OA19001A - Methods and compositions for treating ulcers - Google Patents

Methods and compositions for treating ulcers Download PDF

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OA19001A
OA19001A OA1201600471 OA19001A OA 19001 A OA19001 A OA 19001A OA 1201600471 OA1201600471 OA 1201600471 OA 19001 A OA19001 A OA 19001A
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polypeptide
amino acid
acid sequence
seq
activin
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OA1201600471
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Kenneth M. Attie
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Acceleron Pharma, Inc
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Abstract

The present disclosure provides compositions and methods for treating or preventing ulcers in subjects having low red blood cell levels and/or hemoglobin levels (e.g, anemia). In some embodiments, the compositions of the disclosure may be used to treat or prevent ulcers associated with anemia.

Description

METHODS AND COMPOSITIONS FOR TREATING ULCERS
RELATED APPLICATIONS
Thîs application daims the benefit of priority to ILS. Provisîonal Application Serial Nos., 62/012,109, fîled June 13, 2014, and 62/045,808, filed September 4, 2014. The disclosures of each of the foregoing applications are hereby incorporated in their entirety.
BACKGROUND OF THE INVENTION
The mature red blood cell, or érythrocyte, is responsible for oxygen transport in the circulatory Systems of vertebrates. Red blood cells contain high concentrations of hemoglobin, a protein that binds to oxygen in the lungs at relatively high partial pressure of oxygen (pO2) and delivers oxygen to areas of the body with a relatively low pO2.
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 controls the balance of cell prolifération, différentiation, 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 stîmuli, 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 senes of distinct precursor cell types. The final stages of erythropoiesis occur as réticulocytes are released into the bloodstream and lose their mitochondrîa and ribosomes while assuming the morphology of mature red blood cell. An elevated level of réticulocytes, or an elevated reticulocyte:erythrocyte ratio, in the blood is indicative of increased red blood cell production rates.
In general, anémia is a condition that develops when a subject s blood lacks enough healthy red blood cells or less than the normal quantity of hemoglobin. Anémia may also be diagnosed when there is decreased oxygen-binding capacity of red blood cells, which may resuit from a deformity in one or more hemoglobin subunits. As human cells dépend on oxygen for survival, anémia can resuit in a wide range of clinical complications including, e.g., tissue damage. For example, it has been reported that ulcers are the one of most common cutaneous manifestation of chronic anémia disorders, particularly in hemolytic anémias such as sickle-cell disease and thalassemia. See, e.g., Keast et al, (2004) Ostomy
-i19001
Wound Manage., 50(10): 64-70; Trent et al. (2004) Adv Skin Wound Care, 17(8): 410-416;
J.R. Eckman (1996) Hematol Oncol Clin North Am„ 10(6): 1333-1344; and Rassi et al. (2008) Pédiatrie Annals 37(5): 322-328. The underlying mechanism for ulcer formation in anémie patients has not been completely defined. However, it is believed that multiple 5 complications of anémia contribute to ulcer development including, for example, ischemia, decreased nitric oxide bioavailability, vascular obstruction, thrombosis, and hypoxia. Id.
Ulcer healing in anémie patients is typically a slow process, and such patients are also at a high risk of récurrent ulcération. See, e.g., Keast et al. (2004) Ostomy Wound Manage., 50(10): 64-70; Trent et al. (2004) Adv Skin Wound Care, 17(8): 410-416; J.R. Eckman (1996) 10 Hematol Oncol Clin North Am„ 10(6): 1333-1344; and Rassi et al. (2008) Pédiatrie Annals 37(5): 322-328. Furthermore, most thérapies hâve had limited success in the treatment of ulcers occurring in anémie patients.
Thus, it is an object of the présent disclosure to provide alternative methods for treating or preventing ulcers associated with anémia.
SUMMARY OF THE INVENTION
In part, the présent disclosure demonstrates that ActR.II antagonists can be used to alter various blood parameters (e.g., red blood cell levels, hemoglobin levels, iron levels, bilirubin levels, nitrogen levels, etc.) in patients that hâve anémia as well as treat 20 complications associated with anémia including, for example, ulcers. In particular, the disclosure demonstrates that administration of a GDF Trap polypeptide, which is soluble form of an ActRIIB polypeptide having an acidic amino acid at position 79 with respect to instant SEQ ID NO: 1, increases red blood cell levels and/or hemoglobin levels in patients having various types of hemolytic anémia, particularly the hemoglobinopathic anémias, 25 thalassemia and sickle-cell disease. Surprisingly, in addition to directly affecting various red blood cell parameters, the disclosed ActRH antagonist améliorâtes other complications associated with anémia. For example, treatment with a GDF Trap protein was shown to increase hemoglobin levels and promote wound healing of a cutaneous (skin) ulcer in a human patient having thalassemia. ïn some instances, amelioration of these associated 30 complications is of equal or greater importance to patient health and quality of life as the treatment of the underlying anémia. Therefore, in certain embodiments, the disclosure provides methods of using one or more ActRII antagonists to increase red blood cell levels
-219001 and/or hemoglobin levels in patients in need thereof and to treat or prevent one or more complications associated with low red blood cell levels and/or hemoglobin levels in these patients. In particular, the disclosure provides methods for treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject in need thereof that has low levels of red blood cells and/or hemoglobin or is otherwise classified as a subject having an anémia [e.g., hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseôphosphate dehydrogenase deficiency, sickle-cell disease, thalassemia (both alpha and beta), and paroxysmal noctumal hemoglobinuria] by administering one or more ActRII antagonists. In some embodiments, the disclosure provides methods for treating an ulcer, particularly a cutaneous ulcer, in a subject in need thereof that has low levels of red blood cells and/or hemoglobin or is otherwise classified as a subject having an anémia [e.g., hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseô-phosphate dehydrogenase deficiency, sickle-cell disease, thalassemia (both alpha and beta), and paroxysmal noctumal hemoglobinuria] by administering one or more ActRII antagonists. In some embodiments, the disclosure provides methods for preventing an ulcer, particularly a cutaneous ulcer, in a subject in need thereof that has low levels of red blood cells and/or hemoglobin or is otherwise classified as a subject having an anémia [e.g., hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseô-phosphate dehydrogenase deficiency, sickle-cell disease, thalassemia (both alpha and beta), and paroxysmal noctumal hemoglobinuria] by administering one or more ActRII antagonists. In some embodiments, the methods of the disclosure relate to treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a hemolytic anémia by administering one or more ActRII antagonists. In some embodiments, the methods of the disclosure relate to treating an ulcer, particularly a cutaneous ulcer, in a subject that has a hemolytic anémia by administering one or more ActRII antagonists. In some embodiments, the methods of the disclosure relate to preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a hemolytic anémia by administering one or more ActRII antagonists. In particular, the methods of the disclosure relate, in part, to methods of treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a hemoglobinopathy anémia by administering one or more ActRII antagonists. In some embodiments, the methods of the disclosure relate to methods of treating an ulcer, particularly a cutaneous ulcer, in a subject that has a hemoglobinopathy anémia by administering one or more ActRII antagonists. In some embodiments, the methods of the disclosure relate to methods of preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a hemoglobinopathy anémia by
-319001 administering one or more ActRII antagonists. For example, the présent disclosure relates, in part, to methods of treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a thalassemia syndrome by administering one or more ActRII antagonists. In some embodiments, the présent disclosure relates to methods of treating an ulcer, particularly a cutaneous ulcer, in a subject that has a thalassemia syndrome by administering one or more ActRII antagonists. In some embodiments, the présent disclosure relates to methods of preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a thalassemia syndrome by administering one or more ActRII antagonists. In some embodiments, the présent disclosure relates to methods of treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject that has sîckle-cell disease by administering one or more ActRII antagonists. In some embodiments, the présent disclosure relates to methods of treating an ulcer, particularly a cutaneous ulcer, in a subject that has sickle-cell disease by administering one or more ActRII antagonists. In some embodiments, the présent disclosure relates to methods of preventing an ulcer, particularly a cutaneous ulcer, in a subject that has sickle-cell disease by administering one or more ActRII antagonists. In certain aspects, one or more ActRII antagonists can be used in combination with one or more existing supportive thérapies for treating or preventing ulcers and/or treating anémia (e.g., supportive thérapies for treating sickle-cell disease, thalassemia, etc.). Examples of such supportive thérapies are well known in the art and also described herein. In some embodiments, the subject is a transfusion dépendent subject having anémia. In some embodiments, the subject is a non-transfusion dépendent subject having anémia.
In part, the disclosure provides methods of treating ulcers associated with anémia, particularly treating or preventing cutaneous (skin) ulcers, with one or more ActRII antagonists. In some embodiments, the disclosure provides methods of treating ulcers associated with anémia, particularly treating cutaneous (skin) ulcers, with one or more ActRII antagonists. In part, the disclosure provides methods of preventing ulcers associated with anémia, particularly preventing cutaneous (skin) ulcers, with one or more ActRII antagonists. ActRII antagonists of the disclosure include, for example, agents that can inhibit ActRII receptor (e.g., an ActRIIA and/or ActRIIB receptor) mediated activation of a signal transduction pathway (e.g., activation of signal transduction via intracellular mediators, such as SMAD l, 2, 3, 5, and/or 8); agents that can inhibit one or more ActRII ligands (e.g., activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, Nodal, etc.) from, e.g., binding to and/or activating an ActRII receptor; agents that inhibit expression
-419001 (e.g., transcription, translation, cellular sécrétion, or combinations thereof) of an ActRII ligand and/or an ActRII receptor; and agents that can inhibit one or more intracellular mediators of the ActRII signaling pathway (e.g·, SMADs l, 2, 3, 5, and/or 8).
In certain embodiments, the disclosure relates to one or more ActRII antagonists for use in a method to increase red blood cell levels and/or hemoglobin levels in patients in need thereof and to treat or prevent one or more complications associated with low red blood cell levels and/or hemoglobin levels in these patients. In particular, the disclosure provides ActRII antagonists for use in treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject in need thereof that has low levels of red blood cells and/or hemoglobin or is otherwise classified as a subject having an anémia [e.g., hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseô-phosphate dehydrogenase deficiency, sicklecell disease, thalassemia (both alpha and beta), and paroxysmal nocturnal hemoglobinuria]. In some embodiments, the disclosure provides ActRII antagonists for use in treating an ulcer, particularly a cutaneous uicer, in a subject in need thereof that has low levels of red blood cells and/or hemoglobin or is otherwise classified as a subject having an anémia [e.g., hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseôphosphate dehydrogenase deficiency, sickle-cell disease, thalassemia (both alpha and beta), and paroxysmal nocturnal hemoglobinuria]. In some embodiments, the disclosure provides ActRII antagonists for use in preventing an ulcer, particularly a cutaneous ulcer, in a subject in need thereof that has low levels of red blood cells and/or hemoglobin or is otherwise classified as a subject having an anémia [e.g., hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseô-phosphate dehydrogenase deficiency, sicklecell disease, thalassemia (both alpha and beta), and paroxysmal nocturnal hemoglobinuria]. In some embodiments, the ActRII antagonists of the disclosure are for use in treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a hemolytic anémia. In some embodiments, the ActRII antagonists of the disclosure are for use in treating an ulcer, particularly a cutaneous ulcer, in a subject that has a hemolytic anémia. In some embodiments, the ActRII antagonists of the disclosure are for use in preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a hemolytic anémia. In particular, the
ActRII antagonists of the disclosure are for use in, in part, treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a hemoglobinopathy anémia. In some embodiments, the ActRII antagonists of the disclosure are for use in treating an ulcer, particularly a cutaneous ulcer, in a subject that has a hemoglobinopathy anémia. In some
-519001 embodiments, the ActRII antagonists of the disclosure are for use in preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a hemoglobinopathy anémia. For example, the présent disclosure relates, in part, to one or more ActRII antagonists for use in treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a thalassemia syndrome. In some embodiments, the présent disclosure relates to one or more ActRII antagonists for use in treating an ulcer, particularly a cutaneous ulcer, in a subject thaï has a thalassemia syndrome. In some embodiments, the présent disclosure relates to one or more ActRII antagonists for use in preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a thalassemia syndrome. In some embodiments, the présent disclosure relates to one or more ActRII antagonists for use in treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject that has sickle-cell disease. In some embodiments, the présent disclosure relates to one or more ActRII antagonists for use in treating an ulcer, particularly a cutaneous ulcer, in a subject that has sickle-cell disease. In some embodiments, the présent disclosure relates to one or more ActRII antagonists for use in preventing an ulcer, particularly a cutaneous ulcer, in a subject that has sickle-cell disease. In certain aspects, one or more ActRII antagonists can be used in combination with one or more existing supportive thérapies for treating or preventing ulcers and/or treating anémia (e.g., supportive thérapies for treating sickle-cell disease, thalassemia, etc.). Examples of such supportive thérapies are well known in the art and also described herein.
In part, the disclosure provides one or more ActRII antagonists for use in treating ulcers associated with anémia, particularly treating or preventing cutaneous (skin) ulcers. In some embodiments, the disclosure provides one or more ActRII antagonists for use in treating ulcers associated with anémia, particularly treating cutaneous (skin) ulcers, with one or more ActRII antagonists. In part, the disclosure provides one or more ActRII antagonists for use in preventing ulcers associated with anémia, particularly preventing cutaneous (skin) ulcers. ActRII antagonists of the disclosure include, for example, agents that can inhibit ActRII receptor (e.g., an ActRlIA and/or ActRIIB receptor) mediated activation of a signal transduction pathway (e.g., activation of signal transduction via intracellular mediators, such as SMAD l, 2, 3, 5, and/or 8); agents that can inhibit one or more ActRII ligands (e.g., activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, Nodal, etc.) from, e.g., binding to and/or activating an ActRII receptor; agents that inhibit expression (e.g., transcription, translation, cellular sécrétion, or combinations thereof) of an ActRII
-619001 ligand and/or an ActRII receptor; and agents that can inhibit one or more intracellular mediators of the ActRII signaling pathway (e.g., SMADs l, 2, 3, 5, and/or 8).
In certain embodiments, the disclosure relates to the use of one or more ActRII antagonists in the manufacture of a médicament for increasing red blood cell levels and/or 5 hemoglobin levels in patients in need thereof and for treating or preventing one or more complications associated with low red blood cell levels and/or hemoglobin levels in these patients. In particular, the disclosure provides the use of one or more ActRII antagonists in the manufacture of a médicament for treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject in need thereof that has low levels of red blood cells and/or hemoglobin or 10 is otherwise classified as a subject having an anémia [e.g., hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseô-phosphate dehydrogenase deficiency, sickle-cell disease, thalassemia (both alpha and beta), and paroxysmal nocturnal hemoglobinuria], In some embodiments, the disclosure provides the use of one or more ActRII antagonists in the manufacture of a médicament for treating an ulcer, particularly a 15 cutaneous ulcer, in a subject in need thereof that has low levels of red blood cells and/or hemoglobin or is otherwise classified as a subject having an anémia [e.g., hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseô-phosphate dehydrogenase deficiency, sickle-cell disease, thalassemia (both alpha and beta), and paroxysmal nocturnal hemoglobinuria]. In some embodiments, the disclosure provides the 20 use of one or more ActRII antagonists in the manufacture of a médicament for preventing an ulcer, particularly a cutaneous ulcer, in a subject in need thereof that has low levels of red blood cells and/or hemoglobin or is otherwise classified as a subject having an anémia [e.g., hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseôphosphate dehydrogenase deficiency, sickle-cell disease, thalassemia (both alpha and beta), 25 and paroxysmal nocturnal hemoglobinuria]. In some embodiments, the disclosure provides the use of one or more ActRII antagonists in the manufacture of a médicament for treating or preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a hemolytic anémia. In some embodiments, the disclosure provides the use of one or more ActRII antagonists in the manufacture of a médicament for treating an ulcer, particularly a cutaneous ulcer, in a 30 subject that has a hemolytic anémia. In some embodiments, the disclosure provides the use of one or more ActRII antagonists in the manufacture of a médicament for preventing an ulcer, particularly a cutaneous ulcer, in a subject that has a hemolytic anémia. In particular, the disclosure provides the use of one or more ActRII antagonists in the manufacture of a
-719001 médicament for, in part, treating or preventing an ulcer, particuiarly a cutaneous ulcer, in a subject that has a hemoglobinopathy anémia. In some embodiments, the disclosure provides the use of one or more ActRIl antagonists in the manufacture of a médicament for treating an ulcer, particuiarly a cutaneous ulcer, in a subject that has a hemoglobinopathy anémia. In 5 some embodiments, the disclosure provides the use of one or more ActRIl antagoniste m the manufacture of a médicament for preventing an ulcer, particuiarly a cutaneous ulcer, in a subject that has a hemoglobinopathy anémia. For example, the présent disclosure relates, in part, to the use of one or more ActRIl antagonists in the manufacture of a médicament for treating or preventing an ulcer, particuiarly a cutaneous ulcer, in a subject that has a thalassemia syndrome. In some embodiments, the disclosure provides the use of one or more ActRIl antagonists in the manufacture of a médicament for treating an ulcer, particuiarly a cutaneous ulcer, in a subject that has a thalassemia syndrome by administering one or more ActRIl antagonists. In some embodiments, the disclosure provides the use of one or more ActRIl antagonists in the manufacture of a médicament for preventing an ulcer, particuiarly a cutaneous ulcer, in a subj ect that has a thalassemia syndrome. In some embodiments, the disclosure provides the use of one or more ActRIl antagonists in the manufacture of a médicament for treating or preventing an ulcer, particuiarly a cutaneous ulcer, in a subject that has sickle-cell disease. In some embodiments, the disclosure provides the use of one or more ActRIl antagonists in the manufacture of a médicament for treating an ulcer, particuiarly a cutaneous ulcer, in a subject that has sickle-cell disease. In some embodiments, the disclosure provides the use of one or more ActRIl antagonists in the manufacture of a médicament for preventing an ulcer, particuiarly a cutaneous ulcer, in a subject that has sickle-cell disease. In certain aspects, one or more ActRIl antagonists can be used in combination with one or more existing supportive thérapies for treating or preventing ulcers and/or treating anémia (e.g., supportive thérapies for treating sickle-cell disease, thalassemia, etc.). Examples of such supportive thérapies are well known in the art and also described herein.
In part, the disclosure provides the use of one or more ActRIl antagonists in the manufacture of a médicament for treating ulcers associated with anémia, particuiarly treating 30 or preventing cutaneous (skin) ulcers. In some embodiments, the disclosure provides the use of one or more ActRIl antagonists in the manufacture of a médicament for treating ulcers associated with anémia, particuiarly treating cutaneous (skin) ulcers, with one or more ActRIl antagonists. In part, the disclosure provides the use of one or more ActRIl antagonists in the
-819001 manufacture of a médicament for preventing ulcers associated with anémia, particularly preventing cutaneous (skin) ulcers. ActR.II antagonists of the disclosure include, for example, agents that can inhibit ActRII receptor (e.g., an ActRIlA and/or ActRIIB receptor) mediated activation of a signal transduction pathway (e.g., activation of signal transduction via intracellular mediators, such as SMAD 1,2,3, 5, and/or 8); agents that can inhibit one or more ActRII ligands (e.g., activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, Nodal, etc.) from, e.g., binding to and/or activating an ActRII receptor; agents that inhibit expression (e.g., transcription, translation, cellular sécrétion, or combinations thereof) of an ActRII ligand and/or an ActRII receptor; and agents that can inhibit one or more intracellular mediators of the ActRII signaling pathway (e.g., SMADs 1, 2, 3, 5, and/or 8).
In certain embodiments, ActRII antagonists to be used in accordance with the methods disclosed herein are agents that bind to and/or inhibit GDF11 and/or GDF8 (e.g., an agent that inhibits GDF11- and/or GDF8-mediated activation of ActRIlA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). Such agents are referred to collectively as GDF-ActRII antagonists. Optionally, such GDF-ActRII antagonists may further inhibit one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal. Therefore, in some embodiments, the disclosure provides methods of using one or more ActRII antagonists, including, for example, soluble ActRIlA polypeptides, soluble
ActRIIB polypeptides, GDF Trap polypeptides, anti-ActRIIA antibodles, anti-ActRIIB antibodies, anti-ActRII ligand antibodies (e.g, anti-GDFl 1 antibodies, anti-GDF8 antibodies, anti-activin A antibodies, anti-activin B antibodies, anti-activin AB antibodies, anti-activin C antibodies, anti-activin E antibodies, anti-BMP6 antibodies, anti-BMP7 antibodies, and antiNodal antibodies), small molécule inhibitors of ActRIlA, small molécule inhibitors of
ActRIIB, small molécule inhibitors of one or more ActRII ligands (e.g., activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, Nodal, etc.), inhibitor nucléotides of ActRIlA, inhibitor nucléotides of ActRIIB, inhibitor nucléotides of one or more ActRII ligands (e.g., activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, Nodal, etc.), or combinations thereof, to increase red blood cell levels and/or hemoglobin levels in a subject in need thereof, treat or prevent an anémia in a subject in need thereof, and/or treat or prevent ulcers, particularly cutaneous ulcers, in a subject that has anémia. In certain embodiments, ActRII antagonists to be used in accordance with the methods disclosed herein bind activin A or acitivin B. In certain embodiments, ActRII
-919001 antagonists to be used in accordance with the methods disclosed herein bind activîn A . In certain embodiments, ActRII antagonists to be used in accordance with the methods disclosed herein bind activin B. In certain embodiments, ActRII antagonists to be used in accordance with the methods disclosed herein do not substantially bind to and/or inhibit activin A (e.g., activin A-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
In part, the présent disclosure demonstrates that an ActRII antagonist comprising a variant, extracellular (soluble) ActRIIB domain that binds to and inhibits GDFl l activity (e.g., GDFl l-mediated ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling) may be used to increase red blood cell levels in vivo, treat anémia resulting from varîous conditions/disorders, and treat a cutaneous ulcer in a patient with anémia. Therefore, in certain embodiments, ActRII antagonists to be used in accordance with the methods disclosed herein [e.g., methods of increasing red blood cell levels in a subject in need thereof, methods of treating anémia in a subject in need thereof, methods of treating or preventing one or more complications of anémia (particularly ulcers) in subject in need thereof, etc.] are soluble ActRII polypeptides (e.g., soluble ActRIIA or ActRIIB polypeptides) that bind to and/or inhibit GDFl l (e.g., GDFl 1-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). While soluble ActRIIA and soluble ActRIIB ActRII antagonists may affect red blood cell formation and ulcers through a mechanism other than GDFl 1 antagonism, the disclosure nonetheless demonstrates that désirable therapeutic agents, with respect to the methods disclosed herein, may be selected on the basis of GDFl 1 antagonism or ActRII antagonism or both. Optionally, such soluble ActRII polypeptide antagonists may further bind to and/or inhibit GDF8 (e.g. inhibit GDF8mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). In some embodiments, soluble ActRIIA and ActRIIB polypeptides of the disclosure that bind to and/or inhibit GDFl 1 and/or GDF8 may further bind to and/or inhibit one or more additional ActRII ligands selected from: activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal.
In certain aspects, the présent disclosure provides GDF Traps that are variant ActRII polypeptides (e.g., ActRIIA and ActRIIB polypeptides), including ActRII polypeptides having amino- and carboxy-terminal truncations and/or other sequence alterations (one or more amino acid substitutions, additions, délétions, or combinations thereof). Optionally, GDF Traps of the invention may be designed to preferentially antagonize one or more ligands
-1019001 of ActRII receptors, such as GDF8 (also called myostatin), GDFl l, Nodal, BMP6, and BMP7 (also called OP-l). As disclosed herein, examples of GDF Traps include a set of variants derived from ActRIIB that hâve greatly diminished affinity for activin, particularly activîn A. These variants exhibit désirable 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, GDF Traps to be used in accordance with the methods disclosed herein [e.g., methods of increasing red blood cell levels in a subject in need thereof, methods of treating anémia in a subject in need thereof, methods of treating or preventing one or more complications of anémia (particularly ulcers) in subject in need thereof, e/c.] bind to and/or inhibit GDFl 1. Optionally, such GDF Traps may further bind to and/or inliibit GDF8. In some embodiments, GDF Traps that bind to and/or inhibit GDFl 1 and/or GDF8 may further bind to and/or inhibit one or more additional ActRII ligands (e.g., activin B, activin E, BMP6, BMP7, and Nodal). In some embodiments, GDF Traps to be used in accordance with the methods disclosed herein do not substantially bind to and/or inhibit activin A (e.g., activin A-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). In certain embodiments, a GDF Trap polypeptide comprises an amino acid sequence that comprises, consists of, or consists essentially of, the amino acid sequence of SEQ ID NOs: 36, 37, 41, 44, 45, 50 or 51, and polypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing. In other embodiments, a GDF Trap polypeptide comprises an amino acid sequence that comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, 10, 11, 22, 26, 28, 29, 31, or 49, and polypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing. In still other embodiments, a GDF Trap polypeptide comprises an amino acid sequence that comprises of the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, 29, 31, or 49, and polypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing, wherein the position corresponding to 79 in SEQ ID NO: 1, 4, or 50 is an acidic amino acid. A GDF Trap may include a functional fragment of a naturel ActRII polypeptide, such as one comprising at least 10, 20, or 30 amino acids of a sequence selected from SEQ ID NOs: 1, 2, 3,4, 5, 6, 9, 10,11, or 49 or a sequence of SEQ ID NO: 2, 5, 10, 11, or 49 lacking the C-terminal 1,2, 3, 4, 5 or 10 to 15 amino acids and lacking 1, 2, 3, 4 or 5 amino acids at the N-terminus. In some embodiments, a polypeptide will comprise a truncation relative to SEQ ID NO: 2 or 5 of between 2 and 5 amino acids at the N-terminus and no more than 3 amino acids at the C-terminus. In some
-1119001 embodiments, a GDF Trap for use in accordance with the methods disclosed herein consists of, or consists essentially of, the amino acid sequence of SEQ ID NO.36.
