereof, the method comprising administering to the patient an effective amount of a polypeptide comprising an amino acid sequence that is at least 95% identical to the sequence of amino acids 29-109 of SEQ ID NO: 1, wherein the polypeptide comprises an acidic amino acid at the position corresponding to position 79 of SEQ ID NO:1, and wherein the polypeptide is capable of inhibiting signaling by myostatin and/or GDF11 in a cell-based assay.
  2. 2. The method of claim 1, wherein the hemoglobinopathy is sickle-cell disease.
  3. 3. The method of claim 1, wherein the hemoglobinopathy is thalassemia.
  4. 4. The method of claim 1, wherein the acidic amino acid an aspartic acid.
  5. 5. The method of claim 1, wherein the acidic amino acid is a glutamic acid.
  6. 6. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the sequence of amino acids 29-109 of SEQ ID NO: 1.
  7. 7. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of amino acids 25-118 of SEQ ID NO: 1.
  8. 8. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of amino acids 25-128 of SEQ ID NO: 1.
  9. 9. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 99% identical to the sequence of amino acids 25-131 of SEQ ID NO: 1.
  10. 10. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of amino acids 25-131 of SEQ ID NO: 1.
  11. 11. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the sequence of amino acids 25-131 of SEQ ID NO: 1.
  12. 12. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 29.
  13. 13. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 29.
  14. 14. The method of any one of claims 1-4, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 29.
  15. 15. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 32.
  16. 16. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 32.
  17. 17. The method of any one of claims 1 -5, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:32.
  18. 18. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 37.
  19. 19. The method of any one of claims 1 -5, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 37.
  20. 20. The method of any one of claims 1 -5, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 37.
  21. 21. The method of any one of claims 1 -20, wherein said polypeptide further comprises one or more polypeptide portions that enhance one or more of: in vivo stability, in vivo half life, administration, tissue localization or distribution, formation of protein complexes, and purification.
  22. 22. The method of claim 21, wherein said polypeptide portion comprises a constant region derived from an IgG heavy chain.
  23. 23. The method of claim 22, wherein the constant region derived from an IgG heavy chain is an Fc domain.
  24. 24. The method of any one of claims 21-23, wherein the polypeptide comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7.
  25. 25. The method of any one of claims 21-23, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7.
  26. 26. The method of any one of claims 21-23, wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 7.
  27. 27. The method of any one of claims 21-23, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 7.
  28. 28. The method of any one of claims 21-23, wherein the polypeptide comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 28.
  29. 29. The method of any one of claims 21-23, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 28.
  30. 30. The method of any one of claims 21-23, wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 28.
  31. 31. The method of any one of claims 21-23, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 28.
  32. 32. The method of any one of claims 21-23, wherein the polypeptide comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 38.
  33. 33. The method of any one of claims 21-23, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 38.
  34. 34. The method of any one of claims 21-23, wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 38.
  35. 35. The method of any one of claims 21-23, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 38.
  36. 36. The method of any one of claims 1 -35, wherein the polypeptide further comprises one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent.
  37. 37. The method of any one of claims 1 -36, wherein the polypeptide binds to GDF11.
  38. 38. The method of any one of claims 1 -36, wherein the polypeptide binds to GDF8.
  39. 39. The method of any one of claims 1 -36, wherein the polypeptide binds to GDF11 and GDF8.
  40. 40. Use of a polypeptide comprising an amino acid sequence that is at least 95% identical to the sequence of amino acids 29-109 of SEQ ID NO: 1, wherein the polypeptide comprises an acidic amino acid at the position corresponding to position 79 of SEQ ID NO:1, and wherein the polypeptide is capable of inhibiting signaling by myostatin and/or GDF11 in a cell-based assay, for the manufacture of a medicament for treating a hemoglobinopathy.
  41. 41. The use of claim 40, wherein the hemoglobinopathy is sickle-cell disease.
  42. 42. The use of claim 40, wherein the hemoglobinopathy is thalassemia.
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Citations (2)

* Cited by examiner, † Cited by third party
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WO2008076437A2 (en) * 2006-12-18 2008-06-26 Acceleron Pharma Inc. Activin-actrii antagonists and uses for increasing red blood cell levels
WO2008097541A2 (en) * 2007-02-02 2008-08-14 Acceleron Pharma Inc. Variants derived from actriib and uses therefor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008076437A2 (en) * 2006-12-18 2008-06-26 Acceleron Pharma Inc. Activin-actrii antagonists and uses for increasing red blood cell levels
WO2008097541A2 (en) * 2007-02-02 2008-08-14 Acceleron Pharma Inc. Variants derived from actriib and uses therefor

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