Optionally, a GDF Trap comprising an altered ActRIl ligand-binding domain has a ratio of Kd for activin A binding to Kd for GDFl 1 and/or GDF8 binding that is at least 2-, 5-, 10-, 20, 50-, 100- or even 1000-fold greater relative to the ratio for the wild-type ligandbinding domain. Optionally, the GDF Trap comprising an altered ligand-binding domain has a ratio of IC50 for inhibiting activin A to IC50 for inhibiting GDFl 1 and/or GDF8 that is at least 2-, 5-, 10-, 20-, 25- 50-, 100- or even 1000-fold greater relative to the wild-type ActRIl ligand-binding domain. Optionally, the GDF Trap comprising an altered ligand-binding domain inhibits GDFl 1 and/or GDF8 with an IC50 at least 2, 5, 10, 20, 50, or even 100 times less than the IC50 for inhibiting activin A. These GDF Traps can be fusion prolcins that include an immunoglobulin Fc domain (either wild-type or mutant). In certain cases, the subject soluble GDF Traps are antagonists (inhibitors) of GDF8 and/or GDFl 1.
In certain aspects, the disclosure provides GDF Traps which are soluble ActRIIB polypeptides comprising an altered ligand-binding (e.g., GDFl 1-binding) 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, K55, R56, Y60, A64, K74, W78, L79, D80, F82 and Fl01 of human ActRIIB (numbering is relative to SEQ ID NO: 1). Optionally, the altered ligand-binding domain can hâve increased selectivity for a ligand such as GDF8/GDF11 relative to a wild-type ligand-binding domain of an ActRIIB receptor. To illustrate, these mutations are demonstrated herein to încrease the selectivity oi the altered ligand-binding domain for GDFl 1 (and therefore, presumably, GDF8) over activin: K.74Y, K74F, K.741, L79D, L79E, and D80I. The following mutations hâve the reverse effect, increasing the ratio of activin binding over GDFl 1 : D54A, K55A, L79A and F82A. The overall (GDFl 1 and activin) binding activity can be increased by inclusion of the “tail” région or, presumably, an unstructured linker région, and also by use of a K74A mutation. Other mutations that caused an overall decrease in ligand binding affinity include: R40A, E37A, R56A, W78A, D80K, D80R. D80A. D80G, D80F, D80M and D80N. Mutations max be combined to achieve desîred effects. For example, many of the mutations that aliect the ratio of GDFl 1 :Activin binding hâve an overall négative 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
-1219001
GDF Trap is an ActRIIB polypeptide comprising an L79D or L79E mutation, optionally in combination with additional amino acid substitutions, additions or délétions.
In certain embodiments, ActRII antagonists to be used in accordance with the methods disclosed herein are ActRIIB polypeptides or ActRIIB-based GDF Trap polypeptides. In general such ActRIIB polypeptides and ActRIIB-based GDF Trap polypeptides are soluble polypeptides that comprise a portion/domain derived from the ActRIIB sequence of SEQ ID NO: l, 4, or 49, particularly an extracellular, ligand-binding portion/domain derived from the ActRIIB sequence of SEQ 1D NO:l, 4, or 49. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 2I-29 (e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO:l or 4 [optionally beginning at 22-25 (e.g., 22, 23, 24, or 25) of SEQ ID NO:l or 4] and ending at any one of amino acids 109-134 (e.g., 109, HO, lll, H2, H3, H4, H5, H6, H7, 118, H9, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1 or 4. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-29 (e.g., 20, 21,22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 or 4 [optionally beginning at 22-25 (e.g., 22, 23, 24, or 25) of SEQ ID NO:1 or 4] and ending at any one of amino acids 109-133 (e.g., 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-24 (e.g., 20, 21, 22, 23, or 24) of SEQ ID NO: 1 or 4 [optionally beginning at 22-25 (e.g., 22, 23, 24, or 25) of SEQ ID NO: 1 or 4] and ending at any one of amino acids 109-133 (e.g., 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133) of SEQ ID NO; 1 or 4. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 21-24 (e.g., 21, 22, 23, or 24) of SEQ ID NO: 1 or 4 and ending at any of amino acids 109-134 (e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131. 132, 133, or 134) of SEQ ID NO: 1 or 4. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-24 (e.g., 20, 21, 22, 23, or 24) of SEQ ID NO: I or 4 and ending at any one of amino acids 118-133 (e.g., 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 21-24 (e.g., 21,22, 23, or 24) of SEQ ID NO: 1 or 4 and ending at any
-1319001 one of amino acids 118-134 (e.g., Il 8, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1 or 4. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-24 (e.g., 20, 21,22, 23, or 24) of SEQ ID NO: 1 or 4 and ending at any one of amino acids 128133 (e.g, 128, 129,130, 131, 132, or 133) of SEQ ID NO: 1 or 4, In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any of amino acids 2024 (e.g., 20,21, 22, 23, or 24) ofSEQ ID NO: 1 or 39 and ending at any of amino acids 128-133 (e.g., 128, 129, 130, 131, 132, or 133) ofSEQ ID NO: I or 39. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any one ofamino acids 21-29 (e.g, 21,22, 23, 24, 25, 26, 27, 28, or 29) ofSEQ ID NO: 1 or 4 and ending at any one of amino acids 118-134 (e.g., 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) ofSEQ ID NO: 1 or 4. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-29 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) ofSEQ ID NO: 1 or 4 and ending at any one of amino acids 118-133 (e.g., 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133) ofSEQ ID NO: lor4. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at one any of amino acids 21-29 (e.g., 21,22, 23, 24, 25, 26, 27, 28, or 29) ofSEQ ID NO: 1 or 4 and ending at any one of amino acids 128-134 (e.g., 128, 129, 130, 131, 132, 133, or 134) ofSEQ ID NO: I or4. In some embodiments, the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-29 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) ofSEQ ID NO: 1 or 4 and ending at any one of amino acids 128-133 (e.g., 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4. Surprisingly, ActRIIB and ActRIIB-based GDF Trap constructs beginning at 22-25 (e.g., 22, 23, 24, or 25) ofSEQ ID NO: 1 or 4 hâve activity levels greater than proteins having the full extracellular domain of human ActRIIB. In some embodiments, the ActRIIB polypeptides and ActRIIB-based GDF Trap polypeptides comprises, consists essentially of, or consists of, an amino acid sequence beginning at amino acid position 25 of SEQ ID NO: 1 or 4 and ending at amino acid position 131 ofSEQ ID NO: 1 or 4. Any of the foregoing ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptides may be produced as a homodimer. Any of the foregoing ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptides may further comprise a heterologous portion that comprises a constant région from an IgG heavy chain, such as an Fc domain. Any of the above ActRIIBbased GDF Trap polypeptides may comprise an acidic amino acid at the position corresponding to position 79 ofSEQ ID NO: 1, optionally in combination with one or more
-1419001 additional amino acid substitutions, délétions, or insertions relative to SEQ ID NO: l. Any of the above ActRIIB polypeptides ActRIIB-based GDF Trap polypeptides, including homodimer and/or fusion proteins thereof, may bind to and/or inhibit signaling by activîn (e.g., activin A, activin B, activîn C, or activin AB) in a cell-based assay. Any of the above ActRIIB polypeptides ActRIIB-based GDF Trap polypeptides, including homodimer and/or fusion proteins thereof, may bind to and/or inhibit signaling by GDFl l and/or GDF8 in a cell based assay. Optionally, any of the above ActRIIB polypeptides ActRIIB-based GDF Trap polypeptides, including homodimer and/or fusion proteins thereof, may bind to and/or inhibit signaling of one or more of activin B, activin C, activin E, BMP6, BMP7, and Nodal in a cell-based assay.
Other ActRIIB polypeptides and ActRIIB-based GDF Trap polypeptides are contemplated, such as the following. An ActRIIB polypeptide or GDF Trap polypeptide comprising an amino acid sequence that is at least 80% (e.g, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO: 1 or 4, wherein the position corresponding to 64 of SEQ ID NO: 1 is an R or K, and wherein the ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide inhibits signaling by activin. GDF8, and/or GDFl 1 in a cell-based assay. The ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide as above, wherein at least one alteration with respect to the sequence of SEQ ID NO: 1 or 4 is positioned outside of the ligand-binding pocket, The ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide as above, wherein at least one alteration with respect to the sequence of SEQ ID NO: 1 or 4 is a conservative alteration positioned within the ligand-binding pocket. The ActRIIB polypeptide or ActRIIB-based GDl· I rap polypeptide as above, wherein at least one alteration with respect to the sequence of SEQ ID NO: 1 or 4 is an alteration at one or more positions selected from the group consisting of K.74, R40, Q53, K55, F82, and L79.
Other ActRIIB polypeptides and ActRIIB-based GDF Trap polypeptides are contemplated, such as the following. An ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide comprising an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO: I or 4, 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 ActRIIB, and at a position outside of the ligand binding pocket. The ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide as above, wherein the ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide comprises
-1519001 an N at the position corresponding to position 24 of SEQ ID NO: l or 4 and an S or T al lhe position corresponding to position 26 of SEQ ID NO: I or 4, and wherein the ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide inliibits signaling by activin, GDF8, and/or GDFl l in a cell-based assay. The ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide as above, wherein the ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide comprises an R or K at the position corresponding to position 64 of SEQ ID NO: I or 4. The ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide as above, wherein ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide comprises a D or E at the position corresponding to position 79 of SEQ ID NO: l or 4, and wherein the ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide inliibits signaling by activin, GDF8, and/or GDFl l in a cell-based assay. The ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide as above, wherein at least one alteration with respect to the sequence of SEQ ID NO: l or 4 is a conservative alteration positioned within the ligand-binding pocket. The ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide as above, wherein at least one alteration with respect to the sequence ofSEQIDNO: l or4isan alteration at one or more positions selected from the group consisting of K74, R40, Q53, K55, F82, and L79. The ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide above, wherein the ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptide is a fusion protein further comprising one or more heterologous portion. Any of the above ActRIIB polypeptides or ActRIIB-based GDF Trap polypeptides, or fusion proteins thereof, may be produced as a homodimer. Any of the above ActRIIB fusion proteins or ActRIIB-basedGDF Trap fusion proteins may hâve a heterologous portion that comprises a constant région from an IgG heavy chain, such as an Fc domain.
In certain embodiments, an ActRIIB polypeptide, for use in accordance with the methods disclosed herein, comprises an amino acid sequence that comprises, consists of, or consists essentially of, the amino acid sequence of SEQ ID NOs: 2, 3, 5, 6, 29, 3I, or 49, and polypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any ol’the foregoing. An ActRIIB polypeptide may include a functional fragment of a natural ActRIIB polypeptide, such as one comprising at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: 2, 3, 5, 6, 29, 31, or 49 or a sequence of SEQ ID NO: 2 or 5. lacking the C-terminal l, 2, 3, 4, 5 or 10 to 15 amino acids and lacking l, 2, 3, 4 or 5 amino acids at the N-terminus. In some embodiments, a polypeptide will comprise a truncation relative to SEQ ID NO: 2 or 5 of between 2 and 5 amino acids at the N-terminus and no more
-1619001 than 3 amino acids at the C-terminus. In some embodiments, a GDF Trap for use in accordance with the methods described herein consists of, or consists essentially of, the amino acid sequence of SEQ ID NO:29.
A general formula for an active (e.g., ligand binding) ActRIlA polypeptide is one that comprises a polypeptide that starts at amino acid 30 and ends at amino acid 110 of SEQ ID NO:9. Accordingly, ActRIlA polypeptides and ActRIIA-based GDF Traps of the présent disclosure may comprise, consist, or consist essentially of a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO:9. Optionally, ActRIlA polypeptides and ActRIIA-based GDF Trap polypeptides of the présent disclosure comprise, consists, or consist essentially of a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids amino acids 12-82 of SEQ ID NO:9 optionally beginning at a position ranging from 1-5 (e.g., 1, 2, 3, 4, or 5) or 3-5 (e.g., 3, 4, or 5) and ending at a position ranging from 110-116 (e.g., 110, 111, 112, 113, 114, 115, or 116) or 110-115 (e.g., 110, 111, 112, 113, 114, or 115) or SEQ ID NO:9, respectively, and comprising no more than 1, 2, 5, 10 or 15 conservative amino acid changes in the ligand binding pocket, and zéro, one or more non-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82 in the ligand-binding pocket with respect to SEQ ID NO:9. Any of the foregoing ActRIlA polypeptide or ActRIIA-based GDF Trap polypeptides may be produced as a homodimer. Any of the foregoing ActRIlA polypeptide or ActRIIA-based GDF Trap polypeptides may further comprise a heterologous portion that comprises a constant région from an IgG heavy chain, such as an Fc domain. Any of the above ActRIlA polypeptides ActRIIA-based GDF Trap polypeptides, including homodimer and/or fusion proteins thereof, may bind to and/or inliibit signaling by activin (e.g., activin A, activin B, activin C, or activin AB) in a cell-based assay. Any of the above ActRIlA polypeptides ActRIIA-based GDF Trap polypeptides, including homodimer and/or fusion proteins thereof, may bind to and/or inhibit signaling by GDF11 and/or GDF8 in a cell based assay. Optionally, any of the above ActRIlA polypeptides ActRIIB-based GDF Trap polypeptides, including homodimer and/or fusion proteins thereof, may bind to and/or inhibit signaling of one or more of activin B, activin C, activin E, GDF7, and Nodal in a cell-based assay.
In certain embodiments, ActRIlA polypeptides and ActRIIA-based GDF-Trap polypeptides, for use in accordance with the methods disclosed herein, comprises an amino acid sequence that comprises, consists of, or consists essentially of. the amino acid sequence of SEQ ID NOs: 9, 10, 22, 26, or 28, and polypeptides that are at least 80%, 85%, 90%, 95%,
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96%, 97%, 98%, or 99% identical to any of the foregoing. An ActRIIA polypeptide or ActRIlA-based GDF-Trap polypeptide may include a functional fragment of a natural ActRIIA polypeptide, such as one comprising at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: 9, 10, 22, 26, or 28 or a sequence of SEQ ID NO: 10, lacking the C-terminal 1,2, 3, 4, 5 or 10 to 15 amino acids and lacking 1, 2, 3, 4 or 5 amino acids at the N-terminus. In some embodiments, a polypeptide will comprise a truncation relative to SEQ ID NO: 10 of between 2 and 5 amino acids at the N-terminus and no more than 3 amino acids at the C-terminus. In some embodiments, an ActRIIA polypeptide for use in the methods described herein consîsts of, or consists essentially of, the amino acid sequence of SEQ ID NO: 26 or 28.
An ActRII polypeptide (e.g. an ActRIIA or ActRIIB polypeptide) or GDF Trap polypeptide of the disclosure may include one or more alterations (e.g., amino acid additions, délétions, substitutions, or combinations thereof) in the amino acid sequence ofan ActRII polypeptide (e.g., in the ligand-binding domain) relative to a naturally occurring ActRII 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 ActRII polypeptide.
Optionally, ActRII polypeptides (e.g. an ActRIIA or ActRIIB polypeptides) and GDF Trap polypeptides of the disclosure comprise one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a famesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent.
In some embodiments, an ActRII polypeptide (e.g. an ActRIIA or ActRIIB polypeptide) or GDF Trap polypeptide of the disclosure may be a fusion proteîn thaï has, as one domain, an ActRII polypeptide or GDF Trap polypeptide (e.g., a ligand-binding domain of an ActRII receptor, optionally with one or more sequence variations) and one or more additional heterologous domains that provide a désirable 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/administration, tissue localization or distribution, formation of protein complexes, multimerization of the fusion protein, and/or purification. ActRII polypeptide and GDF Trap fusion proteins may include a heterologous polypeptide domain such as but not limited to, an immunoglobulin Fc domain (wild-type or mutant) or a sérum albumin. In some embodiments,
-1819001 the immunoglobulin Fc domain is an IgGl Fc domain. In some embodiments, the IgGl Fc domain is a human IgGl Fc domain. In some embodiments, the IgGl Fc domain is a mouse IgGl Fc domain. In certain embodiments, an ActRII polypeptide and GDF Trap polypeptide fusion protein comprises a relatively unstructured linker positioned between the ActRII or GDF Trap polypeptide domain and the heterologous domain. In certain embodiments, an ActRII polypeptide and GDF Trap fusion protein comprises a relatively unstructured linker positioned between the Fc domain and the ActRII or GDF Trap domain. This unstructured linker may correspond to the roughly 15 amino acid unstructured région at the C-terminal end of the extracellular domain of ActRII or GDF Trap (the tail”), or il may be an artificial sequence of between 3 and 5, I5, 20, 30, 50 or more amino acids 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:52), SG4 (SEQ ID NO:54), or TG3 (SEQ ID NO:53) singlets or repeats] or a sériés of three glycines. A fusion protein may include a purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion. In certain embodiments. an ActRII fusion protein or GDF Trap fusion comprises a leader sequence. The leader sequence may be a native ActRII leader sequence (e.g, a native ActRIIA or ActRIIB leader sequence) or a heterologous leader sequence. In certain embodiments, the leader sequence is a Tissue Plasminogen Activator (TPA) leader sequence. In some embodiments, an ActRII fusion protein or GDF Trap fusion protein comprises an amino acid sequence as set forth in the formula A-B-C. The B portion is an N- and C-terminally truncated ActRII or GDF Trap polypeptide as described herein. The A and C portions may be independently zéro, one or more than one amino acids, and both A and C portions are heterologous to B. The A and/or C portions may be attached to the B portion via a linker sequence.
Optionally, ActRII polypeptides (e.g., ActRlIA and ActRIIB polypeptides) GDF Trap polypeptides, including variants and fusion proteins thereof, to be used in accordance with the methods disclosed herein bind to one or more ActRIIB ligand (e.g., activin A, activin B, activin AB, activin C, activin E, GDFl l, GDF8, BMP6, BMP7, and/or Nodal) with a Kd less than IO micromolar, less than l micromolar, less than IOO nanomolar, less than IO nanomolar, or less than I nanomolar. Optionally, such ActRII polypeptides GDF Trap polypeptides inhibit ActRII signaling, such as ActRlIA and/or ActRIIB intracellular signal transduction events triggered by an ActRII ligand (e.g., SMAD 2/3 and/or SMAD l/5/8 signaling).
-1919001 ln certain aspects, the disclosure provides pharmaceutical préparations or compositions comprising an ActRII antagonist of the présent disclosure (e.g., an ActRIIA polypeptide, and ActRIIB polypeptide, a GDF Trap polypeptide) and a pharmaceutically acceptable carrier. A pharmaceutical préparation or composition may also include one or more additional compounds such as a compound that is used to treat a disorder or condition described herein (e.g., an addition compound that increases red blood cell levels and/or hemoglobin levels in a subject in need thereof, treats or prevents anémia in a subject in need thereof, treat or prevents an ulcer, particularly a cutaneous ulcer, a subject in need thereof). Preferably, a pharmaceutical préparation or composition of the disclosure is substantially pyrogen-free.
In general, it is préférable that an ActRHA polypeptide, and ActRIIB polypeptide, or a GDF Trap polypeptide be expressed in a mammalian cell line that médiates suitably natural glycosylation of the polypeptide so as to diminish the likelihood of an untavorable immune response in a patient. Human and CHO cell lines hâve been used successfully, and it is expected that other common mammalian expression vectors will be usefuL In some embodiments, préférable ActRIIA polypeptides, ActRIIB polypeptides, and GDF Trap polypeptides are glycosylated and hâve a glycosylation pattern that is obtainable from a mammalian cell, preferably a CHO cell.
In certain embodiments, the disclosure provides packaged pharmaceuticals comprising a pharmaceutical préparation or composition described herein and labeled for use in one or more of increasing red blood cell levels and/or hemoglobin in a mammal (preferably a human), treating or preventing anémia in a mammal (preferably a human), treating or preventing sickle cell disease in a mamamal (preferably a human). and/or treating or preventing one or more complications of sickle-cell disease (e.g., anémia, vaso-occlusive crisis, ulcers (such as cutaneous ulcers), etc.) in a mammal (preferably a human). In certain embodiments, the disclosure provides packaged pharmaceuticals comprising a pharmaceutical préparation or composition described herein and labeled for use in treating anémia in a mammal (preferably a human), treating sickle cell disease in a mamamal (preferably a human), and/or treating one or more complications of sickle-cell disease (e.g., anémia, vaso-occlusive crisis, ulcers (such as cutaneous ulcers), etc.) in a mammal (preferably a human). In certain embodiments, the disclosure provides packaged pharmaceuticals comprising a pharmaceutical préparation or composition described herein and labeled for use in preventing anémia in a mammal (preferably a human), preventing
-2019001 sickle cell disease in a mamamal (preferably a human), and/or treating or preventing one or more complications of sickle-cell disease (e.g.t anémia, vaso-occlusive crisis, ulcers (such as cutaneous ulcers), etc.) in a mammal (preferably a human).
In certain aspects, the disclosure provides nucleic acids encoding an ActRII polypeptide (e.g., an ActRIIA or ActRIIB polypeptide) or GDF Trap polypeptide. An isolated polynucleotide may comprise a coding sequence for a soluble ActRII polypeptide or GDF Trap polypeptide, such as described herein. For example, an isolated nucleic acid may include a sequence coding for an ActRII polypeptide or GDF Trap comprising an extracellular domain (e.g., ligand-binding domain) of an ActRII polypeptide having one or more sequence variations and a sequence that would code for part or ail of the transmembrane domain and/or the cytoplasmic domain of an ActRII polypeptide, but for a stop codon positioned within the transmembrane domain or the cytoplasmic domain, or positioned between the extracellular domain and the transmembrane domain or cytoplasmic domain. For example, an isolated polynucleotide coding for a GDF Trap may comprise a full-length ActRII polynucleotide sequence such as SEQ ID NO: l, 4, or 9 or having one or more variations, or a partially truncated version, said isolated polynucleotide further comprising a transcription termînation codon at least six hundred nucléotides before the j’-terminus or otherwise positioned such that translation of the polynucleotide gives rise to an extracellular domain optionally fused to a truncated portion of a full-length ActRII. Nucleic acids disclosed herein may be operably linked to a promotcr for expression, and the disclosure provides cells transformed with such recombinant polynucleotides. Preferably the cell is a mammalian cell, such as a CHO cell.
In certain aspects, the disclosure provides methods for making an ActRII polypeptide or GDF Trap. Such a method may include expressing any of the nucleic acids disclosed herein (e.g., SEQ ID NO: 8, 13, 27, 32, 39, 42, 46, or 48) in a suitable cell, such as a Chinese hamster ovary (CHO) cell. Such a method may comprise: a) culturing a cell 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, the présent disclosure relates to an antibody, or combination of antibodies, that antagonize ActRII activity (e.g., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling). In particular, the
-2l19001 disclosure provides methods of using an antibody ActRII antagonist, or combination of antibody ActRII antagonists, to, e.g., increase red blood cell levels in a subject in need thereof, treat or prevent an anémia in a subject in need thereof, and/or treat or prevent an ulcer, particularly a cutaneous ulcer, in a subject that has anémia. In some embodiments, the disclosure provides methods of using an antibody ActRII antagonist, or combination of antibody ActRII antagonists to treat an ulcer, particularly a cutaneous ulcer, in a subject that has anémia. In some embodiments, the disclosure provides methods of using an antibody ActRII antagonist, or combination of antibody ActRII antagonists to prevent an ulcer, particularly a cutaneous ulcer, in a subject that has anémia.
In certain embodiments, an antibody ActRII antagonist of the disclosure is an antibody, or combination of antibodies, that binds to and/or inhibits activity of at least GDFl l (e.g., GDFl l-mediated activation of ActRIlA and/or ActRIIB signaling transduction, such as SM AD 2/3 signaling). Optionally, the antibody, or combination of antibodies, further binds to and/or inhibits activity of GDF8 (e.g., GDF8-mediated activation of ActRIlA and/or ActRIIB signaling transduction, such as SM AD 2/3 signaling), particularly in the case of a multi-specifïc antibody that has binding affïnity for both GDFl l and GDF8 or in the context of a combination of one or more anti-GDFl l antibody and one or more anti-GDF8 antibody. Optionally, an antibody, or combination of antibodies, of the disclosure does not substantially bind to and/or inhibit activity of activin A (e.g., activin Λ-mcdiated activation of ActRIlA or ActRIIB signaling transduction, such as SMAD 2/3 signaling). In some embodiments, an antibody, or combination of antibodies, of the disclosure that binds to and/or inhibits the activity of GDFl l and/or GDF8 further binds to and/or inhibits activity of one of more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal (e.g., activation of ActRIlA or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling), particularly in the case of a multi-speciiic antibody that has binding affïnity for multiple ActRII ligands or in the context of a combination multiple antibodies - each having binding affïnity for a different ActRII ligand.
In part, the disclosure demonstrates that ActRII antagonists may be used in combination (e.g., administered at the same time or different times, but generally in such a manner as to achieve overlapping pharmacological effects) with EPO receptor activators to increase red blood cell levels (erythropoiesis) or treat anémia in patients in need thereof. In part, the disclosure demonstrates that a GDF Trap can be administered in combination with an EPO receptor activator to synergistically increase formation of red blood ceils in a patient.
-2219001 particuiarly in sickle-cell patients. Thus, the effect of this combinée! treatment can be significantly greater than the sum of the effects of the ActRIl antagonists and the EPO receptor activator when administered separately at their respective doses. In certain embodiments, this synergism may be advantageous since it enables target levels of red blood cells to be attained with lower doses of an EPO receptor activator, thereby avoiding potential adverse effects or other problems associated with higher levels of EPO receptor activation. Accordingly, in certain embodiments, the methods of the présent disclosure (e.g, methods of increasing red blood cell levels and/or hemoglobin in a subject in need thereof, treating or preventing anémia in a subject in need thereof, and/or treating or preventing an ulcer in a subject that has anémia) comprise administerîng a patient in need thereof one or more ActRIl antagonists (e.g., ActRIIA polypeptides, ActRIIB polypeptides, and/or GDF Trap polypeptides) in combination with one or more EPO receptor activators.
An EPO receptor activator may stimulate erythropoiesis by directly contacting and activating EPO receptor. In certain embodiments, the EPO receptor activator is one of a class of compounds based on the 165 amino-acid sequence of native EPO and generally known as erythropoiesis-stimulating agents (ESAs), examples of which are epoetin alfa, epoetin beta (NeoRecormon®), epoetin delta (Dynepo™), and epoetin oméga. In other embodiments, ESAs include synthetic EPO proteins (SEPs) and EPO dérivatives with nonpeptîdic modifications conferring désirable pharmacokinetic properties (lengthened circulating halflife), examples of which are darbepoetin alfa (Aranesp®) and methoxy-polycthylene-glycol epoetin beta (Mircera®). In certain embodiments, an EPO receptor activator may be an EPO receptor agonist that does not incorporate the EPO polypeptide backbonc or is not generally classified as an ESA. Such EPO receptor agonists may include, but are not limited to, peptidic and nonpeptîdic mimelics of EPO, agonistîc antibodies targeting EPO receptor, fusion proteins comprising an EPO mimetic domain, and erytliropoietin receptor extendedduration limited agonists (EREDLA),
In certain embodiments, an EPO receptor activator may stimulate erythropoiesis indirectly, without contacting EPO receptor itself, by enhancing production of endogenous EPO. For example, hypoxia-inducible transcription factors (HIFs) are endogenous stimulators of EPO gene expression that are suppressed (destabilized) under normoxic conditions by cellular regulatory mechanisms. In part, the disclosure provides increased erythropoiesis in a patient by combined treatment with a GDF Trap and an indirect EPO receptor activator with HIF stabilizing properties, such as a prolyl hydroxylase inhibitor.
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ActRII antagonists, particularly ActRII polypeptides and GDF Trap polypeptides, may also be used for treating or preventing other disorders and conditions such as promoting muscle growth and/or treating or preventing a muscle-related disorder, promoting bone growth and/or treating or preventing a bone-related disorder, treating or preventing cancer (particularly multiple myeloma and/or breast cancer), See, e.g., U.S. Patent Nos: 7,612,041; 8,173,601; 7,842,663 as well as U.S. Patent Application Publication No. U.S. 2009/0074768. In certain instances, when administering a GDF Trap polypeptide for thèse other therapeutic indications, it may be désirable to monitor the effects on red blood cells during administration of the ActRII antagonist, or to déterminé or adjust the dosing of the ActRII antagonist, 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.
BR1EF DESCRIPTION OF THE DRAWINGS
The patent or patent application file contains at least one drawing executcd in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Figure 1 shows an alignment of extracellular domains of human ActRIIA (SEQ ID NO:56) and human ActRIIB with the residues that are deduced herein, based on composite analysis of multiple ActRIIB and ActRIIA crystal structures, to directly contact ligand indicated with boxes (SEQ ID NOs: 57-64).
Figure 2 shows a multiple sequence alignment of various vertebrate ActRIIB proteins and human ActRIIA.
Figures 3A and 3B shows the purification of ActRIIA-hFc expressed in CHO cells. The protein purifies as a single, well-defined peak as visualized by sizing column (top panel) and Coomassie stained SDS-PAGE (bottom panel) (left lane: molecular weight standards; right lane: ActRIIA-hFc).
Figures 4A and 4B shows the binding of ActRIIA-hFc to activin and GDF-11, as measured by BiacoreIM assay.
Figures 5A and 5B show the effects of ActRIIA-hFc on red blood cell counts in female non-human primates (NHPs). Female cynomolgus monkeys (four groups of five
-2419001 monkeys each) were treated with placebo or Img/kg, 10 mg/kg or 30 mg/kg of ActRIIA-hFc on day 0, day 7, day 14 and day 21. Figure 5A shows red blood cell (RBC) counts. Figure 5B shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group. At day 57, two monkeys remained in each group.
Figures 6A and 6B shows the effects of ActRIIA-hFc on red blood cell counts in male non-human primates. Male cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or l mg/kg, 10 mg/kg, or 30 mg/kg of ActRIIA-hFc on day 0, day 7, day 14, and day 21. Figure 6A shows red blood cell (RBC) counts. Figure 6B shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group. At day 57, two monkeys remained in each group.
Figures 7A and 7B shows the effects of ActRIIA-hFc on réticulocyte counts in female non-human primates. Cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or l mg/kg, 10 mg/kg, or 30 mg/kg of ActRIIA-hFc on day 0, day 7, day 14, and day 21. Figure 7A shows absolute réticulocyte counts. Figure 7B shows the percentage of réticulocytes relative to RBCs. Statistical significance is relative to baseline for each group. At day 57, two monkeys remained in each group.
Figures 8A and 8B shows the effects of ActRIIA-hFc on réticulocyte counts in male non-human primates. Cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or l mg/kg, 10 mg/kg or 30 mg/kg of ActRIIA-hFc on day 0, day 7, day 14 and day 21. Figure 8A shows absolute réticulocyte counts. Figure 8B shows the percentage of réticulocytes relative to RBCs. Statistical significance is relative to baseline for each group. At day 57, two monkeys remained in each group.
Figure 9 shows results from the human clinical trial described in Example 5, where the area-under-curve (AUC) and administered dose of ActRIIA-hFc hâve a linear corrélation, regardless of whether ActRIIA-hFc was administered intravenously (IV) or subcutaneously (SC).
Figure 10 shows a comparison of sérum levels of ActRIIA-hFc in patients administered IV or SC.
Figure 11 shows bone alkaline phosphatase (BAP) levels în response to different dose levels of ActRIIA-hFc. BAP is a marker for anabolic bone growth.
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Figure 12 depicts the médian change from baseline of hematocrit levels from the human clinical trial described in Example 5. ActRIIA-hFc was administered intravenously (IV) at the indicated dosage.
Figure 13 depicts the médian change from baseline of hemoglobin levels from the human clinical trial described in Example 5. ActRIIA-hFc was administered intravenously (IV) at the indicated dosage.
Figure 14 depicts the médian change from baseline of RBC (red blood cell) count from the human clinical trial described in Example 5. ActRIIA-hFc was administered intravenously (IV) at the indicated dosage.
Figure 15 depicts the médian change from baseline of réticulocyte count from the human clinical trial described in Example 5. ActRIIA-hFc was administered intravenously (IV) at the indicated dosage.
Figure 16 shows the full amino acid sequence for the GDF Trap ActRllB(L79D 20l34)-hFc (SEQ ID NO:38), including the TPA leader sequence (double underlined), ActRIIB extracellular domain (residues 20-134 in SEQ ID NO: l ; underlined). and hFc domain. The aspartate substituted at position 79 in the native sequence is double underlined and highlighted. as is the glycine revealed by sequencing to be the N-terminal residue in the mature fusion protein.
Figures 17A and 17B show a nucléotide sequence encoding ActRIIB(L79D 20-134)hFc. SEQ ID NO:39 corresponds to the sense strand. and SEQ ID NO:40 corresponds to the antisense strand. The TPA leader (nucléotides 1-66) is double underlined, and the ActRIIB extracellular domain (nucléotides 76-420) is underlined.
Figure 18 shows the full amino acid sequence for the truncated GDF Trap ActRIIB(L79D 25-131)-hFc (SEQ ID NO:41), including the TPA leader (double underlined), truncated ActRIIB extracellular domain (residues 25-131 in SEQ ID NO:1; underlined), and hFc domain. The aspartate substituted at position 79 in the native séquence is double underlined and highlighted, as is the glutamate revealed by sequencing to be the N-terminal residue in the mature fusion protein.
Figures 19A and 19B show a nucléotide sequence encoding ActRlIB(L79D 25-131)hFc. SEQ ID NO:42 corresponds to the sense strand, and SEQ ID NO:43 corresponds to the antisense strand. The TPA leader (nucléotides 1-66) is double underlined, and the truncated
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ActRIIB extracellular domain (nucléotides 76-396) is underlined. The amino acid sequence for the ActRIIB extracellular domain (residues 25-131 in SEQ ID NO: l) is also shown.
Figure 20 shows the amino acid sequence for the truncated GDF Trap ActRIIB(L79D 25-l31)-hFc without a leader (SEQ ID NO:44). The truncated ActRIIB 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-terminal residue in the mature fusion protein.
Figure 21 shows the amino acid sequence for the truncated GDF Trap AclRIlB(L79D 25-131) without the leader, hFc domain, and linker (SEQ ID NO:45). 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-terminal residue in the mature fusion protein.
Figures 22A and 22B show an alternative nucléotide sequence encoding ActRIIB(L79D 25-13l)-hFc. SEQ ID NO:46 corresponds to the sense strand, and SEQ ID NO:47 corresponds to the antisense strand. The TPA leader (nucléotides l-66) is double underlined, the truncated ActRIIB extracellular domain (nucléotides 76-396) is underlined, and substitutions in the wild-type nucléotide sequence of the extracellular domain are double underlined and highlighted (compare with SEQ ID NO:42, Figure 19). Ί he amino acid sequence for the ActRIIB extracellular domain (residues 25-131 in SEQ ID NO:1) is also shown.
Figure 23 shows nucléotides 76-396 (SEQ ID NO:48) of the alternative nucléotide sequence shown in Figure 22 (SEQ ID NO:46). The same nucléotide substitutions indicated in Figure 22 are also underlined and highlighted here. SEQ ID NO:48 encodes only the truncated ActRIIB extracellular domain (corresponding to residues 25-131 in SEQ ID NO:1) with a L79D substitution, e.g., ActRIIB(L79D 25-131).
Figure 24 shows the effect of treatment with ActRIIB(L79D 20-134)-hFc (gray) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change in red blood cell concentration from baseline in cynomolgus monkey. VEH = vehicle. Data are means + SEM. n = 4-8 per group.
Figure 25 shows the effect of treatment with ActRIIB(L79D 20-134)-hFc (gray) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change in hematocrit from baseline in cynomolgus monkey. VEH = vehicle. Data are means + SEM. n = 4-8 per group.
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Figure 26 shows the effect of treatment with ActRIIB(L79D 20-l34)-hFc (gray) or ActRIIB(L79D 25-l31)-hFc (black) on the absolute change in hemoglobin concentration from baseline in cynomolgus monkey. VEH — vehicle. Data are means + SEM. n = 4-8 per group.
Figure 27 shows the effect of treatment with ActRIIB(L79D 20-l34)-hFc (gray) or ActRIIB(L79D 25-131 )-hFc (black) on the absolute change in circulating réticulocyte concentration from baseline in cynomolgus monkey. VEH = vehicle. Data are means + SEM. n = 4-8 per group.
Figure 28 shows the effect of combined treatment with erythropoietin (EPO) and ActRIIB(L79D 25-13I)-hFc for 72 hours on hematocrit in mice. Data arc means 1 SEM (n ~ 4 per group), and means that are significantly different irom each other (p < 0.05, unpaired ttest) are designated by different letters. Combined treatment increased hematocrit by 23% compared to vehicle, a synergistic increase greater than the sum of the separate effects of EPO and ActRIIB(L79D 25-131)-hFc.
Figure 29 shows the effect of combined treatment with EPO and ActRIIB(L79D 25131 )-hFc for 72 hours on hemoglobin concentrations in mice. Data are means ± SEM (n = 4 per group), and means that are significantly different from each other (p < 0.05) are designated by different letters. Combined treatment increased hemoglobin concentrations by 23% compared to vehicle, which was also a synergistic effect.
Figure 30 shows the effect of combined treatment with EPO and ActRIIB(L79D 25131 )-hFc for 72 hours on red blood cell concentrations in mice. Data are means ± SEM (n = 4 per group), and means that are significantly different from each other (p < 0.05) are designated by different letters. Combined treatment increased red blood cell concentrations by 20% compared to vehicle, which was also a synergistic effect.
Figure 31 shows the effect of combined treatment with EPO and ActRIIB(L79D 25131 )-hFc for 72 hours on numbers of erythropoietic precursor cells in mouse spleen. Data are means ± SEM (n = 4 per group), and means that are significantly different from each other (p <0.01) are designated by different letters. Whereas EPO alone increased the number of basophilie erythroblasts (BasoE) dramatically at the expense of late-stage precursor maturation, combined treatment increased BasoE numbers to a lesser but still significant extent while supporting undiminished maturation of late-stage precursors.
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Figures 32A-C compares RBC parameters in an Hbb'1' mouse model of β-thalassemia with those in wildtype (WT) mice. Blood samples from untreated mice at 2-6 months of agc were analyzed to détermine red biood cell number (RBC; A), hematocrit (HCT; B), and hemoglobin concentration (Hgb; C). Data are means ± SEM (n = 4 per group), ***, p<0.001. Hbb'1' mice were confirmed to be severely anémie.
Figure 33 shows the effect of ActRIIB(L79D 25-131)-mFc on RBC number in an Hbb'1' mouse model of β-thalassemia. Blood samples were collected after 4 weeks of treatment. Data are means of 2 per group, with bars indicating the range. Treatment with ActRIIB(L79D 25-131 )-mFc reduced by half the RBC déficit présent in Hbb'1' mice.
Figure 34 shows the effect of ActRIIB(L79D 25-131 )-mFc on RBC morphology in an Hbb'1' mouse mode! of β-thalassemia. Images of Giemsa-stained blood smears from mice treated for 4 weeks were obtained at lOOx magnification. Note hemolysis, cellular débris, and many small or irregularly shaped RBCs in blood from the vehicle-treated Hbb'1' mouse. By comparison, ActRIIB(L79D 25-131)-mFc treatment greatly reduced hemolysis, débris, and the occurrence of irregularly shaped RBCs while increasing the number of normally shaped RBCs.
Figure 35 shows the effect of ActRIIB(L79D 25-13 l)-mFc treatment for 2 months on RBC number in an Hbb’1' mouse model of β-thalassemia, with data from vehiclc-dosed wildtype mice included for comparison. Data are means ± SEM; n = 7 per group. **, 1’ < 0.01 vs. vehicle-treated Hbb'1' mice. Treatment with ActRIIB(L79D 25-131)-mFc reduced the mean RBC déficit in Hbb'1' mice by more than 50%.
Figure 36 shows the effect of ActRIIB(L79D 25-131 )-mFc treatment for 2 months on sérum bilirubin levels in an Hbb1 mouse model of β-thalassemia, with data from vehicledosed wildtype mice included for comparison. Data are means ± SEM. # # il·, P < 0.001 vs. vehicle-treated wildtype mice; *, P < 0.05 vs. vehicle-treated Hbb'^mïce . Treatment with ActRIIB(L79D 25-131)-mFc reduced total bilirubin levels significantly in Hbb' mice.
Figure 37 shows the effect of ActRlIB(L79D 25-13 l)-mFc treatment for 2 months on sérum EPO level in an Hbb'1' mouse model of β-thalassemia, with data from vehicle-dosed wildtype mice included for comparison. Data are means ± SEM. # # #. P < 0.001 vs. vehicle-treated wildtype mice; *, P < 0.05 vs. vehicle-treated Hbb' mice. Treatment with ActRUB(L79D 25-131 )-mFc reduced mean circulating EPO levels by more than 60% in Hbb' '' mice.
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Figures 38A and 38B show the effect of ActRlIB(L79D 25-13 l)-mFc on splenomegaly in an Hbb'1' mouse mode! of β-thalassemia, with data from vehicle-dosed wildtype mice included for comparison. A. Means ± SEM from mice starting at 3 months of âge after treatment with 1 mg/kg twice weekly for 2 months. # # #, P < 0.001 vs. vehicletreated wildtype mice; *, P < 0.05 vs. vehicle-treated Hbh'!' mice. B. Représentative spleen sizes, as observed in a separate study in mice starting at 6-8 months of âge after treatment with 1 mg/kg twice weekly for 3 months. Treatment with ActRIIB(L79D 25-13 l)-mFc reduced spleen weight significantly in Hbb'1' mice.
Figure 39 shows the effect of ActRIIB(L79D 25-131)-mFc treatment for 2 months on bone minerai density in an Hbb'1' mouse model of β-thalassemia, with data from vehicledosed wildtype mice included for comparison. Means ± SEM based on fémur analysis. #, P < 0.05 vs. vehicle-treated wildtype mice; *, P < 0.05 vs. vehicle-treated Hbb'1' mice. Treatment with ActRIlB(L79D 25-131)-mFc normalized bone minerai density in Hbb'1' mice.
Figures 40A-C show the effect of ActRIIB(L79D 25-131)-mFc treatment for 2 months on parameters of iron homeostasis in an Hbb'1' mouse model of β-thalassemia. Means ± SEM for sérum iron (A), sérum ferritin (B), and transferin saturation (C). *, P < 0.05; **, P < 0.01 vs.'vehicle-treated Hbb'1' mice. Treatment with ActRIIB(L79D 25-131)-mFc reduced each measure of iron overload significantly in Hbb'1' mice.
Figure 41 shows the effect of ActRHB(L79D 25-13 l)-mFc treatment for 2 months on tissue iron overload in an Hbb'1' mouse model of β-thalassemia. Iron levels in tissue sections (200 pm) from spleen (A-C), liver (D-F), and kidney (G-I) were determincd by staining with Perl’s Prussian blue. Iron staining in wildtype spleen (A) was abundant in red pulp (arrows) but absent in white pulp (*). Increased iron staining in spleen of Hbb'1' mice (B) reflects expansion of red pulp régions due to extramedullary erythropoiesis. ActRIlB(L79D 25-131)mFc in Hbb'1' mice decreased splenic erythropoiesis and restored the wildtype pattern of splenic iron staining (C) In addition, abnormal iron staining in hepatic Kupffer cells (H, arrows) and rénal cortex (E, arrows) of Hbb' mice was normalized by ActRIIB(L79D 25131)-mFc (F and I). Magnification, 200x.
Figure 42 shows the effect of ActRIIB(L79D 25-13 l)-mFc treatment for 2 months on hepatic levels of hepcidin mRNA in a Hbb'1' mouse model of β-thalassemia. Means ± SEM; *, P < 0.05 vs. vehicle-treated Hbb'1' mice. Treatment with ActRIIB(L79D 25-13 l)-mFc increased expression of hepcidin mRNA significantly in Hbb'1' mice.
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Figure 43 shows the effect of ActRIIB(L79D 25-l3l)-mFc on circulating levels of reactive oxygen species (ROS) in an Hbb’1' mouse model of β-thalasscmia, with data from vehicle-dosed wildtype mice included for comparison. Data are géométrie means ± SEM. # # #, P < 0.001 vs. vehicle-treated wildtype mice; ***, P < 0.001 vs. vehicle-treated Hbb'1' mice. Treatment with ActRIIB(L79D 25-131)-mFc reduced ROS significantly in Hbb'1' mice.
Figure 44 shows the effect of ActRIIB(L79D 25-131)-mFc on the absolute change in red blood cell concentration in sickle-cell disease (SCD) mice. Data are means ± SEM (n = 5 per group). Wt = wild-type mice. which were non-symptomatic compound hétérozygote (β/β8) mice. ActRIIB(L79D 25-131)-mFc treatment resulted in a significant increase in red blood cell levels in sickle-cell mice (P<0.001) in comparision control mice (sickle-cell mice administered vehicle alone).
Figure 45 shows the effect of ActRIIB(L79D 25-131)-mFc on red blood cell levels. hematocrit levels, and hemoglobin levels in sickle-cell mice. Data arc mean changes from baseline over 4 weeks (± SEM) vs. sickle-cell control mice. ActRIIB(L79D 25-131)-mFc treatment resulted in a significant increase in red blood cell levels, hematocrit levels, and hemoglobin levels in sickle-cell mice in comparision to control mice.
Figure 46 shows the effect of ActRIIB(L79D 25-131)-mFc on various blood parameters (Le., mean corpuscular volume, red blood cell (RDC) distribution width, réticulocytes, and reactive oxygen species in sickle-cell mice). Data are mean changes from baseline over 4 weeks (± SEM) vs. sickle-cell control mice. ActRIlB(L79D 25-131)-mFc treatment resulted in a significant increase in mean corpuscular volume, red blood cell (RDC) distribution width, réticulocytes, and reactive oxygen species in sickle-cell mice in comparision to control mice.
DETAIL DESCRIPTION OF THE INVENTION
1. O ver view
The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence éléments and structural motifs. These proteins are known to exert biologîcal effects on a large variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important fonctions during embryonic development in pattern formation and tissue spécification and can influence a variety of
-3119001 différentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and épithélial cell différentiation, By manipulating the activity of a member of the TGF-beta family, it is often possible to cause significant physiological changes in an organism. For example, the Piedmontese and Belgian Blue cattle breeds carry a loss-of-function mutation in the GDF8 (also called myostatin) gene that causes a marked increase in muscle mass. See, e.g., Grobet et al. (1997) Nat Genet. 17(1):71-4. Furthermore, in humans, inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength. See, e.g., Schuelke et al. (2004) N Engl J Med, 350:2682-8.
TGF-β signais are mediated by heteromeric complexes oftype I and type II serine/threonine kinase receptors, which phosphorylateand activate downstream SMAD proteins (e.g., SMAD proteins 1, 2, 3, 5, and 8) upon ligand stimulation. See, e.g.,Massagué (2000) Nat. Rev. Mol. Cell Biol. 1:169-178. These type I and type II receptors are transmembrane proteins, composed of a ligand-binding extracellular domain with cysteinerich région, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine 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 I receptors by Type II receptors.
Two related Typell receptors (ActRII), ActRIIA and ActRIIB, hâve been identified as theType II receptors for activins. See, e.g., Mathews and Vale (1991) Cell 65:973-982; and Attisanoe/ al. (1992) Cell 68: 97-108. Besides activins, ActRIIA and AclRIlBcan biochemically interact with several other TGF-β family proteinsincluding, for example, BMP6, BMP7, Nodal, GDF8, and GDFl 1. See, e.g., Yamashita ci al. (1995) J. Cell Biol. 130:217-226;Lee and McPherron (2001) Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman (2001) Mol. Cell 7: 949-957; and Oh et al. (2002) Genes Dev. 16:2749-54. ALK4 is the primary type I receptor for activins, particularly for activin A, and ALK-7 may serve as a receptor for other activins as well, particularly for activin B. In certain embodiments, the présent disclosure relates to antagonizing a ligand of an ActRII receptor (also referred to as an ActRII ligand) with one or more inhibitor agents disclosed herein, particularly inhibitor agents that can antagonize GDFl 1 and/or GDF8.
Activins are dimeric polypeptide growth factors that belong to the TGF-beta superfamily. There are three principal activin forms (A, B, and ΛΒ) that are homo/heterodimers of two closely related β subunits (βΛβΛ, βυβιι, and β/χβΐΐ, respective!}).
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The human genome also encodes an aclivin C and an activin E, which are primarily expressed in lhe liver, and heterodimeric forms containing Pc or Pe are also known.
In the TGF-beta superfamily, activins are unique and multifunctional factors that can stimulate hormone production in ovarian and placental cells, support neuronal cell survival, influence cell-cycle progress positively or negatively depending on cell type, and induce mesodermal différentiation at least in amphibian embryos. DePaolo et al. (1991) Proc Soc Ep Biol Med. 198:500-512; Dyson et a/.(1997) Curr BioL 7:81-84; and Woodruff (1998) Biochem Pharmacol. 55:953-963. Moreover, erythroid différentiation factor (EDF) isolated from the stimulaled human monocytic leukemic cells was found to be identical to activin A. Murata et al. (1988) PNAS, 85:2434. It has been suggested that activin A promûtes erythropoiesis in the bone marrow. In several tissues, activin signaling is antagonized by its related heterodimer, inhibin. For example, during the release of follicle-stimulating hormone (FSH) from the pituitary, activin promotes FS1T sécrétion and synthesis, while inhibin prevents FSH sécrétion and synthesis. Other proteins that may regulate activin bioactivity and/or bind to activin include follistatin (FS), follistatin-related protein (FSRP, also known as FLRG or FSRL3), and aj-macroglobulin.
As described herein, agents that bind to “activin A” are agents that specifically bind to the Pasubunit, whelher in the context oi an isolated βΑ subunit or as a dimeric complex (e.g., a PaPa homodimer or a ρΛββ heterodimer). In the case of a heterodimer complex (e.g., a βΛβΒ heterodimer), agents that bind to “activin A” are spécifie for epitopes présent within the Pa subunit, but do not bind to epitopes présent within the ηοη-βΑ subunit of the complex (e.g., the βΒ subunit of the complex). Similarly, agents disclosed herein that antagonize (inhibit) “activin A” are agents that inhibit one or more activities as mediated by a βΑ subunit, whether in the context of an isolated βΛ subunit or as a dimeric complex (e.g.. a βΛβΛ homodimer or a βΛβΒ heterodimer). In the case ofβΛββ heterodimers, agents thaï inhibit “activin A” are agents that specifically inhibit one or more activities of the βΛ subunit, but do not inhibit the activity of the ηοη-βΑ subunit of the complex (e.g., the ββ subunit of the complex). This principle applies also to agents that bind to and/or inhibit “activin B”, “activin C”, and “activin E”. Agents disclosed herein that antagonize “activin AB” are agents that inhibit one or more activities as mediated by the βΛ subunit and one or more activities as mediated by the βΒ subunit.
Nodal proteins hâve functions in mesoderm and endoderm induction and formation, as well as subséquent organization of axial structures such as heart and stomach in early embryogenesis. 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. Studies support the idea that ActRIIA and ActRIIB serve as type II receptors for Nodal. See, e.g., Sakuma et al. (2002) Genes 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 phosphorylate SMAD2. Nodal proteins are implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patteming, and left-right axis spécification. Experimental evidence has demonstrated that Nodal signaling activâtes pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TGF-beta. However, Nodal is unable to induce pTlx2-Lux. a reporter specifically responsive to bone morphogenetic proteins. Recent results provide direct biochemical evidence that Nodal signaling is mediated by both activin-TGF-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 Différentiation Factor-8 (GDF8) is also known as myostatin. GDF8 is a négative regulator of skeletal muscle mass. GDF8 is highly expressed in the developing and adult skeletal muscle. The GDF8 null mutation in transgenic mice is characterized by a marked hypertrophy and hyperplasia of the skeletal muscle. McPherron et al., Nature (1997) 387:83-90. Similar increases in skeletal muscle mass are évident in naturally occumng mutations of GDF8 in cattle [yee, e.g., Ashmore et al. (1974) Growth. 38:501-507: Swatland and Kieffer (1994) J. Anim. Sci. 38:752-757; McPherron and Lee (1997) Proc. Natl. Acad. Sci. USA 94:12457-12461; and Kambadur et al. (1997) Genome Res. 7:910-915] and, strikingly, in humans [see, e.g., Schuelke et cd. (2004) N Engl J Med 350:2682-8]. Studies hâve also shown that muscle wasting associated with HlV-infection in humans is accompanied by increases in GDF8 protein expression. See, e.g., Gonzalez-Cadavid et al. (1998) PNAS 95:14938-43. In addition, GDF8 can modulate the production of musclespecîfic enzymes (e.g., creatine kinase) and modulate myoblast cell prolifération. See, e.g. international patent application publication no. WO 00/43781. The GDF8 propeptide can noncovalently bind to the mature GDF8 domain dimer, inactivating its biological activity. See, e.g., Miyazono el al. (1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J.
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Biol. Chem., 263: 7646-7654; and Brown et al. (1990) Growth Factors, 3: 35-43]. Other proteins which bind to GDF8 or structurally related proteins and inhibit their biological activity include follistatin, and potentially, follistatin-related proteins. See. e.g.. Gainer et al (1999) Dev. Biol., 208: 222-232.
Growth and Différentiation Factor-11 (GDF11), also known as BMP11, is a secreted protein. McPherron et al. (1999) Nat. Genet. 22: 260-264. GDF11 is expressed in the tail bud, limb bud, maxillary and mandibular arches, and dorsal root ganglia during mouse development See, e.g., Nakashima et al. (1999) Mech. Dev. 80: 185-189. GDF11 plays a unique rôle in patterning both mesodermal and neural tissues. See, e.g., Gamer et al. (1999) Dev Biol., 208:222-32. GDF11 was shown to be a négative regulator of chondrogenesis and myogenesis in developing chick limb. See, e.g., Gamer et al. (2001 ) Dev Biol. 229:407-20. The expression of GDF11 in muscle also suggests its rôle in regulating muscle growth in a similar way to GDF8. In addition, the expression of GDF11 in brain suggests that GDF11 may also possess activities that relate to the function of the nervous system. Interestingly. GDFl 1 was found to inhibit neurogenesis in the olfactory epithelium. See, e.g., Wu et al. (2003) Neuron. 37:197-207.
Bone morphogenetic protein (BMP7), also called ostéogénie protein-1 (OP-1), is well known to induce cartilage and bone formation. In addition, BMP7 régulâtes a wide array of physiological processes. For example, BMP7 may be the osteoinductive factor responsible for the phenomenon of épithélial osteogenesis. It is also found that BMP7 plays a rôle in calcium régulation and bone homeostasîs. Like activin, BMP7 binds to Type II receptors, ActRIlA and ActRIIB. However, BMP7 and activin recruit distinct Type I receptors into heteromeric receptor complexes. The major BMP7 Type I receptor observed was ALK2, while activin bound exclusively to ALK.4 (ActRIIB). BMP7 and activin elîcited distinct biological responses and activated different SMAD pathways. See, e.g., Macias-Silva et al. (1998) J Biol Chem. 273:25628-36.
As demonstrated herein, ActRII polypeptides (e.g., ActRIlA and ActRIIB polypeptidesjcan be used to increase red blood cell levels in vivo. In certain examples, it is shown that a GDF Trap polypeptide (specifically a variant ActRIIB polypeptide) is characterized by unique biological properties in comparison to a corresponding sample of a wild-type (unmodified) ActRII polypeptide. This GDF Trap is characterized. in part, by substantial loss of binding affinity for activin A, and therefore significantly diminished capacity to antagonize activin A activity, but retains near wild-type levels of binding and
-3519001 inhibition of GDFl l. Invivo, the GDF Trap is more effective at increasing red blood cell levels as compared to the wild-type ActRIl polypeptide and has bénéficiai effects in patients with anémia induding, e.g., patients with sickle-cell disease and patients with thalassemia. For example, it is shown herein that GDF Trap therapy results in increased hemoglobin levels in human patients that hâve thalassemia. In addition to improvements in red blood cell parameters, certain thalassemia patients were observed to hâve substantial resolution ol a leg ulcer (which is a common cutaneous complication of anémia, particularly in hemolytic anémias such as thalassemia and sickle-cell disease) during the course of GDF Trap therapy. These data indicate a much broader use for ActRIl antagonists in the treatment of various complications of anémie disorders beyond the positive effects on red blood cell parameters.
Accordingly, the methods of the présent disclosure, in general, are directed to the use of one or more ActRIl antagonist agents described herein, optionally in combination with one or more supportive thérapies, to încrease in red blood cell levels in a subject in need thereof, treat or prevent an anémia in a subject in need thereof, and/or to treat or prevent one or more complications of anémia induding, for example, ulcers, particularly cutaneous ulcers.
Furthermore, the data of the présent disclosure indicates that the observed biological activity of an ActRIl polypeptide, with respect to red blood cell parameters and ulcers, is not dépendent on activin A inhibition. However, it is to be noted that the unmodified ActRIIB polypeptide, which retains activin A binding, still demonstrates the capacity to increase red blood cells in vivo. Furthermore, an ActRIIB or ActRIIA polypeptide that retains activin A inhibition may be better suited in some applications, in comparîson to a GDF Trap having diminished binding affmity for activin A, where more modest gains in red blood cell levels are désirable and/or where some level of off-target activity is acceptable (or even désirable).
Il should be noted that hematopoiesis is a complex process, regulated by a variety of factors, induding erythropoietin, G-CSF and iron homeostasis. The tenus increase red blood cell levels” and “promote red blood cell formation” refer to clinically observable metrics, such as hematocrit, red blood cell counts, and hemoglobin measurements, and are intended to be neutral as to the mechanism by which such changes occur.
EPO is a glycoprotein hormone involved in the growth and maturation of erythroid progenitor cells into érythrocytes. EPO is produced by the liver during fêtai life and by the kidney in adults. Decreased production of EPO, which commonly occurs in adults as a conséquence of rénal failure, leads to anémia. EPO has been produced by genetic
-3619001 engineering techniques based on expression and sécrétion of the protein from a host cell transfected with the EPO gene. Administration of such recombinant EPO has been effective in the treatment of anémia. For example, Eschbach et al. ( 1987, N Engl J Med 316:73) describe the use of EPO to correct anémia caused by chronic rénal failure.
Effects of EPO are mediated through its binding to, and activation of, a cell surface receptor belonging to the cytokine receptor superfamily and designated the EPO receptor. The human and murine EPO receptors hâve been cloned and expressed. See, e.g., D’Andrea et al. (1989) Cell 57:277; Jones et al. (1990) Blood 76:31; Winkelman et al.(\Wty Blood 76:24; and U.S. Pat. No. 5,278,065. The human EPO receptor gene encodes a 483 amino acid transmembrane protein comprising an extracellular domain of approximately 224 amino acids and exhibits approximately 82% amino acid sequence identity with the murine EPO receptor. See, e.g., U.S. Pat.No. 6,319,499. The cloned, full-length EPO receptor expressed in mammalian cells (66-72 kDa) binds EPO with an affinity (Kd ~ 100-300 nM) similar to that of the native receptor on erythroid progenitor cells. Thus, this form is thoughl to contain the main EPO binding déterminant and is referred to as the EPO receptor. By analogy with other closely related cytokine receptors, the EPO receptor is thought to dimerize upon agonist binding. Nevertheless, the detailed structure of the EPO receptor, which may be a multimeric complex, and its spécifie mechanism of activation are not completely understood. See, e.g., U.S. Pat.No. 6,319,499.
Activation of the EPO receptor results in several biological effects. Thcse include increased prolifération of immature erythroblasts, increased différentiation of immature erythroblasts, and decreased apoptosis in erythroid progenitor cells. See, e.g., Lîboi et al. (1993) Proc Natl Acad Sci USA 90:11351-11355; Koury et al. (1990) Science 248:378-381. The EPO receptor signal transduction pathways medîating prolifération and différentiation appear to be distinct. See, e.g., Noguchi et al. (1988) Mo] Cell Biol 8:2604: Patel et al. (1992) J Biol Chem, 267:21300; and Liboi et al. ( 1993) Proc Natl Acad Sci USA 90:11351 -11355). Some results suggest that an accessory protein may be required for médiation of the différentiation signal. See, e.g., Chiba et al. (1993) Nature 362:646; and Chiba et al. (1993) Proc Natl Acad Sci USA 90:11593. However, there is controversy regarding the rôle of accessory proteins in différentiation since a constitutively activated form of the receptor can stimulate both prolifération and différentiation. See, e.g., Pharr et al. (1993) Proc Natl Acad Sci USA 90:938.
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EPO receptor activators include small molécule erythropoiesis-stimulating agents (ESAs) as well as EPO-based compounds. An example of the former is a dimeric peptidebased agonist covalently linked to polyethylene glycol (proprietary name Hematide™ and Omontys®), which has shown erythropoiesis-stimulating properties in healthy volunteers and in patients with both chronic kidney disease and endogenous anti-EPO antibodies. See. e.g.. Stead et al. (2006) Blood 108:1830-1834; and Macdougall et al. (2009) N Engl J Med 361:1848-1855. Other examples include nonpeptide-based ESAs. See, e.g., Qureshi et al. (1999) Proc Natl Acad Sci USA 96:12156-12161.
EPO receptor activators also include compounds that stimulate erythropoiesis indirectly, without contacting EPO receptor itself, by enhancing production of endogenous EPO. For example, hypoxia-inducible transcription factors (HIFs) are endogenous stimulators of EPO gene expression that are suppressed (destabilized) under normoxic conditions by cellular regulatory mechanisms. Therefore, inhibitors of HIF prolyl hydroxylase enzymes are being investigated for EPO-inducing activity in vivo. Other indirect activators of EPO receptor include inhibitors of GATA-2 transcription factor [see, e.g., Nakano et al. (2004) Blood 104:4300-4307], which tonically inhibits EPO gene expression, and inhibitors of hemopoietic cell phosphatase (HCP or SHP-1), which functions as a négative regulator of EPO receptor signal transduction [see, e.g.,Klingmuller et al. (1995) Cell 80:729-738.
The terms used in this spécification generally hâve their ordinary meanîngs in the art, within the context of this disclosure and in the spécifie context where each term is used. Certain terms are discussed below or elsewhere in the spécification, to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them. The scope or meaning of any use of a term will be apparent from the spécifie context in which they are used.
“Homologous,” in ail its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evolutionary origin,” including proteins from superfainilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) hâve sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of spécifie residues or motifs and conserved positions.
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The term “sequence similarity,” in ail its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutîonary 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 evolutîonary origin.
Percent (%)sequence identity with respect to a référencé polypeptide (or nucléotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to the amino acid residues (or nucleic acids) in the référencé polypeptide (nucléotide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achîevcd in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can détermine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid (nucleic acid) sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is regîstered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating System, including digital UNIX V4.0D. Ail sequence comparison parameters are set by the ALIGN-2 program and do not vary.
As used herein “does not substantially bind to JC’ is intended to mean that an agent has a Kd that is greater than about ΙΟ'7, ΙΟ'6, ΙΟ'5, ΙΟ'4 or greater (e.g., no détectable binding by the assay used to détermine the Kd) for “X”.
2. ActRII Antagonist
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The data presented herein demonstrates that antagonists (inhibitors) of ActRII (e.g., antagonist of ActRlIA and/or ActRIIB SMAD 2/3 and/or SMAD 1/5/8 signaling) can be used in increasing red blood cell levels m vivo. In particular, such ActRII antagonists are shown herein to be effective in treating various anémias as well as various complications (e.g, disorders/conditions) of anémia including, for example, cutaneous ulcers. According!y, the present disclosure provides, in part, various ActRII antagonist agents that can be used, alone or in combination with one or more erythropoiesis stimulating agents (e.g., EPO) or other supportive thérapies [e.g., treatment with hydroxyurea, blood transfusion, iron chélation therapy, and/or pain management (e.g, treatment with one or more of opioid analgésie agents, non-steroidal anti-inflammatory drugs, and/or corticosteroids)], to treat or prevent an anémia in a subject in need thereof and/or to treat or prevent a cutaneous ulcer in a patient that has anémia.
In certain embodiments, the ActRII antagonists to be used in accordance with the methods disclosed herein are GDF-ActRII antagonists (e.g., antagonists of GDF-mediated ActRlIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling), particularly GDFl l- and/or GDF8-mediated ActRII signaling. In some embodiments. ActRII antagonists of the present disclosure are soluble ActRII polypeptides (e.g, soluble ActRlIA and ActRIIB polypeptides) and GDF Trap polypeptides, such as ActRIIA-Fc fusion proteins, ActRIIB-Fc fusion proteins, and GDF Trap-Fc fusion proteins.
Although soluble ActRII polypeptides and GDF Trap polypeptides of the disclosure may affect red blood cell levels and/or cutaneous ulcers through a mechanism other than GDF (e.g GDFl l and/or GDF8) antagonism [e.g, GDFl l and/or GDF8 inhibition may be an indicator of the tendency of an agent to inliibit the activities of a spectrum of additional agents, including, perhaps, other members of the TGF-beta superfamily (e.g, activin B, activin C, activin E, BMP6, BMP7, and/or Nodal) and such collective inhibition may lead to the desired effect on, e.g., hematopoiesis], other types of GDF-ActRII antagonist are expected to be useful including, for example, anti-GDFl l antibodics: anti-GDF8 antibodics: anti-ActRIIA antibodies; anti-ActRIIB antibodies; antisense, RNAi, or ribozyme nucleic acids that inhibit the production of one or more of GDFl 1, GDF8, ActRlIA, and/or ActRIIB; and other inhibitors (e.g, smali molécule inliibitors) of one or more of GDFl 1, GDF8, ActRlIA, and/or ActRIIB, particularly agents that disrupt GDFl 1- and/or GDF8-ActRIIA binding and/or GDFl 1- and/or GDF8-ActRIIB binding as well as agents that inhibit expression of one or more ofGDFl 1, GDF8, ActRlIA, and/or ActRIIB. Optionally, GDF-4019001
ActRII antagonists of the present disclosure may bind to and/or inhibit the activity (or expression) of other ActRII ligands including, for example, activin A, activin AB, activin B, activin C, activin E, BMP6, BMP7, and/or Nodal. Optionally, a GDF-ActRII antagonist of the present disclosure may be used in combination with at least one additional ActRII antagonist agent that binds to and/or inhibits the activity (or expression) of one or more additional ActRII ligands including, for example, activin A, activin AB, activin B, activin C, activin E, BMP6, BMP7, and/or Nodal. In some embodiments, ActRII antagonists to be used in accordance with the methods disclosed herein do not substantially bind to and/or inhibit activin A (e.g., activin A-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
A. ActRII polypeptides and GDF Traps
In certain aspects, the present disclosure relates to ActRII polypeptides. In particular, the disclosure provides methods of using ActRII polypeptides to, e.g., treat or prevent an anémia in a subject in need thereof and/or treat or prevent one or more complication of anémia including, for example, cutaneous ulcers. As used herein the term “ActRII” refers to the family of type II activin receptors. This family includes both the activin receptor type IIA and the activin receptor type IIB. In some embodiments, the disclosure provides methods of using ActRII polypeptides to treat an anémia in a subject in need thereof and/or treat one or more complications of anémia including, for example, cutaneous ulcers, in a subject having anémia. In some embodiments, the disclosure provides methods of using ActRII polypeptides to prevent an anémia in a subject in need thereof and/or prevent one or more complications of anémia including, for example, cutaneous ulcers in a subject having anémia. In some embodiments, the ActRII polypeptides are ActRIIA polypeptides. In some embodiments, the ActRII polypeptides are ActRIIB polypeptides.
As used herein, the term “ActRIIB” refers to a family of activin receptor type IIB (ActRIIB) proteins from any species and variants derived from such ActRIIB proteins by mutagenesis or other modification. Reference to ActRIIB herein is understood to be a reference to any one of the currently identîfied forms. Members ofthe ActRIIB family are generally transmembrane proteins, composed of a ligand-bînding extracellular domain comprising a cysteine-rich région, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
The term “ActRIIB polypeptide” includes polypeptides comprising any naturally occurring polypeptide of an ActRIIB family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Examples of such variant ActRIIA polypeptides are provided throughout the présent disclosure as well as in International Patent Application Publication No. WO 2006/012627, which is incorporated herein by reference in its entirety. Optionally, ActRIIB polypeptides of the présent disclosure can be used to increase red blood cell levels in a subject. Numbering of amino acids for ail ActRIIB-related polypeptides described herein is based on the numbering of the human ActRIIB precursor protein sequence provided below (SEQ ID NO:1), unless specifically designated otherwise.
The human ActRIIB precursor protein sequence is as follows:
1 MTAPWVALAL LWGSLCAGSG RGEAETRECI YYNANWELER TNQSGLERCE
51 GEQDKRLHCY ASWR^SSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY
101 FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS
151 LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR
201 FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA
251 EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY
301 LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK
351 PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC
401 KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL
451 AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV
501 TNVDLPPKES SI (SEQ ID NO: 1)
The signal peptide is indicated with single underlined; the extracellular domain is indicated in bold font; and the potential, endogenous N-linked glycosylation sites arc indicated with double underline.
The processed soluble (extracellular) human ActRIIB polypeptide sequence is as follows:
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GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKK GCWEDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPT APTiSEQ ID NO:2).
In some embodiments, the protein may be produced with an “SGR...” sequence at the N-terminus. The C-terminal “tail” of the extracellular domain is indicated by single underline. The sequence with the “tail” deleted (a Al 5 sequence) is as follows:
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKK GCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA (SEQ ID N0:3).
A form of ActRIIB with an alanine at position 64 of SEQ ID NO:1 (A64) is also reported in the literature. See, e.g., Hilden et al. (1994) Blood. 83(8): 2163-2170. Applicants hâve ascertained that an ActRIIB-Fc fusion protein comprising an extracellular domain of ActRIIB with the A64 substitution has a relatively low affinity for activin and GDF11. By contrast, the same ActRIIB-Fc fusion protein with an arginine at position 64 (R64) has an affinity for activin and GDF11 in the low nanomolar to high picomolar range. Therefore, sequences with an R64 are used as the “wild-type” reference sequence for human ActRIIB in this disclosure.
The form of ActRIIB with an alanine at position 64 is as follows:
1MTAPWVALAL LWGSLCAGSG RGEAETRECI YYNANWELER TNQSGLERCE
51 GEQDKRLHCY ASWANSSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY
101 FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS
151 LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR
201 FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA
251 EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY
301 LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK
351 PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC
401 KAADGPVDEY MLPFEEEIGQ HPSLEELQEV WHKKMRPTI KDHWT.KHPGI.
451 AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV
501 TNVDLPPKES SI(SEQ ID NO:4).
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The signal peptide is indicated by single underline and the extracellular domain is indicated by bold font.
The processed soluble (extracellular) ActRIIB polypeptide sequence of the alternative A64 form is as follows:
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKK GCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPT APT(SEO ID N0:5).
In some embodiments, the protein may be produced with an “SGR...” sequence at the N-terminus. The C-terminal “tail” of the extracellular domain is indicated by single underline. The sequence with the “tail” deleted (a Δ15 sequence) is as follows: GRGEAETRECIYYNANWELERTNQSGLERCEGEQDRRLHCYASWANSSGTIELVKK GCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA (SEQ ID N0:6).
The nucleic acid sequence encoding a human ActRIIB precursor protein is shown below (SEQ ID NO:7), consisting of nucléotides 25-1560 of Genbank Référencé Sequence NM 001106.3, which encode amino acids 1-513 of the ActRIIB precursor. The sequence as shown provides an arginine at position 64 and may be modifîed to provide an alanine instead. The signal sequence is underlined.
1 ATGACGGCGC CCTGGGTGGC CCTCGCCCTC CTCTGGGGAT_ CGCTGTGCGC
51 CGGCTCTGGG CGTGGGGAGG CTGAGACACG GGAGTGCATC TACTACAACG
ICI CCAACTGGGA GCTGGAGCGC ACCAACCAGA GCGGCCTGGA gcgctgcgaa
151 GGCGAGCAGG ACAAGCGGCT GCACTGCTAC GCCTCCTGGC GCAACAGCTC
201 TGGCACCATC GAGCTCGTGA AGAAGGGCTG CTGGCTAGAT GACTTCAACT
251 GCTACGATAG GCAGGAGTGT GTGGCCACTG AGGAGAACCC CCAGGTGTAC
301 TTCTGCTGCT GTGAAGGCAA CTTCTGCAAC GAACGCTTCA CTCATTTGCC
351 AGAGGCTGGG GGCCCGGAAG TCACGTACGA GCCACCCCCG ACAGCCCCCA
401 CCCTGCTCAC GGTGCTGGCC TACTCACTGC TGCCCATCGG GGGCCTTTCC
451 CTCATCGTCC TGCTGGCCTT TTGGATGTAC CGGCATCGCA AGCCCCCCTA
501 CGGTCATGTG GACATCCATG AGGACCCTGG GCCTCCACCA CCATCCCCTC
551 TGGTGGGCCT GAAGCCACTG CAGCTGCTGG AGATCAAGGC TCGGGGGCGC
601 TTTGGCTGTG TCTGGAAGGC CCAGCTCATG AATGACTTTG TAGCTGTCAA
651 GATCTTCCCA CTCCAGGACA AGCAGTCGTG GCAGAGTGAA CGGGAGATCT
701 TCAGCACACC TGGCATGAAG CACGAGAACC TGCTACAGTT CATTGCTGCC
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751 GAGAAGCGAG GCTCCAACCT CGAAGTAGAG CTGTGGCTCA TCACGGCCTT
801 CCATGACAAG GGCTCCCTCA CGGATTACCT CAAGGGGAAC ATCATCACAT
851 GGAACGAACT GTGTCATGTA GCAGAGACGA TGTCACGAGG CCTCTCATAC
901 CTGCATGAGG ATGTGCCCTG GTGCCGTGGC GAGGGCCACA AGCCGTCTAT
951 TGCCCACAGG GACTTTAAAA GTAAGAATGT ATTGCTGAAG AGCGACCTCA
1001 CAGCCGTGCT GGCTGACTTT GGCTTGGCTG TTCGATTTGA GCCAGGGAAA
1051 CCTCCAGGGG ACACCCACGG ACAGGTAGGC ACGAGACGGT ACATGGCTCC
1101 TGAGGTGCTC GAGGGAGCCA TCAACTTCCA GAGAGATGCC TTCCTGCGCA
1151 TTGACATGTA TGCCATGGGG TTGGTGCTGT GGGAGCTTGT GTCTCGCTGC
1201 AAGGCTGCAG ACGGACCCGT GGATGAGTAC ATGCTGCCCT TTGAGGAAGA
1251 GATTGGCCAG CACCCTTCGT TGGAGGAGCT GCAGGAGGTG GTGGTGCACA
1301 AGAAGATGAG GCCCACCATT AAAGATCACT GGTTGAAACA CCCGGGCCTG
1351 GCCCAGCTTT GTGTGACCAT CGAGGAGTGC TGGGACCATG ATGCAGAGGC
1401 TCGCTTGTCC GCGGGCTGTG TGGAGGAGCG GGTGTCCCTG ATTCGGAGGT
1451 CGGTCAACGG CACTACCTCG GACTGTCTCG TTTCCCTGGT GACCTCTGTC
1501 ACCAATGTGG ACCTGCCCCC TAAAGAGTCA AGCATC (SEQ ID NO; 7).
A nucleic acid sequence encoding processed soluble (extracellular) human ActRIIB polypeptide is as follows (SEQ ID NO:8). The sequence as shown provides an arginine at position 64 and may be modified to provide an alanine instead.
1 GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG
51 GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC
101 AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC
151 ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA
201 TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT
251 GCTGTGAAGG CAACTTCTGC AACGAACGCT TCACTCATTT GCCAGAGGCT
301 GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACC (SEQ ID
NO:8).
In certain embodiments, the présent disclosure relates to ActRIIA polypeptides. As used herein, the term “ActRIIA” refers to a family of activin receptor type IIA (ActRIIA) proteins from any species and variants derived from such ActRIIA proteins by mutagcncsis or other modification. Reference to ActRIIA herein is understood to be a reference to any one of the currently identified forms. Members of the ActRIIA family are generally transmembrane proteins, composed of a ligand-binding extracellular domain comprising a
-4519001 cysteine-rich région, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
The term “ActRIIA polypeptide” includes polypeptides comprising any naturally occurring polypeptide of an ActRIIA family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Examples of such variant ActRIIA polypeptides are provided throughout the présent disclosure as well as in International Patent Application Publication No. WO 2006/012627, which is incorporated herein by reference in its entirety. Optionally, ActRIIA polypeptides of the présent disclosure can be used to increase red blood cell levels in a subject. Numbering of amino acids for ail ActRIIA-related polypeptides described herein is based on the numbering of the human ActRIIA precursor protein sequence provided below (SEQ ID NO:9), unless specifically designated otherwise.
The human ActRIIA precursor protein sequence is as follows:
MGAAAKLAFA VFLISCSSGAILGRSETQEC LFFNANWEKD RT^QTGVEPC
51 YGDKDKRRHC FATWK^ISGS IEIVKQGCWL DDINCYDRTD CVEKKDSPEV
101 YFCCCEGNMC NEKFSYFPEM EVTQPTSNPV TPKPPYYNIL LYSLVPLMLI
151 AGIVICAFWV YRHHKMAYPP VLVPTQDPGP PPPSPLLGLK PLQLLEVKAR
201 GRFGCVWKAQ LLNEYVAVKI FPIQDKQSWQ NEYEVYSLPG MKHENILQFI
251 GAEKRGTSVD VDLWLITAFH EKGSLSDFLK ANWSWNELC HIAETMARGL
301 AYLHEDIPGL KDGHKPAISH RDIKSKNVLL KNNLTACIAD FG1.ALKFEAG
351 KSAGDTHGQV GTRRYMAPEV LEGAINFQRD AFLRIDMYAM GLVLWELASR
401 CTAADGPVDE YMLPFEEEIG QHPSLEDMQE VVVHKKKRPV LRDYWQKHAG
451 MAMLCETIEE CWDHDAEARL SAGCVGERIT QMQRLTNIIT TEDIVTVVTM
501 VTNVDFPPKE SSL(SEQ ID NO:9)
The signal peptide is indicated by single underline; the extracellular domain is indicated in bold font; and the potential, endogenous N-linked glycosylation sites are indicated by double underline.
The processed soluble (extracellular) human ActRIIA polypeptide sequence is as follows:
-4619001
ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRIICFATWKNISGSIEIVKQG CWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPK PP(SEQ ID NO:lO)
The C-terminal “tail” of the extracellular domain is indicated by single underline.
The sequence with the “taîl” deleted (a Δ15 sequence) is as follows:
1LGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDK.RRHCFATWKNISGSIEIVK.QG
CWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM (SEQ ID NO:l l)
The nucleic acid sequence encoding human ActRIIA precursor protein is shown below (SEQ ID NO:l2), as follows nucléotides 159-1700 of Genbank Reference NM_001616.4. The signal sequence is underlined.
1 atgggagctg ctgcaaagtt ggcgtttgcc gtcLl tcL t a_ 1ctcclg L Lc
51 ttcaggtgct atacttggta gatcagaaac tcaggagtgt cttttcttta
101 atgctaattg ggaaaaagac agaaccaatc aaactggtgt tgaaccgtgt
151 tzatggtgaca aagataaacg gcggcattgt tttgctacct ggaagaatat
201 ttctggttcc attgaaatag tgaaacaagg ttgttggctg gatgatatca
251 actgctatga caggactgat tgtgtagaaa aaaaagacag ccctgaagta
301 tatttttgtt gctgtgaggg caatatgtgt aatgaaaagt tttcttatt t
351 tccggagatg gaagtcacac agcccacttc aaatccagtt acacctaagc
401 caccctatta caacatcctg ctctattcct tggtgccact tatgttaatt
451 gcggggattg tcatttgtgc attttgggtg tacaggcatc acaagatggc
501 ctaccctcct gtacttgttc caactcaaga cccaggacca cccccacctt
551 ctccattact aggtttgaaa ccactgcagt tattagaagt gaaagcaagg
601 ggaagatttg gttgtgtctg gaaagcccag ttgcttaacg aatatgtggc
651 tgtcaaaata tttccaatac aggacaaaca gtcatggcaa aatgaatacg
701 aagtctacag tttgcctgga atgaagcatg agaacatatt acagttcatt
751 ggtgcagaaa aacgaggcac cagtgttgat gtggatcttt ggctgatcac
801 agcatttcat gaaaagggtt cactatcaga ctttcttaag gctaatgtgg
851 tctcttggaa tgaactgtgt catattgcag aaaccatggc tagaggattg
901 gcatatttac atgaggatat acctggccta aaagatggcc acaaacctgc
951 catatctcac agggacatca aaagtaaaaa tgtgctgttg aaaaacaacc
1001 tgacagcttg cattgctgac tttgggttgg ccttaaaatt tgaggctggc
1051 aagtctgcag gcgataccca tggacaggtt ggtacccgga ggtacatggc
-4719001
1101 tccagaggta ttagagggtg ctataaactt ccaaagggat gcatttttga 1151 ggatagatat gtatgccatg ggattagtcc tatgggaact ggcttctcgc 1201 tgtactgctg cagatggacc tgtagatgaa tacatgttgc catttgagqa 1251 ggaaattggc cagcatccat ctcttgaaga catgcaggaa gttgttgtgc 1301 ataaaaaaaa gaggcctgtt ttaagagatt attggcagaa acatgctgga 1351 atggcaatgc tctgtgaaac cattgaagaa tgttgggatc acgacgcaga 1401 agccaggtta tcagctggat gtgtaggtga aagaattacc cagatgcaga 1451 gactaacaaa tattattacc acagaggaca ttgtaacagt ggtcacaatg 1501 gtgacaaatg ttgactttcc tcccaaagaa tctagtcta (SEQIDNO:12)
The nucleic acid sequence encoding processed soluble (extracellular human ActRIIA polypeptide îs as follows:
1 atacttggta gatcagaaac tcaggagtgt cttttcttta atgctaattg
51 ggaaaaagac agaaccaatc aaactggtgt tgaaccgtgt tatggtgaca
101 aagataaacg gcggcattgt tttgctacct ggaagaatat ttctggttcc
151 attgaaatag tgaaacaagg ttgttggctg gatgatatca aetgetatga
201 caggactgat tgtgtagaaa aaaaagacag ccctgaagta tatttttgtt
251 gctgtgaggg caatatgtgt aatgaaaagt tttcttattt tccggagatg
301 gaagtcacac agcccacttc aaatccagtt acacctaagc caccc (SEQ ID
NO: 13 )
An aligninent of the amino acid sequences of human ActRIIB soluble extraccllular domain and human ActRIIA soluble extracellular domain are illustrated in Figure 1. This alignment indicates amino acid residues within both receptors that are believed to directly contact ActRII ligands. Figure 2 depicts a multiple sequence alignment of varions vertebrate ActRIIB proteins and human ActRIIA. From these alignments is it possible to predict key amino acid positions within the ligand-binding domain that are important for normal ActRIIligand binding activities as well as to predict amino acid positions that are likely to be tolérant to substitution without significantly altering normal ActRll-Iigand binding activities.
In other aspects, the présent disclosure relates to GDF Trap polypeptides (also referred to as “GDF Traps”). In particular, the disclosure provides methods of using GDF Trap polypeptides to, e.g., treat or prevent an anémia in a subject in need thereof, treat sickle cell disease in a subject in need thereof and/or treat or prevent one or more complications of anémia including, for example, cutaneous ulcers. In some embodiments, the disclosure
-4819001 provides methods of using GDF Trap polypeptides to treat an anémia in a subject in need thereof and/or treat one or more complications of anémia including, for example, cutaneous ulcers, in a subject having anémia. In some embodiments, the disclosure provides methods of using GDF Trap polypeptides to prevent an anémia in a subject in need thereof and/or prevent one or more complications of anémia including, for example, cutaneous ulcers, in a subject having anémia.
In some embodiments, GDF Traps of the présent disclosure are soluble, variant ActRII polypeptides (e.g., ActRIlA and ActRIIB polypeptides) that comprise one or more mutations (e.g., amino acid additions, délétions, substitutions, and combinations thereof) in the extracellular domain (also referred to as the ligand-binding domain) of an ActRII polypeptide (e.g., a “wild-type” ActRII polypeptide) such that the variant ActRII polypeptide has one or more altered ligand-binding activities than the corresponding wild-type ActRII polypeptide. In some embodiments, GDF Trap polypeptides of the présent disclosure retain at least one similar activity as a corresponding wild-type ActRII polypeptide (e.g., an ActRIlA or ActRIIB polypeptide). For example, a GDF Trap may bind to and/or inhibit (e.g. antagonize) the function of one or more ActRII ligands (e.g., inhibit ActRII ligand-mediated activation of the ActRIlA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling pathway). In some embodiments, GDF Traps of the présent disclosure bind to and/or inhibit one or more of activin A, activin B, activin AB, activin C, activin E, Nodal, GDF8, GDF11, BMP6 and/or BMP7).
In certain embodiments, GDF Trap polypeptides of the disclosure hâve elevated binding affmity for one or more spécifie ActRII ligands (e.g.. GDF8, GDF11, BMP6. Nodal. and/or BMP7). In other embodiments, GDF Trap polypeptides of the disclosure hâve decreased binding affmity for one or more spécifie ActRII ligands (e.g., activin A, activin B, activin AB, activin C, and/or activin E). In still other embodiments, GDF Trap polypeptides of the disclosure hâve elevated binding affmity for one or more spécifie ActRII ligands and decreased binding affmity for one or more different/other ActRII ligands. Accordingly, the présent disclosure provides GDF Trap polypeptides that hâve an altered binding specificity for one or more ActRII ligands.
In certain embodiments, GDF Traps of the présent disclosure are designed to preferentially bind to and antagonize GDF11 and/or GDF8 (also known as myostatin), e.g., in comparison to a wild-type ActRII polypeptide. Optionally, such GDFI1 and/or GDF8
-4919001 binding Traps may further bind to and/or antagonize one or more of Nodal, GDF8, GDF11, BMP6 and/or BMP7. Optionally, such GDF11 and/or GDF8-binding Traps may further bind to and/or antagonize one or more of activin B, activin C, activin E, Nodal, GDl'8. GDl· 11. BMP6 and/or BMP7. Optionally, such GDF11 and/or GDF8-binding Traps may further bind to and/or antagonize one or more of activin A, activin A/B, activin B, activin C, activin E, Nodal, GDF8, GDF11, BMP6 and/or BMP7. In certain embodiments, GDF Traps of the present disclosure hâve diminished binding affinity for activins (e.g., activin A, activin A/B, activin B, activin C, activin E ), e.g., in comparision to a wild-type ActRII polypeptide. In certain embodiments, a GDF Trap polypeptide of the present disclosure has diminished binding affinity for activin A.
For example, the disclosure provides GDF Trap polypeptides that preferentially bind to and/or antagonize GDF8/GDF11 relative to activin A. As demonstrated by the Examples ofthe disclosure, such GDF Trap polypeptides are more potent activators oferythropoiesis in vivo in comparision to ActRII polypeptides that retain high binding affinity for activin A. Furthermore, these non-activin A-bînding GDF Traps polypeptides demonstrate decreased effects on other tissues. Therefore, such GDF Traps may be useful for increasing red blood cell levels in a subject while reducing potential off-target effects associated with binding/antagonizing activin A. However, such sélective GDF Trap polypeptides may be less désirable in some applications wherein more modest gains in red blood cell levels may be needed for therapeutic effect and wherein some level of off-target effect is acceptable (or even désirable).
Amino acid residues of the ActRIIB proteins (e.g., E39, K55, Y60, K74, W78, L79, D80, and Fl01) are in the ActRIIB ligand-binding pocket and help médiate binding to its ligands including, for example, activin A, GDF11, and GDF8. Thus the present disclosure provides GDF Trap polypeptides comprising an altered-Iigand binding domain (e.g., a GDF8/GDF11-binding domain) of an ActRIIB receptor which comprises one or more mutations at those amino acid residues.
Optionally, the altered ligand-binding domain can hâve increased selectivity for a ligand such as GDF11 and/or GDF8 relative to a wild-type ligand-binding domain of an ActRIIB receptor. To illustrate, one or more mutations may be selected that încrease the selectivity of the altered ligand-binding domain for GDF11 and/or GDF8 over one or more activins (activin A, activin B, activin AB, activin C, and/or activin A), particularly activin A.
-5019001
Optionally, the altered ligand-binding domain has a ratio of K<i for activin binding to IQ for GDFl 1 and/or GDF8 binding that is at least 2-, 5-, 10-, 20-, 50-, 100- or even 1000-fold greater relative to the ratio for the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has a ratio of IC50 for inhibiting activin to 1C5O for inhibiting GDFl 1 and/or GDF8 that is at least 2-, 5-, 10-, 20-, 50-, 100- or even 1000-fold greater relative to the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain inhibits GDFl 1 and/or GDF8 with an IC50 at least 2-, 5-, 10-, 20-, 50-, 100- or even 1000-times less than the IC50 for inhibiting activin.
As a spécifie example, the positively-charged amino acid residue Asp (D80) of the ligand-binding domain of ActRIIB can be mutated to a different amino acid residue to produce a GDF Trap polypeptide that preferentially binds to GDF8, but not activin. Preferably, the D80 residue with respect to SEQ ID NO:1 is changed to an amino acid residue selected from the group consistîng of: an uncharged amino acid residue, a négative amino acid residue, and a hydrophobie amino acid residue. As a further spécifie example, the hydrophobie residue L79 of SEQ ID NO:1 can be altered to confier altered activinGDF11/GDF8 binding properties. For example, an L79P substitution reduces GDFl 1 binding to a greater extent than activin binding. In contrast, replacement of L79 with an acidic amino acid [an aspartic acid or glutamic acid; an L79D or an L79E substitution] greatly reduces activin A binding affinity while retaining GDFl 1 binding affinity. In exemplary embodiments, the methods described herein utilize a GDF Trap polypeptide which is a variant ActRIIB polypeptide comprising an acidic amino acid (e.g., D or E) at the position correspondîng to position 79 of SEQ ID NO: 1, optionally in combination with one or more additional amino acid substitutions, additions, or délétions.
As will be recognized by one of skill in the art, most of the described mutations, variants or modifications described herein may be made at the nucleic acid level or, in some cases, by post-translational modification or chemical synthesis. Such techniques are well known in the art and some of which are described herein.
In certain embodiments, the présent disclosure relates to ActRII polypeptides (ActRIIA and ActRIIB polypeptides) which are soluble ActRII polypeptides. As described herein, the term “soluble ActRII polypeptide” generally refers to polypeptides comprising an extracellular domain of an ActRII protein. The term “soluble ActRII polypeptide,” as used herein, includes any naturally occurring extracellular domain of an ActRII protein as well as any variants thereof (including mutants, fragments, and peptidomimetic forms) that retain a
-5119001 useftil activity (e.g., a GDF Trap polypeptide as described herein). Other examples of soluble ActRII polypeptides comprise a signal sequence in addition to the extracellular domain of an ActRII or GDF Trap protein. For example, the signal sequence can be a native signal sequence of an ActRIIA or ActRIIB protein, or a signal sequence from another protein including, for example, a tissue plasminogen activator (TPA) signal sequence or a honey bee melittin (HBM) signal sequence.
In part, the présent disclosure identifies functionally-active portions and variants of ActRII polypeptides that can be used as guidance for generating and using ActRIIA polypeptides, ActRIIB polypeptides, and GDF Trap polypeptides within the scope of the methods described herein.
ActRII proteins hâve been characterized in the art in ternis of structural and functional characteristics, particularly with respect to ligand-binding. See, e.g., Attisano et al. (1992) Cell 68(1):97-108; Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al. (2006) PNAS 103(20: 7643-7648; Thompson et al. (2003) The EMBO Journal 22(7): 1555-1566; and U.S. Patent Nos: 7,709,605, 7,612,041, and 7,842,663.
For example, Attisano et al. showed that a délétion of the proline knot at the Cterminus of the extracellular domain of ActRIIB reduced the affinity of the receptor for activin. An ActRIIB-Fc fusion protein containing amino acids 20-119 of présent SEQ ID NO:1, “ActRIIB(20-l 19)-Fc”, has reduced binding to GDF-11 and activin relative to an ActRIIB(20-134)-Fc, which includes the proline knot région and the complété juxtamembrane domain. See, e.g., U.S. Patent No. 7,842,663. However, an ActRIIB(20129)-Fc protein retains similar but somewhat reduced activity relative to the wild-type, even though the proline knot région is disrupted. Thus, ActRIIB extracellular domains that stop at amino acid 134, 133, 132, 131, 130 and 129 (with respect to présent SEQ ID NO:1 ) are ail expected to be active, but constructs stopping at 134 or 133 may be most active. Similarly, mutations at any of residues 129-134 (with respect to SEQ ID NO: 1 ) are not expected to alter ligand-binding affïnity by large margins. In support of this, mutations of P129 and P130 (with respect to SEQ ID NO:1) do not substantially decrease ligand binding.Therefore, an ActRIIB polypeptide or an ActRIIB-based GDF Trap polypeptide of the présent disclosure may end as early as amino acid 109 (the final cysteine), however, forms ending at or between 109 and 119 (e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119) are expected to hâve reduced ligand binding. Amino acid 119 (with respect to présent SEQ ID NO:1) is
-5219001 poorly conserved and so is readily altered or truncated. ActRIIB polypeptides and ActRIIBbased GDF Traps ending at 128 (with respect to présent SEQ ID NO:1) or later should retain ligand binding activity. ActRIIB polypeptides and ActRIIB-based GDF Traps ending at or between 119 and 127 (e.g., 119, 120, 121, 122, 123, 124, 125, 126, or 127), with respect to SEQ ID NO: 1, will hâve an intermediate binding ability. Any of these forms may be désirable to use, depending on the clinical or experimental setting.
At the N-terminus of ActRIIB, it is expected that a protein beginning at amino acid 29 or before (with respect to présent SEQ ID NO:l) will retain ligand-binding activity. Amino acid 29 représente the initial cysteine. An alanine-to-asparagine mutation at position 24 (with respect to présent SEQ ID NO:1) întroduces an N-linked glycosylation sequence without substantially affecting ligand-binding. See, e.g., U.S. Patent No. 7,842,663. This confirms that mutations in the région between the signal cleavage peptide and the cysteine cross-linked région, corresponding to amino acids 20-29 are well tolerated. In particular, ActRIIB polypeptides and ActRIIB-based GDF Traps beginning at position 20, 21, 22, 23, and 24 (with respect to présent SEQ ID NO:1) should retain general ligand-biding activity, and ActRIIB polypeptides and ActRIIB-based GDF Traps beginning at positions 25, 26, 27, 28, and 29 (with respect to présent SEQ ID NO: 1 ) are also expected to retain ligand-biding activity. Data shown herein as well as in, e.g., U.S. Patent No. 7,842,663 demonstrates that, surprisingly, an ActRIIB construct beginning at 22, 23, 24, or 25 will hâve the most activity.
Taken together, an active portion (e.g., ligand-binding activity) of ActRIIB comprises amino acids 29-109 of SEQ ID NO:1. Therefore ActRIIB polypeptides and ActRIIB-based GDF Traps of the présent disclosure may, for example, comprise an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIB beginning at a residue corresponding to amino acids 20-29 (e.g., beginning at amino acid 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 and ending at a position corresponding to amino acids 109-134 (e.g., ending at amino acid 109, 110, 111, 112, 113, 114, 115, 116, 117, 118. 1 19, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1. In some embodiments, ActRIIB-based GDF Trap polypeptides of the présent disclosure do not comprise or consist of amino acids 29-109 of SEQ ID NO: 1. Other examples include polypeptides that begin at a position from 20-29 (e.g., position 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) or 21-29 (e.g., position 21, 22, 23, 24, 25, 26, 27, 28, or 29) and end at a position from 119-134 (e.g, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131. 132. 133.
-5319001 or 134), 119-133 (e.g., 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133), 129-134 (e.g. 129, 130, 131, 132, 133, or 134), or 129-133 (e.g., 129, 130, 131, 132, or 133) of SEQ ID NO: 1. Other examples include construis that begin at a position from 20-24 (e.g, 20, 21,22, 23, or 24), 21-24 (e.g., 21, 22, 23, or 24), or 22-25 (e.g, 22, 22, 23, or 25) and end at a position from 109-134 (e.g, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134), 119134 (e.g., 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) or 129-134 (e.g, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1. Variants within these ranges are also contemplated, partîcularly those having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding portion of SEQ ID NO: 1. In some embodiments, the ActRIIB polypeptides and ActRIIB-based GDF Traps comprise a polypeptide having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid residues 25131 of SEQ ID NO: 1. In certain embodiments, ActRIIB-based GDF Trap polypeptides do not comprise or consist of amino acids 25-131 of SEQ ID NO : 1.
The disclosure includes the results of an analysis of composite ActRIIB structures, shown in Figure 1, demonstrating that the ligand-binding pocket is deiined, in part, by residues Y31, N33, N35, L38 through T41, E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87, A92, and E94 through Fl01. At these positions, it is expected that conservative mutations will be tolerated, although a K74A mutation is welltolerated. as are R40A, K55A, F82A and mutations at position L79. R40 is a K in Xenopus, indicating that basic amino acids at this position will be tolerated. Q53 is R in bovine ActRIIB and K in Xenopus ActRIIB, and therefore amino acids including R, K, Q, N and 11 will be tolerated at this position. Thus, a general formula for an ActRIIB polypeptide and ActRIIB-based GDF Trap polypeptide of the disclosure is one that comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 29-109 of SEQ ID NO: 1, optionally beginning at a position ranging from 20-24 (e.g, 20, 21,22, 23, or 24) or 22-25(e.g, 22, 23, 24, or 25) and ending at a position ranging from 129-134 (e.g, 129, 130, 131, 132, 133, or 134), and comprising no more than 1,2, 5, 10 or 15 conservative amino acid changes in the ligand-binding pocket, and zéro, one or more non-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82 in the ligand-binding pocket. Sites
-5419001 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 (with respect to SEQ ID NO:1). An asparagine to alanine alteration at position 65 (N65A) actually improves ligand-binding in the A64 background, and is thus expected to hâve no detrimental effect on ligand-binding in the R64 background. See, e.g., U.S. Patent No. 7,842,663. This change probably éliminâtes glycosylation at N65 in the A64 background, thus demonstrating that a significant change in this région is likely to be tolerated. While an R64A change is poorly tolerated, R64K is well-tolerated, and thus another basic residue, such as H may be tolerated at position 64. See, e.g., U.S. Patent No. 7,842,663.
ActRIIB is well-conserved across nearly ail vertebrates, with large stretches oi the extracellular domain conserved completely. Many of the ligands that bind to ActRIIB are also highly conserved. Accordingly, comparisons of ActRIIB sequences from various vertebrate organisms provide insights into residues that may be altered. Therefore, an active, human ActRIIB variant polypeptide and ActRIIB-based GDF Trap useful in accordance with the presently disclosed methods may include one or more amino acids at corresponding positions from 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 defming an active ActRIIB variant. L46 is a valine in Xenopus ActRIIB, and so this position may be altered, and optionally may be altered to another hydrophobie 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 tolérant of a wide variety of changes, including polar residues, such as E, D, K, R, H, S, T, P, G, Y and probably A. T93 is a K in Xenopus, indicating that a wide structura] variation is tolerated at this position, with polar residues favored, such as S, K, R, E, D, H, G, P, G and Y. F108 is a Y in Xenopus, and therefore Y or other hydrophobie group, such as I, 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 H, as well as Q and N. RI 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.
It has been previously demonstrated that the addition of a further N-linked glycosylation site (N-X-S/T) is well-tolerated relative to the ActRIIB(R64)-Fc form. See, e.g., U.S. Patent No. 7,842,663. Therefore, N-X-S/T sequences may be generally introduced at positions outside the ligand binding pocket defined in Figure 1 in ActRIIB polypeptide and
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ActRIIB-based GDF Traps of the present disclosure. 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, 120-134 or 129-134 (with respect to SEQ ID NO:1). N-X-S/T sequences may also be introduced into the linker between the ActRIIB sequence and an Fc domain or other fusion component. Such a site may be introduced with minimal effort by introducing an N in the correct position with respect to a pre-existing S or T, or by introducing an S or T at a position corresponding to a pre-existing N. Thus, désirable alterations that would create an N-linked glycosylation site are: A24N, R64N, S67N (possibly combined with an N65A alteration), E105N, RI 12N, G120N, E123N, P129N, A132N, RI 12S and RI 12T (with respect to SEQ ID NO:1). 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 glycosylation. Likewise, any T that is predicted to be glycosylated may be altered to an S. Thus the alterations S67T and S44T (with respect to SEQ ID NO: 1) are contemplated. Likewise, in an A24N variant, an S26T alteration may be used. Accordingly, an ActRIIB polypeptide and ActRIIB-based GDF Trap polypeptide of the present disclosure may be a variant having one or more additional, non-endogenous N-linked glycosylation consensus sequences as described above.
The variations described herein may be combined in varions ways. Additionally, the results of the mutagenesîs program described herein indicate that lherc arc amino acid positions in ActRIIB that are often bénéficiai to conserve. With respect to SEQ ID NO:1, these include position 64 (basic amino acid), position 80 (acidic or hydrophobie amino acid), position 78 (hydrophobie, and particularly tryptophan), position 37 (acidic, and particularly aspartic or glutamic acid), position 56 (basic amino acid), position 60 (hydrophobie amino acid, particularly phenylalanine or tyrosine). Thus, in the ActRIIB polypeptides and ActRIIB-based GDF Traps disclosed herein, the disclosure provides a framework of amino acids that may be conserved. Other positions that may be désirable to conserve are as follows: position 52 (acidic amino acid), position 55 (basic amino acid), position 81 (acidic), 98 (polar or charged, particularly E, D, R or K), ail with respect to SEQ ID NO:1.
A general formula for an active (e.g., ligand binding) ActRIIA polypeptide is one that comprises a polypeptide that starts at amino acid 30 and ends at amino acid 110 of SEQ ID NO:9. Accordingly, ActRIIA polypeptides and ActRIIA-based GDF Traps of the present disclosure may comprise a polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO:9. In some embodiments, ActRIIA-based GDF Traps
-5619001 of the présent disclosure do not comprise or consist of amino acids jO-I 10 of SEQ ID NO.9. Optionally, ActRIlA polypeptides and ActRIIA-based GDF Trap polypeptides ot the présent disclosure comprise a polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids amino acids 12-82 of SEQ ID NO:9 optionally beginning at a position ranging from 1-5 (e.g., 1,2, 3, 4, or 5) or 3-5 (e.g., 3, 4, or 5) and ending at a position ranging from 110-116 (e.g., 110, 111, 112, 113, 114, 115, or 116) or 110-115 (e.g., 110, 111, 112, 113, 114, or 115), respectively, and comprising no more than 1,2, 5, 10 or 15 conservative amino acid changes in the ligand binding pocket, and zéro, one or more non-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82 in the ligand-binding pocket with respect to SEQ ID NO:9.
In certain embodiments, functionally active fragments of ActRII polypeptides (e.g. ActRIlA and ActRIIB polypeptides) and GDF Trap polypeptides of the présent disclosure can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an ActRII polypeptide or GDF Trap polypeptide (e.g., SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 46, and 48). In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can fonction as antagonists (inhibitors) of ActRII receptors and/or one or more ActRII ligands (e.g.,GDF 11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and/or Nodal).
In some embodiments, an ActRIlA polypeptide of the présent disclosure is a polypeptide comprising an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, and 28. In certain embodiments. the ActRIlA polypeptide comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10,11, 22, 26, and 28. In certain embodiments, the ActRIlA polypeptide consists essentially of, or consists of, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, and 28.
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In some embodiments, an ActRIIB polypeptide of the présent disclosure is a polypeptide comprising an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: l, 2, 3, 4, 5, 6, 29, 31, and 49. In certain embodiments, the ActRIIB polypeptide comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 31, and 49. In certain embodiments, the ActRIIB polypeptide consists essentially of, or consists of, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 31, and 49.
In some embodiments, a GDF Trap polypeptide of the present disclosure is a variant ActRIIB polypeptide comprising an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 30, 31, 36, 37, 38, 41,44, 45, 49, 50, and 51. In certain embodiments, the GDF Trap comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 30, 31,36, 37, 38, 41,44, 45, 49, 50, and 51. In certain embodiments, the GDF Trap comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 30, 31, 36, 37, 38, 41,44, 45, 49, 50, and 51, wherein the position corresponding to L79 of SEQ ID NO:1,4, or 49 is an acidic amino acids (a D or E amino acid residue). In certain embodiments, the GDF Trap consists essentially of, or consists of, an amino acid sequence that at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 41,44, 45, 50, and 51. In certain embodiments, the GDF Trap does not comprise or consists of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, and 31.
In some embodiments, a GDF Trap polypeptide of the present disclosure is a variant ActRlIA polypeptide comprising an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, 28, 29, and 31. In certain embodiments, the GDF Trap comprises an amino acid sequence that is at least 80%. 81%,
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82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, 28, 29, and 31. In certain embodiments, the GDF Trap consists essentially of, or consists of, an amino acid sequence that at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, 28, 29, and 31. In certain embodiments, the GDF Trap does not comprise or consists of an amino acid sequence selected from SEQ ID NOs: 9, 10, 11,22, 26, 28, 29, and 31.
In some embodiments, the présent disclosure contemplâtes making functional variants by modifying the structure of an ActRII polypeptide (e.g. and ActRIIA or ActRIIB polypeptide) or a GDF Trap for such purposes as enhancing therapeutic effîcacy, or stability (e.g., shelf-life and résistance to proteolytic dégradation in vivo). Variants can be produced by amino acid substitution, délétion, addition, or combinations thereof. For instance, it is reasonable to expect that an isolated replacement of a leucine with an isolcucine or valine. an aspartate with a glutamate, a threonine with a serine, or a sîmilar replacement of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not hâve a major effect on the biological activity of the resulting molécule. 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 polypeptide of the disclosure rcsults in a functional homolog can be rcadily determined by assessing the ability of the variant polypeptide to produce a response in cells in a fashion similar to the wild-type polypeptide, or to bind to one or more ligands, such as GDFl 1, activin A, activin B, activin AB, activin C, activin E, GDF8, BMP6, and BMP7, as compared to the unmodified or a wild-type polypeptide.
In certain embodiments, the présent disclosure contemplâtes spécifie mutations oi ActRII polypeptides and GDF Trap polypeptides of the présent disclosure so as to alter the glycosylation of the polypeptide. Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation récognition sites generally comprise a tripeptide sequence, asparagine-X-threonine or asparagine-X-serine (where “X” is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the polypeptide (for O-linked glycosylation sites). A variety of amino acid
-5919001 substitutions or délétions at one or both of the first or third amino acid positions of a glycosylation récognition site (and/or amino acid délétion at the second position) results in non-glycosylation at the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal of one or more carbohydrate moieties présent on a polypeptide may be accomplished chemically and/or enzymatically. Chemical deglycosylation may involve, for example, exposure of a polypeptide to the compound trifluoromethanesulfonic acid, or an équivalent compound. This treatment results in the cleavage of most or ail sugars except the linking sugar (N-acctylglucosaminc or Nacetylgalactosamine), while leaving the amino acid sequence intact. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. [Meth. EnzymoL (1987) 138:350], The sequence ofa polypeptide may be adjusted, as appropriate, depending on the type of expression System used, as mammalian, yeast, insect, and plant cells may ail introduce differing glycosylation patterns thaï can be affected by the amino acid sequence of the peptide. In general, ActRII polypeptides and GDF Trap polypeptides of the présent disclosure for use in humans may be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are expected to be useful as well.
This disclosure further contemplâtes a method of generating mutants, particularly sets of combinatorial mutants of ActRII polypeptides and GDF Trap polypeptides of the présent disclosure, as well as truncation mutants. Pools of combinatorial mutants are especially useful for identifying ActRII and GDF Trap sequences. The purpose of screening such combinatorial libraries may be to generale, for example, polypeptides variants which hâve altered properties, such as altered pharmacokinetic or altered ligand binding. A variety of screening assays are provided below, and such assays may be used to evaluatc variants. For example, ActRII polypeptides and GDF Trap polypeptides may be screened for ability to bind to an ActRII receptor, to prevent binding of an ActRII ligand (e.g., GDF11, GDF8,
I
-6019001 activin A, activin B, activin AB, activin C, activin E, BMP7, BMP6, and/or Nodai) lo an ActRII polypeptide, or to interfère with signaling caused by an ActRII ligand.
The activity of an ActRII polypeptides or GDF Trap polypeptides may also be tested in a cell-based or in vivo assay. For example, the effect of an ActRII polypeptide or GDF Trap polypeptide 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 ActRII ligand proteins (e.g., GDFl 1, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP7, BMP6, and/or Nodal), and cells may be transfected so as to produce an ActRII polypeptide or GDF Trap polypeptide, and optionally, an ActRII ligand. Likewise, an ActRII polypeptide or GDF Trap polypeptide may be administered to a mouse or other animal, and one or more blood count measurements (e.g., an RBC count, hemoglobin, or réticulocyte) or cutaneous ulcer parameters may be assessed using art recognized methods.
Combinatorial-derived variants can be generated which hâve a sélective or generally increased potency relative to a référencé ActRII polypeptide or GDF Trap polypeptide. Such variants, when expressed from recombinant DNA constructs, can be used in gene therapy protocols. Likewise, mutagenesis can give rise to variants which hâve intracellular half-lives dramatically different than the corresponding unmodified ActRII polypeptide or GDF Trap polypeptide. For example, the altered protein can be rendered either more stable or less stable to proteolytic dégradation or other cellular processes which resuit in destruction of, or otherwise inactivation of an unmodified polypeptide. Such variants, and the genes which encode them, can be utilized to alter ActRII polypeptide or GDF Trap polypeptide levels by modulating the half-life of the polypeptide. 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 ActRII polypeptide or GDF Trap polypeptide levels within the cell. In an Fc fusion protein, mutations may be made in the linker (if any) and/or the Fc portion to alter the half-life of the protein.
A combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential ActRII or GDF Trap sequences. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate sel of potential ActRII or GDF Trap polypeptide encoding nucléotide sequences are expressible as individual polypeptides, or alternat!vely, as a set of larger fusion proteins (e.g., for pliage display).
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There are many ways by which the library of potential homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes can then be ligated into an appropriate vector for expression. The synthesis of degenerate oligonucleotides is well known in the art. See, e.g.,Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques hâve been employed in the directed évolution of other proteins. See, e.g.. Scott et al., (1990) Science 249:386-390; Roberts et al. (1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Patent Nos: 5,223,409, 5,198,346, and 5,096,815).
Altematively, other forms of mutagenesis can be utilized to generate a combinatorial library. For example, ActRIl polypeptides or GDF Trap polypeptides of the présent disclosure can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis [see, e.g., Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601; Nagashima étal. (1993) J. Biol. Chem. 268:28882892; Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science 244:1081-1085], by linker scanning mutagenesis (see, e.g., Gustin et al. (1993) Virology 193:653-660; and Brown et al. (1992) Mol. Cell Biol. 12:2644-2652; McKnighl et al. (1982) Science 232:316), by saturation mutagenesis [see, e.g., Meyers et al., (1986) Science 232:613]; by PCR mutagenesis [see, e.g., Leung et al. (1989) Method Cell Mol Biol 1:11-19]; or by random mutagenesis, including chemical mutagenesis [see, e.g., Miller et al. (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al. (1994) Strategies in Mol Biol 7:32-34]. Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method for identifying truncated (bioactive) forms of ActRIl polypeptides.
A wide-range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ActRIl polypeptides or GDF Trap polypeptides of the
-6219001 disclosure. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, iransforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which détection of a desired activity facilitâtes relatively easy isolation of the vector encoding the gene whose product was detected. In some embodiments, assays include ActRIl ligand (e.g., GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP7, BMP6, and/or Nodal) binding assays and/or ActRIl ligand-mediated cell signaling assays.
In certain embodiments, ActRIl polypeptides or GDF Trap polypeptides ol the présent disclosure may further comprise post-translational modifications in addition to any that are naturally présent in the ActRIl (e.g., an ActRIIA or ActRIIB polypeptide) or GDF Trap polypeptide. Such modifications include, but are not limited to, acétylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a resuit, the ActRIl polypeptide or GDF Trap polypeptide may contain non-amino acid éléments, such as polyethylene glycols, lipids, polysaccharide- or mono-saccharide, and phosphates. Effects of such nonamino acid éléments on the functionality of a ligand Trap polypeptide may be tested as described herein for other ActRIl or GDF Trap variants. When a polypeptide of the disclosure is produced in cells by cleaving a nascent form of the polypeptide, posttranslational processing may also be important for correct folding and/or function of the protein. Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) hâve spécifie cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the ActRIl polypeptides or GDF Trap polypeptides.
In certain aspects, ActRIl polypeptides or GDF Trap polypeptides ot the présent disclosure include fusion proteins having at least a portion (domain) of an ActRIl polypeptide (e.g., an ActRIIA or ActRIIB polypeptide) or GDF Trap polypeptide and one or more heterologous portions (domains). Well known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain Fc région, maltose binding protein (MBP), or human sérum albumin. A fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particuiarly useful for isolation of the fusion proteins by affmity chromatography. For the purpose of affmity purification, relevant matrices for affmity chromatography, such as glutathione-, amylase-, and nickel- or cobalt
-6319001 conjugated resins are used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification System and the QIAexpress™ System (Qiagen) useful with (HISe) fusion partners. As another example, a fusion domain may be selected so as to facilitate détection of the ligand Trap polypeptides. Examples of such détection domains include the varions fluorescent proteins (e.g., GFP) as well as “epitope tags,” which are usually short peptide sequences for which a spécifie antibody is available. Well known epitope tags for which spécifie monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags. In some cases, the fusion domains hâve 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 subséquent chromatographie séparation. In certain embodiments, an ActRII polypeptide or a GDF Trap polypeptide is fused with a domain that stabilizes the polypeptide in vivo (a “stabilizer” domain). By “stabilizing” is meant anything that increases sérum 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 immunoglobulîn are known to confer désirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human sérum albumin can confer désirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains (that confer an additional biological fonction, such as further stimulation of muscle growth).
In certain embodiments, the present disclosure provides ActRII or GDF Trap fusion proteins comprising an immunoglobulîn Fc domain. In some embodiments. the immunoglobulîn Fc domain is a mammalian immunoglobulîn domain. In some embodiments, the immunoglobulîn Fc domain is a human immunoglobulîn domain. In some embodiments, the immunoglobulîn Fc domain is a mouse immunoglobulîn domain. In certamin embodiments, the immunoglobulîn Fc domain is an IgA, IgD, IgE, IgG, or IgM Fc domain. In certain embodiments, the immunoglobulîn Fc domain is an IgG[, IgGj, IgGj, IgÛ4, IgA], or IgA? Fc domain. In some embodiments, the immunoglobulîn Fc domain is a human IgG l Fc domain, or a human IgG2 Fc domain.
In certain embodiments, the present disclosure provides ActRII or GDF Trap fusion proteins comprising the following IgGl Fc domain sequence:
THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV WDVSHEDPE
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VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK
101 VSNKALPVPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ ID NO:14).
In other embodiments, the present disclosure provides ActRII or GDF Trap fusion proteins comprising the following variants of the IgGl Fc domain:
1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE
51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK
101 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV
201 FSCSVMHEAL HNHYTQKSLS LSPGK (SEC ) ID NO:64)
1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV WD (A) VSHEDPE
51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK(A)
101 VSNKALPVPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF
151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG PFFLYSKLTV DKSRWQQGNV
201 FSCSVMHEAL HN(A)HYTQKSLS LSPGK (SEQ ID NO: 15).
Optionally, the IgGl Fc domain has one or more mutations at residues such as Asp265, lysine 322, and Asn-434. In certain cases, the mutant IgGl Fc domain having one or more of these mutations (e.g., Asp-265 mutation) has reduced ability of binding to the Fcy receptor relative to a wild-type 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 MFIC class 1-related Fc-receptor (FcRN) relative to a wild-type IgGl Fc domain.
In certain other embodiments, the present disclosure provides ActRII or GDF trap fusion proteins comprising variants of the lgG2 Fc domain, including the following:
1 VECPPCPAPP VAGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSHEDPEVQ
51 FNWYVDGVEV HNAKTKPREE QFNSTFRWS VLTWHQDWL NGKEYKCKVS
101 NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP
151 SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS
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201 CSVMHEALHN HYTQKSLSLS PGK (SEQ ID NO:65)
It is understood that different éléments of the fusion proteins may be arranged in any manner that is consistent with the desired functionality. For example, an ActRII polypeptide domain or GDF Trap polypeptide domain may be placed C-terminal to a heterologous domain, or alternatively, a heterologous domain may be placed C-terminal to an ActRII polypeptide domain or GDF Trap polypeptide domain. The ActRII polypeptide domain or 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.
For example, an ActRII or GDF Trap fusion protein may comprises an amino acid sequence as set forth in the formula A-B-C. The B portion corresponds to an ActRII polypeptide domain or a GDF Trap polypeptide domain. The A and C portions may be independently zéro, one or more than one amino acids, and both the A and C portions when présent are heterologous to B. The A and/or C portions may be 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 [e.g., a TGGG linker (SEQ ID NO:53)]. In certain embodiments, an ActRII or GDF Trap fusion protein comprises an amino acid sequence as set forth in the formula A-B-C, wherein A is a leader (signal) sequence, B consists of an ActRII or GDF polypeptide domain, 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, an ActRII or GDF Trap fusion protein comprises an amino acid sequence as set forth in the formula A-B-C, wherein A is a TPA leader sequence, B consists of an ActRII or GDF polypeptide domain, and C is an immunoglobulin Fc domain. In some embodiments, fusion proteins comprise the amino acid sequences set forth in any one of SEQ IDNOs: 22, 26, 29, 31, 36, 38, 41,44, and 51.
In certain embodiments, ActRII polypeptides or GDF Trap polypeptides of the présent disclosure contain one or more modifications that are capable of stabilizing the polypeptides. For example, such modifications enhance the in vitro half-life of the polypeptides, enhance circulatory half-life of the polypeptides, and/or reduce proteolytic dégradation of the polypeptides. Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRII polypeptide domain or a GDF Trap polypeptide domain and a stabilizer domain), modifications of a glycosylation site
-6619001 (including, for example, addition of a glycosylation site to a polypeptide of the disclosure), and modifications of carbohydrate moiety (including, for example, removal of carbohydrate moieties from a polypeptide of the disclosure). As used herein, the term “stabilizer domain” not only refers to a fusion domain (e.g., an immunoglobulin Fc domain) as in the case of fusion proteins, but also includes nonproteinaceous modifications such as a carbohydrate moiety, or nonproteinaceous moiety, such as polyethylene glycol.
In some embodiments, ActRII polypeptides and GDF Traps to be used in accordance with the methods described herein are isolated polypeptides. As used herein, an isolated protein or polypeptide is one which has been separated from a component of its natural environment. In some embodiments, a polypeptide of the disclosure is purified to greater than 95%, 96%, 97%, 98%, or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographie (e.g., ion exchange or reverse phase HPLC). Methods for assessment of antibody purity are well known in the art. See, e.g., Flatman et al., (2007) J. Chromatogr. B 848:79-87.
In certain embodiments, ActRII polypeptides and GDF Traps of the disclosure can be produced by a variety of art-known techniques. For example, polypeptides of the disclosure can be synthesized using standard protein chemistry techniques such as those described in Bodansky, M. Prînciples of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (see, e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600). Altematively, the polypeptides of the disclosure, including fragments or variants thereof, may be recombinantly produced using varions expression Systems (e.g., E. coli, Chinese Hamster Ovary (CHO) cells, COS cells, baculovirus) as is well known in the art. In a further embodiment, the modified or unmodified polypeptides of the disclosure may be produced by digestion of recombinantly produced full-length ActRII or GDF Trap polypeptides by using, for example, a protease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE). Computer analysis (using a commercially available software, e.g., MacVector, Oméga, PCGene, Molecular Simulation, Inc.) can be used to identify proteolytic clcavagc sites. Altematively, such polypeptides may be produced from recombinantly produced fulllength ActRII or GDF Trap polypeptides using chemical cleavage (e.g., cyanogen bromide, hydroxylamine, etc. ).
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Any of the ActRII polypeptides disclosed herein (e.g., ActRIIA or ActRIIB polypeptides) can be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., treat or prevent an anémia, treat or prevent one or more complications of anémia such as cutaneous ulcers, etc.). In some embodiments, the desired effect is treating one or more complications of anémia such as cutaneous ulcers. In some embodiments, the desired effect is preventing one or more complications of anémia such as cutaneous ulcers. For example, an ActRII polypeptide disclosed herein can be used in combination with i) one or more additional ActRII polypeptides disclosed herein. ii) one or more GDF Traps disclosed herein; iii) one or more ActRII antagonist antibodics disclosed herein (e.g., an anti-activin A antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl l antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an anti-BMP7 antibody, an anti-ActRIIA antibody, and/or or an anti-ActRIIB antibody); iv) one or more small molécule ActRII antagonists disclosed herein (e.g., a small molécule antagonist of one or more of GDFl l, GDF8, actîvin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); v) one or more of the polynucleotide ActRII antagonists disclosed herein (e.g., a polynucleotide antagonist of one or more of GDFl l, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); vi) one or more follistatin polypeptides disclosed herein; and/or vii) one or more FLRG polypeptides disclosed herein.
Similarly, any of the GDF Traps disclosed herein can be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., treat or prevent an anémia, treat or prevent one or more complications of anémia such as cutaneous ulcers, etc.). In some embodiments, the desired effect is treating one or more complications of anémia such as cutaneous ulcers. In some embodiments, the desired effect is preventing one or more complications of anémia such as cutaneous ulcers. For example, a GDF Trap disclosed herein can be used in combination with i) one or more additional GDF Traps disclosed herein, ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIIA or ActRIIB polypeptides) disclosed herein; iii) one or more ActRII antagonist antibodies disclosed herein (e.g., an anti-activin A antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an antî-GDF8 antibody, an anti-BMP6 antibody, an anti-BMP7 antibody, an anti-ActRIIA antibody, and/or or an antiActRIIB antibody); iv) one or more small molécule ActRII antagonists disclosed herein (e.g., a small molécule antagonist of one or more of GDFl 1, GDF8, activin A, activin B, activin
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AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); v) one or more of the polynucleotide ActRIl antagonists disclosed herein (e.g., a polynucleotide antagonist of one or more of GDFl l, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); vi) one or more follistatin polypeptides disclosed herein; and/or vii) one or more FLRG polypeptides disclosed herein.
B. Nucieic Acids EncodingActRII Polypeptides and GDF Traps
In certain embodiments, the présent disclosure provides isolated and/or recombinant nucieic acids encoding the ActRIl polypeptides and GDF Trap polypeptides (including fragments, iùnctional variants, and fusion proteins thereof) disclosed herein. For example, SEQ ID NO:l2 encodes the naturally occurring human ActRIIA precursor polypeptide, while SEQ ID NO:l3 encodes the processed extracellular domain of ActRIIA. In addition, SEQ ID NO:7 encodes a naturally occurring human ActRIIB precursor polypeptide (the R64 variant described above), while SEQ ID NO:8 encodes the processed extracellular domain of ActRIIB (the R64 variant described above). The subject nucieic acids may be single-stranded or double stranded. Such nucieic acids may be DNA or RNA molécules. These nucieic acids may be used, for example, in methods for making ActRII-based ligand Trap polypeptides of the présent disclosure.
As used herein, isolated nucieic acid(s) refers to a nucieic acid molécule that has been separated from a component of its naturel environment. An isolated nucieic acid includes a nucieic acid molécule contained in cells that ordinarily contain the nucieic acid molécule, but the nucieic acid molécule is présent extrachromosomally or at a chromosomal location that is different from its naturel chromosomal location.
In certain embodiments, nucieic acids encoding ActRIl polypeptides and GDF Traps of the présent disclosure are understood to include nucieic acids that are variants of any one of SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48. Variant nucléotide sequences include sequences that differ by one or more nucléotide substitutions, additions, or délétions including allelic variants, and therefore, wili including coding sequences that differ from the nucléotide sequence designated in any one of SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48.
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In certain embodiments, ActRII polypeptides and GDF Traps of the présent disclosure are encoded by isolated or recombinant nucleic acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48 In some embodiments, GDF Traps of the présent disclosure are not encoded by nucleic acid sequences that comprise or consist of any one of nucléotide sequences corresponding to any one of SEQ ID NOs: 7, 8, 12, 13, 27, and 32. One of ordinary skill in the art will appreciate that nucleic acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences complementary to SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 42, 47, and 48, and variants thereof, are also within the scope of the présent disclosure. In further embodiments, the nucleic acid sequences of the disclosure can be isolated, recombinant, and/or fused with a heterologous nucléotide sequence, or in a DNA library.
In other embodiments, nucleic acids of the present disclosure also include nucléotide sequences that hybridize under highly stringent conditions to the nucléotide sequence designated in SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48, complément sequence of SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43,46, 47, and 48, 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 varicd. 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 chloride/sodium citrate (SSC) at about 45 °C, followed by a wash of 2.0 x SSC at 50 °C. For example, the sait concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 °C to a high stringency of about 0.2 x SSC at 50 °C. In addition, the température in the wash step can be increased from low stringency conditions at room température, about 22 °C, to high stringency conditions at about 65 °C. Boîh température and sait may be varied, or température or sait concentration may be held constant while the other variable is changed. In one embodiment. the disclosure provides nucleic acids which hybridize under low stringency conditions of 6 x SSC at room température followed by a wash at 2 x SSC at room température.
Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48 due to degeneracy in the genetic code are also within the scope of the disclosure. For example, a number of amino acids are designated
-7019001 by more than one triplet. Codons that specify the saine amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may resuit in “silent” mutations which do not affect the amino acid sequence of the protein. 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 nucléotides (up to about 3-5% of the nucléotides) of the nucleic acids encoding a particular protein may exist among individuals of a given species due to naturel allelic variation. Any and ail such nucléotide variations and resulting amino acid polymorphisms are within the scope of this disclosure.
In certain embodiments, the recombinant nucleic acids of the présent disclosure may be operably linked to one or more regulatory nucléotide sequences in an expression construcl. Regulatory nucléotide 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 nucléotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the disclosure. The promoters may be either naturally occurring promoters, or hybrid promoters that combine éléments of more than one promoter. An expression construct may be présent in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In sonie embodiments, the expression veclor conlains a sclcclable marker genc to allow the sélection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.
In certain aspects of the présent disclosure, the subject nucleic acid is provided in an expression vector comprising a nucléotide sequence encoding an ActRII polypeptide or a GDF Trap and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of the ActRII or GDF Trap polypeptide. Accordingly, the term regulatory sequence inciudes promoters, enhancers, and other expression control éléments. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology. Methods in Enzymoîogy, Academie Press, San Diego, CA ( 1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to
-7l19001 express DNA sequences encoding anActRII or GDF Trap polypeptide. Such useful expression control sequences, include, for example, the early and late promoters ol SV4Ü. tel promoter, adenovirus or cytomégalovirus immédiate early promoter, RSV promoters, the lac System, the trp System, the TAC or TRC System, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter régions of phage lambda , the control régions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus System and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and varions combinations thereof. It should be understood that the design of the expression vector may dépend on such factors as the choice of the host cell to be transfonned and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
A recombinantnucleic acid of the présent disclosure can be produced by ligating the cloned gene, or a portion thereof, înto a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinantActRII or GDF Trap polypeptide include plasmids and other vectors. For instance, suitable vectors include plasmids of the following types: pBR322derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E.coli.
Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pllyg derîved vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate réplication and drug résistance sélection in both prokaryotic and eukaryotic cells. Altematively, dérivatives of viruses such as the bovine papîlloma virus (BPV-l), or EpsteinBarr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retrovîral) expression Systems can be found below in the description of gene therapy delivery Systems. The varions methods employed in the préparation of the plasmids and in transformation of host organisms are well
-7219001 known in the art. For other suitable expression Systems for bolh prokaryotic and eukaryotic cells, as well as general recombinant procedures. See, e.g., Molecular Cloning A Laboratory Manual, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some instances, it may be désirable 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 pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the B-gal containing pBlueBac III).
In some embodiments, a vector will be designed for production of the subject ActRII or GDF Trap polypeptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wisc.), As will be apparent, the subject gene constructs can be used to cause expression of the subjectActRII polypeptides in cells propagated in culture, e.g., to produce proteins, including fusion proteins or variant proteins, for purification.
This disclosure also pertains to a host cell transfected with a recombinantgene including a coding sequence for one or more of the subject ActRII or GDF Trap polypeptides. The host cell may be any prokaryotic or eukaryotic cell. For example, an ActRII or GDF Trap polypeptide of the disclosure may be expressed in bacterial cells such as E. coli, însect cells (e.g., using a baculovirus expression System), yeast, or mammalian cells [e.g. a Chinese hamster ovary (CHO) cell line], Other suitable host cells are known to those ski lied in the art.
Accordingly, the présent disclosure further pertains to methods of producing the subjectActRII and GDF Trap polypeptides. For example, a host cell transfected with an expression vector encoding an ActRII or GDF Trap polypeptide can be cultured under appropriate conditions to allow expression of the ActRII or GDF Trap polypeptide to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Altematively, the ActRII or GDF 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 art. The subject polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies spécifie for particular epitopes of theActRII or GDF Trap polypeptides and affinity purification with an agent that binds to a
-7319001 domain fused to the ActRII or GDF Trap polypeptide (e.g., a protein A column may be used to purify an ActRII-Fc or GDF Trap-Fc fusion protein). In some embodiments, the ActRII or GDF Trap polypeptide is a fusion protein containing a domain which facilitâtes its purification.
In some embodiments, purification is achieved by a sériés of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification could be completed with viral filtration and buffer exchange. An ActRII-Fc or GDF Trap-Fc protein may be purified to a purity of >90%, >95%, >96%, >98%, or >99% as determined by size exclusion chromatography and >90%, >95%, >96%, >98%, or >99% as determined by SDS PAGE. The target level of purity should be one that is sufficient to achieve désirable results in mammalian Systems, particularly non-human primates, rodents (mice), and humans.
In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant ActRII or GDF Trap polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni2+ métal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purifiedActRII or GDF Trap polypeptide. See, e.g., Hochulie/ al. (1987) J. Chromatography 411:177; and Janknecht et al. (1991) PNAS USA 88:8972.
Techniques for making fusion genes are well known. Essentially, the joining of varions DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Altematively, PCR amplification of gene fragments can be carried out using anchor prîmers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence. See, e.g., Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992.
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C. Antibody Antagonist
In certain aspects, the présent disclosure relates to an antibody, or combination of antibodies, that antagonize ActRII activity (e.g., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling). In particular, the disclosure provides methods of using an antibody ActRII antagonist, or combination of antibody ActRII antagonists, to, e.g., treat or prevent an anémia in a subject in need thereof and/or treat or prevent one or more complication of anémia including, for example, cutaneous ulcers. In some embodiments, the disclosure provides methods of using an antibody ActRII antagonist, or combination of antibody ActRII antagonists, to treat an anémia in a subject in need thereof and/or treat one or more complications of anémia including, for example, cutaneous ulcers in a subject having anémia. In some embodiments, the disclosure provides methods of using an antibody ActRII antagonist, or combination of antibody ActRII antagonists, to prevent an anémia in a subject in need thereof and/or prevent one or more complications of anémia including, for example, cutaneous ulcers in a subject having anémia.
In certain embodiments, an antibody ActRII antagonist of the disclosure is an antibody, or combination of antibodies, that binds to and/or inhibits activity of at least GDF11 (e.g., GDF11-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). Optionally, the antibody, or combination of antibodies, further binds to and/or inhibits activity of GDF8 (e.g., GDF8-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling), particularly in the case of a multi-specific antibody that has binding affinity for both GDF11 and GDF8 or in the context of a combination of one or more anti-GDFl 1 antibodies and onc or more anli-GDF8 antibodies. Optionally, an antibody, or combination of antibodies, of the disclosure does not substantially bind to and/or tnhibil activity of activin A (e.g., activin A-mediated activation of ActRIIA or ActRIIB signaling transduction, such as SMAD 2/3 signaling). In some embodiments, an antibody, or combination of antibodies, of the disclosure that binds to and/or inhibits the activity of GDF11 and/or GDF8 further binds to and/or inhibits activity of one of more of activin A. activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal (e.g., activation of ActRIIA or ActRIIB SMAD 2/3 and/or SMAD 1/5/8 signaling), particularly in the case of a muiti-specific antibody that has binding affinity for multiple ActRII ligands or in the context of a combination of multiple antibodies - each having binding affinity for a different ActRII ligand.
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In certain aspects, an ActRII antagonîst of the présent disclosure is an antibody, or combination of antibodies, that binds to and/or inhibits activity of at least GDF8 (e.g.. GDF8mediated activation of ActRIlA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). Optionally, the antibody, or combination of antibodies, further binds to and/or inhibits activity of GDF11 (e.g., GDF1 l-mediated activation of ActRIlA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling), particularly in the case of a multispecific antibody that has binding affmity for both GDF8 and GDFl l or in the context of a combination of one or more anti-GDF8 antibodies and one or more anti-GDFl l antibodies. Optionally, an antibody, or combination of antibodies, of the disclosure does not substantially bind to and/or inhibit activity of activin A (e.g., activin A-mediated activation of ActRIlA or ActRIIB signaling transduction, such as SMAD 2/3 signaling). In some embodiments, an antibody, or combination of antibodies, of the disclosure that binds to and/or inhibits the activity of GDF8 and/or GDFl l further binds to and/or inhibits activity of one of more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal (e.g., activation of ActRIlA or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling), particularly in the case of a multi-specific antibody that has binding affmity for multiple ActRII ligands or in the context of a combination multiple antibodies - each having binding affmity for a different ActRII ligand.
In another aspect, an ActRII antagonist of the présent disclosure is an antibody, or combination of antibodies, that binds to and/or inhibits activity of an ActRII receptor (e.g. an ActRIlA or ActRIIB receptor). In some embodiments, an anti-ActRII receptor antibody (e.g. an anti-ActRIIA or anti-ActRIIB receptor antibody), or combination of antibodies, of the disclosure binds to an ActRII receptor and prevents binding and/or activation of the ActRII receptor by at least GDFl l (e.g.,GDFl l-mediated activation of ActRIlA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). Optionally, an anti-ActRII receptor antibody, or combination of antibodies, of the disclosure further prevents binding and/or activation of the ActRII receptor by GDF8. Optionally, an anti-ActRII receptor antibody, or combination of antibodies, of the disclosure does not substantially inhibit activin A from binding to and/or activating an ActRII receptor. In some embodiments, an anti-ActRII receptor antibody, or combination of antibodies, of the disclosure that binds to an ActRII receptor and prevents binding and/or activation of the ActRII receptor by GDFl l and/or GDF8 further prevents binding and/or activation of the ActRII receptor by one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal.
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The term antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An antibody fragment refers to a molécule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molécules (e.g., scFv); and multispecific antibodies formed from antibody fragments. See, e.g., Fludson et al. (2003) Nat. Med. 9:129-134; Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (SpringerVerlag, New York), pp. 269-315 (1994); WO 93/16185; and U.S. Pat. Nos. 5,571,894, 5,587,458, and 5,869,046. Antibodies disclosed herein may be polyclonal antibodies or monoclonal antibodies. In certain embodiments, the antibodies of the présent disclosure comprise a label attached thereto and able to be detected (e.g., the label can be a radioisotope, fluorescent compound, enzyme, or enzyme co-factor). In some embodiments, the antibodies of the présent disclosure are isolated antibodies.
Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; Hudson et al. (2003) Nat. Med. 9:129134 (2003); and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. Triabodies and tetrabodies are also described in Hudson et al. (2003) Nat. Med. 9:129-134.
Single-domain antibodies are antibody fragments comprising ail or a portion of the heavy chain variable domain or ail or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody. See, e.g., U.S. Pat.No. 6,248,516.
Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coii or phage), as described herein.
The antibodies herein may be of any class. The class of an antibody refers to the type of constant domain or constant région possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), for example, IgGt, IgG2, IgGs, IgG4, IgAi, and IgA2. The
-7719001 heavy chain constant domains that correspond to the different classes oi immunoglobulins arc called alpha, delta, epsilon, gamma, and mu.
In general, an antibody for use in the methods disclosed herein specifically binds to its target antigen, preferably with high binding affïnity. Affïnity may be expressed as a Kd value and reflects the intrinsic binding affïnity (e.g., with minimized avidity effects). Typically, binding affïnity is measured in vitro, whether in a cell-free or cell-associated setting. Any of a number of assays known in the art, încluding those disclosed herein, can be used to obtain binding affïnity measurements încluding, for example, surface plasmon résonance (Biacore™ assay), radiolabeled antigen binding assay (RIA), and ELISA. In some embodiments, antibodies of the présent disclosure bind to their target antigens (e.g. GDFl 1, GDF8, ActRIlA, ActRIIB, etc.) with at least a K.D of 1χ 1θ’7 or stronger, IxlO'8 or stronger, IxlO'9 or stronger, IxlO'10 or stronger, Ix 10’11 or stronger, IxlO'12 or stronger. IxlO’13 or stronger. or IxlO'14 or stronger.
In certain embodiments, Kp is measured by RIA performed with the Fab version of an antibody of interest and its target antigen as described by the following assay. Solution binding affïnity of Fabs for the antigen is measured by equilibrating Fab with a minimal concentration of radiolabeled antigen (e.g·., 1_‘I-labeled) in the presence of a titration sériés of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate. See, e.g., Chen et al. (1999) J. Mol. Biol. 293:865-881. To establish conditions for the assay, multi-well plates (e.g., MICROTITER® from Thermo Scientific) are coated (e.g, ovemight) with a capturing anti-Fab antibody (e.g., from Cappel Labs) and subsequently blocked with bovine sérum albumin, preferably at room température (approximately 23°C). In a nonadsorbent plate, radiolabeled antigen are mixed with serial dilutions of a Fab of interest [e.g.. consistent with assessment of the anti-VEGF antibody, Fab-12, in Presla et al., (1997) Cancer Res. 57:4593-4599], The Fab of interest is then incubated, preferably ovemight but the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation, preferably at room température for about one hour. The solution is then removed and the plate is washed times several times, preferably with polysorbate 20 and PBS mixture. When the plates hâve dried, scintillant (e.g, MICROSCINTK’ from Packard) is added, and the plates are counted on a gamma counter (e.g., TOPCOUNT® from Packard).
According to another embodiment, Kd is measured using surface plasmon résonance assays using, for example a BIACORE® 2000 or a BIACORE® 3000 (Biacore, Inc.,
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Piscataway, N.J.) with immobilized antigen CM5 chips at about 10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, Biacore Inc.) are activated with N-cthyl-N'-(3-dimelhylaminopropyl)-carbodiimidc hydrochloride (EDC) and Nhydroxysuccinimide (NHS) according to the supplier's instructions. For example, an antigen can be diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ml (about 0.2 μΜ) before injection at a flow rate of 5 μΐ/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at at a flow rate of approximately 25 μΐ/min. Association rates (kon) and dissociation rates (kOfr) are calculated using, for example, a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fittîng the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k0fl7 kon. See,e.g., Chen et al., (1999) J. Mol. Biol. 293:865-881. If the on-rate exceeds, for example, 106 M'1 s'1 by the surface plasmon résonance assay above, then the on-rate can be delermined by using a fluorescent quenchîng technique that measures the increase or decrease in fluorescence émission intensity (e.g., excitation=295 nm; emission=340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in PBS in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO spectrophotometer (ThermoSpectronic) with a stirred cuvette.
As used herein, anti-GDFl 1 antibody generally refers to an antibody that is capable of binding to GDF11 with sufficient affmity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GDF11. In certain embodiments, the extent of binding of an anti-GDFl 1 antibody to an unrelated, non-GDFl 1 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody to GDF11 as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, an antiGDFl 1 antibody binds to an epitope of GDF11 that is conserved among GDF11 from different species. In certain some embodiments, an anti-GDFl 1 antibody of the présent disclosure is an antagonist antibody that can inhibit GDF11 activity. For example, an antiGDFl 1 antibody of the disclosure may inhibit GDF11 from binding to a cognate receptor (e.g., ActRIIA or ActRIIB receptor) and/or inhibit GDF11-mediatcd signal transduction (activation) of a cognate receptor, such as SMAD2/3 signaling by ActRIIA and/or ActRIIB
-7919001 receptors. In some embodiments, anti-GDFl l antibodies of the présent disclosure do not substantially bind to and/or inhibit activity of activin A. It should be noted that GDFl 1 has high sequence homology to GDF8 and therefore antibodies that bind and/or to GDFl 1, in some cases, may also bind to and/or inhibit GDF8.
An anti-GDF8 antibody refers to an antîbody that is capable of binding to GDF8 with sufficient affinity such that the antibody is useful as a diagnostic and/or lherapeutic agent in targeting GDF8. In certain embodiments, the extent of binding of an anti-GDF8 antibody to an unrelated, non-GDF8 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody to GDF8 as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, an anti-GDF8 antibody binds to an epitope of GDF8 that is conserved among GDF8 from different species. In some embodiments, an anti-GDF8 antibody of the présent disclosure is an antagonist antibody that can inhibit GDF8 activity. For example, an anti-GDF8 antibody of the disclosure may inhibit GDF8 from binding to a cognate receptor (c.g.,|ActRIIA or ActRIIB receptor) and/or inhibit GDF8-mediated signal transduction (activation) of a cognate receptor, such as SMAD2/3 signaling by ActRIIA and/or ActRIIB receptors. In some embodiments, anti-GDF8 antibodies of the présent disclosure do not substantially bind to and/or inhibit activity of activin A. It should be noted that GDF8 has high sequence homology to GDFl 1 and therefore antibodies that bind and/or to GDF8, in many cases, may also bind to and/or inhibit GDF11.
An anti-ActRIIA antibody refers to an antibody that is capable of binding to ActRIIA with sufficient affmity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting ActRIIA. In certain embodiments, the extent of binding of an anti-ActRIIA antibody to an unrelated, non-ActRUA protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody to ActRIIA as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, an anti-ActRIIA antibody binds to an epitope of ActRIIA that is conserved among ActRIIA from different species. In some embodiments, an anti-ActRIIA antibody of the présent disclosure is an antagonist antibody that can inhibit ActRIIA activity. For example, an anti-ActRIIA antibody of the présent disclosure may inhibit one or more ActRIIA ligands selected from activin A, activin B, activin AB, activin C, activin E, GDFl 1, GDF8, activin A, BMP6, and BMP7 from binding to the ActRIIA receptor and/or inhibit one of these ligands from activating ActRIIA signaling (e.g., SMAD2/3 and/or SMAD 1/5/8 ActRIIA signaling). In some embodiments,
-8019001 anti-ActRIIA antibodies of the présent disclosure inhibit GDFl l from binding to the ActRIIA receptor and/or inhibit GDFl l from activating ActRIIA signaling. Optionally, anti-ActRIIA antibodies of the disclosure further inhibit GDF8 from binding to the ActRIIA receptor and/or inhibit GDF8 from activating ActRIIA signaling. Optionally, anti-ActRIIA antibodies of the présent disclosure do not substantially inhibit activin A from binding to the ActRIIA receptor and/or do not substantially inhibit activin A-mediated activation of ActRIIA signaling. In some embodiments, an anti-ActRIIA antibody of the disclosure that inhibits GDFl l and/or GDF8 from binding to and/or activating an ActRIIA receptor further inhibits one or more of activin A, activin B, activin AB, activin C, activin E, activin A, GDF8, BMP6, and BMP7 from binding to and/or activating the ActRIIA receptor.
An anti-ActRIIB antibody refers to an antibody that is capable of binding to ActRIIB with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting ActRIIB. In certain embodiments, the extent of binding of an anti-ActRIIB antibody to an unrelated, non-ActRIIB protein is less than about 10%, 9%, 8%, 7%. 6%. 5%, 4%, 3%, 2%, or less than l% of the binding of the antibody to ActRIIB as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, an anti-ActRIIB antibody binds to an epitope of ActRIIB that is conserved among ActRIIB from different species. In some embodiments, an anti-ActRIIB antibody of the présent disclosure is an antagonist antibody that can inhibit ActRIIB activity. For example, an anti-ActRIIB antibody of the présent disclosure may inhibit one or more ActRIIB ligands selected from activin A, activin B, activin AB, activin C, activin E, GDFl l, GDF8, activin A, BMP6, and BMP7 from binding to the ActRIIB receptor and/or inhibit one of these ligands from activating ActRIIB signaling (e.g., SMAD2/3 and/or SMAD 1/5/8 ActRIIB signaling). In some embodiments, anti-ActRIIB antibodies of the présent disclosure inhibit GDFl l from binding to the ActRIIB receptor and/or inhibit GDFl l from activating ActRIIB signaling. Optionally, anti-ActRIIB antibodies of the disclosure further inhibit GDF8 from binding to the ActRIIB receptor and/or inhibit GDF8 from activating ActRIIB signaling. Optionally, anti-ActRIIB antibodies of the présent disclosure do not substantially inhibit activin A from binding to the ActRIIB receptor and/or do not substantially inhibit activin A-mediated activation of ActRIIB signaling. In some embodiments, an anti-ActRIIB antibody of the disclosure that inhibits GDFl l and/or GDF8 from binding to and/or activating an ActRIIB receptor further inhibits one or more of activin A, activin B, activin AB, activin C, activin E, activin A, GDF8, BMP6, and BMP7 from binding to and/or activating the ActRIIB receptor.
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The nucleic acid and amino acid sequences of human GDFl 1, GDF8, activin A, activin B, activin AB, activin C, activin E, GDF8, BMP6, BMP7, ActRIIB, and ActRIIA or are well known in the art and thus antibody antagonists for use in accordance with this disclosure may be routinely made by the skilled artisan based on the knowledge in the art and teachings provided herein.
In certain embodiments, an antibody provided herein (e.g., an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody) is a chimeric antibody. A chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder ofthe heavy and/or light chain is derived from a different source or species. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl, Acad. Sci. USA, 81:6851-6855. In some embodiments, a chimeric antibody comprises a nonhuman variable région (e.g., a variable région derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant région. In some embodiments, a chimeric antibody is a class switched antibody in which the class or subclass has been changed from that of the parent antibody. In general, chimeric antibodies include antigenbinding fragments thereof.
In certain embodiments, a chimeric antibody provided herein (e.g., an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody) is a humanized antibody. A humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human hypervariable régions (HVRs) and amino acid residues from human framework régions (FRs). In certain embodiments, a humanized antibody will comprise substantially ail of at least one, and typically two, variable domains, in which ail or substantially ail of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and ali or substantially ail of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant région derived from a human antibody. A humanized form of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.
Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson (2008) Front. Biosci. 13:1619-1633 and are further described, for example, in Riechmann et al., (1988) Nature 332:323-329; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34 [describing SDR (a-CDR) grafting];
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Padlan, Mol. Immunol. (1991) 28:489-498 (describing resurfacing); DalI'Acqua et al. (2005) Methods 36:43-60 (describing FR shuffling); Osbourn et al. (2005) Methods 36:61 68; and Klimka et al. Br. J. Cancer (2000) 83:252-260 (describing the guided sélection approach to FR shuffling).
Human framework régions that may be used for humanization include but are not limited to: framework régions selected using the best-fit method [see, e.g., Sims et al. (1993) J. Immunol. 151:2296 ]; framework régions derived from the consensus sequence of human antibodies of a particular subgroup of light-chain or heavy-chain variable régions [see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol., 151:2623]; human mature (somatically mutated) framework régions or human germline framework régions [see, e.g., Almagro and Fransson (2008) Front. Biosci. 13:16191633]; and framework régions derived from screening FR librarîes (see, e.g., Baca et cd., (1997) J. Biol. Chem. 272:10678-10684; and Rosok et cd., (1996) J. Biol. Chem. 271:2261122618). !
In certain embodiments, an antibody provided herein (e.g., an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody) is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel (2001) Curr. Opin.Phamiacol. 5: 368-74 and Lonberg (2008), Curr. Opin.lmmunol. 20:450-459.
Human antibodies may be prepared by administering an immunogen (e.g., a GDF11 polypeptide, GDF8 polypeptide, an ActRIIA polypeptide, or an ActRIIB polypeptide) to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable régions in response to antigenic challenge. Such animais typically contain ail or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are présent extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animais, the endogenous immunoglobulin loci hâve generally been inactivated. For a review of methods for obtaining human antibodies from transgenic animais see, for example, Lonberg (2005) Nat. Biotechnol. 23:1117-1125; U.S. Pat. Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE™ technology); U.S. Pat.No. 5,770,429 (describing HuMab® technology); U.S. Pat.No. 7,041,870 (describing K-M MOUSE® technology); and U.S. Patent Application Publication No. 2007/0061900 (describing VelociMouse® technology). Human variable régions from i
-8319001 intact antibodies generated by such animais may be further modified, for example, by combining with a different human constant région.
Human antibodies provided herein can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies hâve been described. See, e.g., Kozbor J. ImmunoL, (1984) 133: 3001; Brodeur et al. (1987) Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York; and Boemer et al. (1991) J. ImmunoL, I47: 86. Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., (2006) Proc. Natl. Acad. Sci. USA, 103:3557-3562. Additional methods include thosc described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue (2006) 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein (2005) Histol. HistopathoL, 20(3):927-937 (2005) and Vollmers and Brandlein (2005) Methods Find Exp. Clin. Pharmacol., 27(3): 185-91.
Human antibodies provided herein (e.g., an anti-GDFl 1 antibody, an anti-activin B antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody) may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries arc described herein.
For example, antibodies of the présent disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. A variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed. for example, in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:137, O'Brien et al., ed., Human Press, Totowa, N.J, and further described, for example, in the McCafferty et al. (1991) Nature 348:552-554; Clackson et al., (1991) Nature 352: 624-628; Marks et al. (1992) J. Mol. Biol. 222:581-597; Marks and Bradbury (2003) in Methods in Molecular Biology 248:161-175, Lo, ed., Human Press, Totowa, N.J.; Sidhu et al. (2004) J. Mol. Biol. 338(2):299-310; Lee et al. (2004) J. Mol. Biol. 340(5):1073-1093: Fellouse (2004) Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472; and Lee et al. (2004) J. ImmunoL Methods 284(1-2): 119-132.
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In certain phage display methods, répertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. ( 1994) Ann. Rev. ImmunoL, 12: 433-455. Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen (e.g., GDF11, activin B, ActRIIA. or ActRIIB) without the requirement of constructing hybridomas. Altematively, the naive répertoire can be cloned (e.g., from human) to provide a single source of antibodies directed against a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al. (1993) EMBO J, 12: 725-734. Finally, naive libraries can also be made synthetically by cloning un-rearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 régions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter (1992) J. Mol. Biol., 227: 381-388. Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
In certain embodiments, an antibody provided herein is a multi-specific antibody, for example, a bispecific antibody. Multi-specific antibodies (typically monoclonal antibodies) hâve binding specificities for at least two different epitopes (e.g., two, three, four, five, or six or more) on one or more (e.g., two, three, four, five, six or more) antigens.
In certain embodiments, a multi-specific antibody of the present disclosure comprises two or more binding specificities, with al least one ofthe binding specificities being for a GDF11 epitope, and optionally one or more additional binding specificities being for an epitope on a different ActRII ligand (e.g., GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6 BMP7 and/or Nodal) and/or an ActRII receptor (e.g., an ActRIIA and/or ActRIIB receptor). In certain embodiments, multi-specific antibodies may bind to two or more different epitopes of GDF11. Preferably a multi-specific antibody ofthe disclosure that has binding affinity, in part, for an GDF11 epitope can be used to inhibit a GDF11 activity (e.g., the ability to bind to and/or activate an ActRIIA and/or ActRIIB receptor), and optionally inhibit the activity of one or more different ActRII ligands (e.g., GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal) and/or an ActRII receptor (e.g., an ActRIIA or ActRIIB receptor). In certain embodiments, multi-specific
-8519001 antibodies of the présent disclosure that bind to and/or inhibit GDFl l forther bind to and/or inhibit at least GDF8. Optionally, multi-specific antibodies of the disclosure that bind to and/or inhibit GDFl l do not substantiaily bind to and/or substantially inhibit activin A. In some embodiments, multi-specific antibodies of the disclosure that bind to and/or inhibit GDFl l and GDD8 further bind to and/or inhibit one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal.
In certain embodiments, a multi-specific antibody of the présent disclosure comprises two or more binding specificities, with at least one of the binding specificities being for a GDF8 epitope, and optionally one or more additional binding specificities being for an epitope on a different ActRII ligand (e.g., GDFl 1, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal) and/or an ActRII receptor (e.g, an ActRIIA and/or ActRIIB receptor). In certain embodiments, multi-specific antibodies may bind to two or more different epitopes of GDF8. Preferably a multi-specific antibody of the disclosure that has binding affmily, in part, for an GDF8 epitope can be used to inhibit an GDF8 activity (e.g., the ability to bind to and/or activate an ActRIIA and/or ActRIIB receptor), and optionally inhibit the activity of one or more different ActRII ligands (e.g, GDFl 1, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal) and/or an ActRII receptor (e.g., an ActRIIA or ActRIIB receptor). In certain embodiments, multi-specific antibodies of the présent disclosure that bind to and/or inhibit GDF8 further bind to and/or inhibit at least GDFl 1. Optionally, multi-specific antibodies of the disclosure that bind to and/or inhibit GDF8 do not substantially bind to and/or substantially inhibit activin A. In some embodiments, multi-specific antibodies of the disclosure that bind to and/or inhibit GDF8 and GDFl 1 further bind to and/or inhibit one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal.
Engineered antibodies with three or more functional antigen binding sites, including Octopus antibodies, are also included herein. See, e.g., US 2006/0025576A1.
In certain embodiments, the antibodies disclosed herein (e.g., an anti-GDFl 1 antibody, an anti-activin B antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody) are monoclonal antibodies. Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, Le., the individual antibodies comprising the population are identical and/or bind the same epitope, cxcept ior possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody préparation, such variants generally being présent in minor
-8619001 amounts. In contrast to polyclonal antibody préparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody préparation is directed against a single epitope on an antigen. Thus, the modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animais containing ail or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
For exampie, by using immunogens derived from GDFl l or GDF8, anti-protein/antipeptide antisera or monoclonal antibodies can be made by standard protocols. See, e.g., i
Antibodies: A Laboratory Manual (1988) ed. by Harlow and Lane, Cold Spring Flarbor Press: 1988. A mammal, such as a mouse, a hamster, or rabbit can be immunized with an immunogenic form of the GDFl 1 or GDF8 polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein. Techniques for conferring immunogénieity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of a GDFl 1 or GDF8 polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by détection of antibody titers in plasma or sérum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibody production and/or level of binding affinity.
Following immunization of an animal with an antigenic préparation of GDFl 1 or GDF8, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the sérum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard Somalie cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma technique [.see, e.g., Kohler and Milstein (1975) Nature, 256: 495-497], the human B cell hybridoma technique [see, e.g.,Kozbar et al. (1983) Immunology Today, 4:72], and the EBV-hybridoma technique to produce human monoclonal antibodies [Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96], Hybridoma cells can be screened
-8719001 immunochemically for production of antibodies specifically reactive with a GDF11 or GDF8 polypeptide, and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
In certain embodiments, one or more amino acid modifications may be introduced into the Fc région of an antibody provided herein (e.g., an anti-GDFl l antibody, an antiactivin B antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody), thereby generating an Fc région variant. The Fc région variant may comprise a human Fc région sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc région) comprising an amino acid modification (e.g., a substitution, délétion, and/or addition) at one or more amino acid positions.
For example, the présent disclosure contemplâtes an antibody variant that possesses some but not ail effector functions, which make it a désirable candidate for applications in which the half-life of the antibody in vivo is important yet for which certain effector functions [e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)] are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in, for example, Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492. Non-limiting examples of in vitro assays to assess ADCC activity of a molécule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom, L et al. (1986) Proc. Nat'l Acad. Sci. USA 83:70597063]; Hellstrom, I et al. (1985) Proc. Nat'l Acad. Sci. USA 82:1499-1 502; U.S. Pat. No. 5,821,337; and Bruggemann, M. et al. (1987) J. Exp. Med. 166:1351-1361. Alternatively, non-radioactive assays methods may be employed (e.g., ACTI™, non-radioactive cytotoxicity assay for flow cytometry; CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay, Promega, Madison. Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molécule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. (1998) Proc. Nat'l Acad. Sci. USA 95:652-656. Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g.,
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Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complément activation, a CDC assay may be performed. See, e.g., Gazzano-Santoro et ni. (1996) J. ImmunoL Methods 202:163; Cragg, M. S. et al. (2003) Blood 101:1045-1052; and Cragg, M. S, and M. J. Glennie (2004) Blood 103:2738-2743. FcRn binding and in vivo clearance/half-life déterminations can also be performed using methods known in the art. See, e.g., Petkova, S. B. et al. (2006) Int'LImmunol. 18(12):1759-1769.
Antibodies of the présent disclosure (e.g., an anti-GDFl 1 antibody, an anti-activin B antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody) with reduced effector function include those with substitution of one or more of Fc région residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called DANA Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
In certain embodiments, it may be désirable to create cysteine engineered antibodies, e.g., thioMAbs, in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chaîn; and S400 (EU numbering) of the heavy chain Fc région. Cysteine engineered antibodies may be generated as described, for example, in U.S. Pat. No. 7,521,541.
In addition, the techniques used to screen antibodies in order to identify a désirable antibody may influence the properties of the antibody obtained. For example, if an antibody is to be used for binding an antigen in solution, it may be désirable to test solution binding. A variety of different techniques are available for testing interaction between antibodies and antigens to identify particularly désirable antibodies. Such techniques include ELISAs, surface plasmon résonance binding assays (e.g., the Biacore™ binding assay, Bia-core AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western blots, immunoprécipitation assays, and immunohistochemistry.
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In certain embodiments, amino acid sequence variants of the antibodies and/or the binding polypeptides provided herein are contemplated. For example, it may be désirable to improve the binding affinity and/or other biological properties of the antibody and/or binding polypeptide. Amino acid sequence variants of an antibody and/or binding polypeptides may be prepared by introducing appropriate modifications into the nucléotide sequence encoding the antibody and/or binding polypeptide, or by peptide synthesis. Such modifications include, for example, délétions from, and/or insertions into, and/or substitutions of residues within the amino acid sequences of the antibody and/or binding polypeptide. Any combination of délétion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characterîstics, e.g., target-binding (GDFl l, GDF8, ActRIIA, and/or ActRIIB binding).
Alterations (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such alterations may be made in HVR hotspots, Le., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury (2008) Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described in the art. See, e.g., Hoogenboom et al., in Methods in Molecular Biology 178:1-37, O’Brien et al., ed., Human Press, Totowa, N.J., (2001). In some embodiments of affinity maturation, diversîty is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g.. error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to întroduce diversîty involves HVR-directcd approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
In certain embodiments, substitutions, insertions, or délétions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in ITVRs. Such alterations may be outside of HVR hotspots or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two, or three amino acid substitutions.
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A useful method for identification of residues or régions of the antibody and/or the binding polypeptide that may be targeted for mutagenesis is called alanine scanning mutagenesis as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to déterminé whether the interaction is affected. Further substitutions may be introduced at the amino acid locations demonstratîng functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to détermine whether they contain the desired properties.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Exampies of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molécule include fusion of the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the sérum half-life of the antibody.
In certain embodiments, an antibody and/or binding polypeptide provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody and/or binding polypeptide include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldéhyde may hâve advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody and/or binding polypeptide may vary, and if more than one polymer are attached.
-9119001 they can be the same or different molécules. In general, the number and/or type of polymers used for derivatization can be determined based on considérations including, but not limited to, the particular properties or functions of the antibody and/or binding polypeptide to be improved, whether the antibody dérivative and/or binding polypeptide dérivative will be used in a therapy under defined conditions.
Any of the ActRII antagonist antibodies disclosed herein (e.g,, an anti-activin A antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl l antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an anti-BMP7 antibody, an anti-ActRIIA antibody, and/or or an anti-ActRIIB antibody) can be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., treat or prevent an anémia in a subject in need thereof and/or treat or prevent one or more complications of anémia including, for example, cutaneous ulcers). For example, an ActRII antagonist antibody disclosed herein (e.g., an anti-GDFl l antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl l antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-anti-BMP7 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody) can be used in combination with i) one or more additional ActRII antagonist antibodies disclosed herein, ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIlA and/or ActRIIB polypeptides), iii) one or more GDF Traps disclosed herein; iv) one or more small molécule ActRII antagonist disclosed herein (e.g., a small molécule antagonist of one or more of GDFl l, GDF8, activin A. activin B. activin AB. activin C, activin E, BMP6, BMP7, Nodal, ActRIlA, and/or ActRIIB); v) one or more polynucleotide ActRII antagonists disclosed herein (e.g., a polynucleotide antagonist of one or more of GDFl l, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIlA, and/or ActRIIB); vi) one or more follistatin polypeptides disclosed herein; and/or vii) one or more FLRG polypeptides disclosed herein.
D. Small Molécule Antagonists
In another aspect, the présent disclosure relates to a small molécule, or combination of small molécules, that antagonizes ActRII activity (e.g., inhibition of ActRIlA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling). In particular, the disclosure provides methods of using a small molécule antagonist, or combination of small molécule antagonists, of ActRII to, e.g.,treat or prevent an anémia in a subject in need thereof
-9219001 and/or treat or prevent one or more complications of anémia including, for example, cutaneous ulcers. In some embodiments, the disclosure provides methods of using a small molécule antagonist, or combination of small molécule antagonists of ActRII, to treat an anémia in a subject in need thereof and/or treat one or more complications of anémia including, for example, cutaneous ulcers, in a subject having anémia. In some embodiments, the disclosure provides methods of using a small molécule antagonist, or combination of small molécule antagonists of ActRII, to prevent an anémia in a subject in need thereof and/or prevent one or more complications of anémia including, for example, cutaneous ulcers in a subject having anémia.
i
In some embodiments, an ActRII antagonist of the présent disclosure is a small molécule antagonist, or combination of small molécule antagonists, that direct or indirect inliibits at least GDFl l activity. Optionally, such a small molécule antagonist, or combination of small molécule antagonists, may further inhibit, either directly or indirectly, GDF8. Optionally, a small molécule antagonist, or combination of small molécule antagonists, of the présent disclosure does not substantially inhibit activin A activity. In some embodiments, a small molécule antagonist, or combination of small molécule antagonists, of the présent disclosure that inliibits, either directly or indirectly, GDFl l and/or GDF8 activity further inhibits, either directly or indirectly, activity of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
In certain embodiments, a small molécule antagonist, or combination of small molécule antagonists, of the présent disclosure is an indirect inhibitor of one or more of GDFl l, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, Nodal, ActRIIA, and ActRIIB. For example, a small molécule antagonist, or combination of small molécule antagonists, of the présent disclosure may inhibit the expression (e.g., transcription, translation, cellular sécrétion, or combinations thereof) of at least GDFl l. Optionally, such a small molécule antagonist, or combination of small molécule antagonists, may further inhibit expression of GDF8. Optionally, a small molécule antagonist, or combinations of small molécule antagonists, of the disclosure does not substantially inhibit the expression of activin A. In some embodiments, a small molécule antagonist, or combination of small molécule antagonists, of the disclosure that inhibits expression of GDFl l and/or GDF8 may further inhibit the expression of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
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In other embodiments, a small molécule antagonist, or combination of small molécule antagonists, of the présent disclosure is direct inhibitor of one or more of GDF 11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB. For example, a small molécule antagonist, or combination of small molécule antagonists, of the présent disclosure directly binds to and inhibits at least GDFl l activity (e.g. inhibits the ability GDFl 1 to bind to an ActRIIA and/or ActRIIB receptor; inhibit GDFl 1-mediated activation of the ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). Optionally, a small molécule antagonist, or combinations of small molécule antagonists, of the disclosure may further bind to and inhibit GDF8 activity (e.g. inhibit the ability of GDF8 to bind to an ActRIIA and/or ActRIIB receptor; inhibit GDF8mediated activation of the ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). Optionally, a small molécule antagonist, or combinations of small molécule antagonists, of the disclosure does not substantially bind to or inhibit activin A activity (e.g. the ability of activin A to bind to an ActRIIA and/or ActRIIB receptor; activin A-mediated activation of the ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling pathway). In some embodiments, a small molécule antagonist, or combinations of small molécule antagonists, of the disclosure that binds to and inhibits the activity of GDFl 1 and/or GDF8 further binds to and inhibits the activity of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
In some embodiments, a small molécule antagonist, or combination of small molécule antagonists, of the présent disclosure directly binds to and inhibits at least GDF8 activity (e.g. inhibits the ability GDF8 to bind to an ActRIIA and/or ActRIIB receptor; inhibits GDF8mediated activation of the ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). Optionally, a small molécule antagonist, or combinations of small molécule antagonists, of the disclosure may further bind to and inhibit GDFl 1 activity (e.g. inhibit the ability of GDFl 1 to bind to an ActRIIA and/or ActRIIB receptor; inhibit GDFl 1-mediated activation of the ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). Optionally, a small molécule antagonist, or combinations of small molécule antagonists, of the disclosure does not substantially bind to or inhibit activin A activity (e.g. the ability of activin A to bind to an ActRIIA and/or ActRIIB receptor; activin A-mediated activation of the ActRIIA and/or ActRIIB signaling transduction, SMAD 2/3 signaling). In some embodiments, a small molécule antagonist, or combinations of small molécule antagonists, of the disclosure that binds to and inhibits the activity of GDF8 and/or GDFl 1
-9419001 further binds to and inhibits the activity of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
In some embodiments, a small molécule antagonist, or combination of small molécule antagonists, of the présent disclosure directly binds to and inhibits at least ActRIIA activity (e.g.ActRII ligand-mediated activation of ActRIIA signaling transduction, such as SMAD 2/3 signaling). For example, a small molécule antagonist, or combination of small molécule antagonists, of the disclosure binds to an ActRIIA receptor and inhibits at least GDFl l from binding to and/or activating the ActRIIA receptor. Optionally, such a small molécule antagonist, or combination of small molécule antagonists, may further inhibit GDF8 from binding to and/or activating the ActRIIA receptor. Optionally, a small molécule antagonist, or combination of small molécule antagonists, of the disclosure does not substanlially inhibit activin A from binding to and/or activating an ActRIIA receptor. In some embodiments, a small molécule antagonist, or combination of small molécule antagonists, of the disclosure that inhibits GDFl l and/or GDF8 from binding to and/or activating the ActRIIA receptor further inhibits one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal from binding to/and or activating the ActRIIA receptor.
In some embodiments, a small molécule antagonist, or combination of small molécule antagonists, of the présent disclosure directly binds to and inhibits at least ActRIIB activity (e.g. ActRII ligand-mediated activation of ActRIIB signaling transduction, such as SMAD 2/3 signaling). For example, a small molécule antagonist, or combination of small molécule antagonists, of the disclosure binds to an ActRIIB receptor and inhibits at least GDFl 1 from binding to and/or activating the ActRIIB receptor. Optionally. such a small molécule antagonist, or combination of small molécule antagonists, may further inhibit GDF8 from binding to and/or activating the ActRIIB receptor. Optionally, a small molécule antagonist, or combination of small molécule antagonists, of the disclosure does not substanlially inhibit activin A from binding to and/or activating an ActRIIB receptor. In some embodiments, a small molécule antagonist, or combination of small molécule antagonists, of the disclosure that inhibits GDFl 1 and/or GDF8 from binding to and/or activating the ActRIIB receptor further inhibits one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal from binding to/and or activating the ActRIIB receptor.
Binding organic small molécule antagonists of the présent disclosure may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO 00/00823 and WO 00/39585). In general, small molécules antagonists of the
-9519001 disclosure are usually less than about 2000 daltons in size, altematively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic small molécules that are capable of binding, preferably specifically, to a polypeptide as described herein (e.g., GDF H, GDF8, ActRIIA, and ActRIIB). Such small molécule antagonists may be identified without undue expérimentation using well known techniques. In this regard, it is noted that techniques for screening organic small molécule libraries for molécules that are capable of binding to a polypeptide target are well known in the art. See, e.g., international patent publication Nos.WOÛO/00823 and WOOO/39585.
Binding organic small molécules of the présent disclosure may be. for example, aldéhydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo compounds, and acid chlorides.
Any of the small molécule ActRII antagonists disclosed herein (e.g., a small molécule antagonist of one or more of GDFl 1, GDF8, activin A, activin B, activin AB, activin C, activin E BMP6, BMP7, Nodal, ActRIIA. and/or ActRIIB) can be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., increase red blood cell levels and/or hemoglobin in a subject in need lhereof, treat or prevent an anémia, treat sickle-cell disease, treat or prevent one or more complications of sickle-cell disease). For example, an small molécule ActRII antagonist disclosed herein (e.g., a small molécule antagonist of one or more of GDFl 1, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB) can be used in combination with i) one or more additional small molécule ActRII antagonists disclosed herein, ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIIA and/or ActRIIB polypeptides), iii) one or more GDF Traps disclosed herein; iv) one or more ActRII antagonist antibodies disclosed herein (e.g., an anti-GDFl 1 antibody, an anti-activin B antîbody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-anti-BMP7 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody); v) one or more polynucleotide ActRII antagonists disclosed herein (e.g., a polynucleotide antagonist of one or more of GDFl 1, GDF8, activin
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A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); vi) one or more follistatin polypeptides disclosed herein; and/or vii) one or more FLRG polypeptides disclosed herein.
E. Antagonist Polynuclcotides
In another aspect, the présent disclosure relates to a polynucleotide, or combination of polynuclcotides, that antagonizes ActRII activity (e.g., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling). In particular, the disclosure provides methods of using a polynucleotide ActRII antagonist, or combination of polynucleotide ActRII antagonists, to, e.g., treat or prevent an anémia in a subject in need thereof and/or treat or prevent one or more complication of anémia including, for example, cutaneous ulcers. In some embodiments, the disclosure provides methods of using a polynucleotide ActRII antagonist, or combination of polynucleotide ActRII antagonists, to treat an anémia in a subject in need thereof and/or treat one or more complications of anémia including, for example, cutaneous ulcers, in a subject having anémia. In some embodiments, the disclosure provides methods of using a polynucleotide ActRII antagonist, or combination of polynucleotide ActRII antagonists, to prevent an anémia in a subject in need thereof and/or prevent one or more complications of anémia including, for example, cutaneous ulcers in a subject having anémia. !
In some embodiments, a polynucleotide ActRII antagonist, or combination of polynucleotide ActRII antagonist, of the présent disclosure can be used to inhibit the activity and/or expression of one or more of GDFl 1, GDF8, activin A, activin B, activin AB, activin C, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB. In certain embodiments, a polynucleotide ActRII antagonist, or combination of polynucleotide ActRII antagonist, of the disclosure is a GDF-ActRII antagonist.
In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure inhibits the activity and/or expression (e.g., transcription, translation, sécrétion, or combinations thereof) of at least GDFl 1. Optionally. such a polynucleotide antagonist, or combination of polynucleotide antagonists, may further inhibit the activity and/or expression of GDF8. Optionally, a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure does not substantially inhibit the activity and/or expression of activin A. In some embodiments, a polynucleotide antagonist,
-9719001 or combination of polynucleotide antagonists, of the disclosure that inhibits the activity and/or expression of GDFl l and/or GDF8 may further inhibit the activity and or expression of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6. BMP7, Nodal, ActRIIA, and/or ActRIIB.
In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure inhibits the activity and/or expression (e.g., transcription, translation, sécrétion, or combinations thereof) of at least GDF8. Optionally, such polynucleotide antagonist, or combination of polynucleotide antagonists, may further inhibit the activity and/or expression of GDFl l. Optionally, a polynucleotide antagonist. or combination of polynucleotide antagonists, of the disclosure does not substantially inhibit the activity and/or expression of activin A. In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure that inliibits the activity and/or expression of GDF8 and/or GDFl l may further inhibit the activity and or expression of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB.
In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure inhibits the activity and/or expression (e.g., transcription, translation, sécrétion, or combinations thereof) of at least ActRIIA. Optionally, a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure does not substantially inhibit the activity and/or expression of activin A. In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure that inhibits the activity and/or expression of ActRIIA may further inhibit the activity and or expression of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, and/or ActRIIB.
In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, ofthe disclosure inhibits the activity and/or expression (e.g., transcription, translation, sécrétion, or combinations thereof) of at least ActRIIB. Optionally, a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure does not substantially inhibit the activity and/or expression of activin A. In some embodiments, a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure that inhibits the activity and/or expression of ActRIIB may further inhibit the activity and or expression of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, and/or ActRIIA.
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The polynucleotide antagonists of the présent disclosure may be an antisense nucieic acid, an RNAi molécule (e.g., small interfering RNA (siRNA), small-hairpin RNA (shRNA), microRNA (miRNA)), an aptamer and/or a ribozyme. The nucieic acid and amino acid sequences of human GDFl 1, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB are known in the art and thus polynucleotide antagonists for use in accordance with methods of the présent disclosure may be routinely made by the skilled artisan based on the knowledge in the art and teachings provided herein.
For example, antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix' formation. Antisense techniques are discussed, for example, in Okano (1991) J. Neurochem. 56:560; Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fia. (1988). Triple hélix formation is discussed in, for instance, Cooney et al. (1988) Science 241:456; and Dervan et al., (1991)Science 251:1300. The methods are based on binding of a polynucleotide to a complemcntary DNA or RNA. In some embodiments, the antisense nucieic acids comprise a single-stranded RNA or DNA sequence that is complementary to at least a portion of an RNA transcript of a gene disclosed herein (e.g., GDFl 1, GDF8, activin A, activin B, activin C, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB). Flowever, absolute complementarity, is not required.
A sequence complementary to at least a portion of an RNA, referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucieic acids of a gene disclosed herein (e.g., GDFl 1, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB), a single strand of the duplex DNA may thus be tested. or triplex formation may be assayed. The ability to hybridize will dépend on both the degree of complementarity and the length of the antisense nucieic acid. Generally, the larger the hybridizing nucieic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to détermine the melting point of the hybridized complex.
Polynucleotides that are complementary to the 5' end of the message, for example, the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs hâve been shown to be effective at inhibiting translation of
-9919001 mRNAs as well. See, e.g., Wagner, R., (1994) Nature 372:333-335. Thus, oligonucleotides complementary to either the 5'- or 3'-untranslated, non-coding régions of a gene of the disclosure (e.g., GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB), could be used in an antisense approach to inhibit translation of an endogenous mRNA. Polynucleotides complementary to the 5' untranslated région of the mRNA should include the complément of the AUG start codon. Antisense polynucleotides complementary to mRNA coding régions are less efficient inhibitors of translation but could be used in accordance with the methods of the present disclosure. Whether designed to hybridize to the 5'-untranslated, 3'-untranslated or coding région of an mRNA ofthe disclosure (e.g., an GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB mRNA), antisense nucleic acids should be at least six nucléotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucléotides in length. In spécifie aspects, the oligonucleotide is at least 10 nucléotides, at least 17 nucléotides, at least 25 nucléotides, or at least 50 nucléotides.
In one embodiment, the antisense nucleic acid of the present disclosure (e.g., a GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, or ActRIIB antisense nucleic acid) is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of a gene of the disclosure. Such a vector would contain a sequence encoding the desired antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for réplication and expression in vertebrate cells. Expression of the sequence encoding desired genes of the instant disclosure, or fragments thereof, can be by any promoter known in the art lo act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter région |.«?e . e.g.,Benoist and Chambon (1981) Nature 29:304-310], the promoter contained in the 3’ long terminal repeat of Rous sarcoma virus (see, e.g., Yamamoto et al. (1980) Cell 22:787-797, the herpes thymidine promoter [see, e.g., Wagnér et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445], and the regulatory sequences of the metallothionein gene (see, e.g., Brinster, et al. (1982) Nature 296:39-42.
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I
In some embodiments, the polynucleotide antagonists are interfering RNA or RNAi molécules that target the expression of one or more of: GDFl l, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB. RNAi refers to the expression of an RNA which interfères with the expression of the targeted mRNA. Specifically, RNAi silences a targeted gene via interacting with the spécifie mRNA through a siRNA (small interfering RNA). The ds RNA complex is then targeted for dégradation by the cell. An siRNA molécule is a double stranded RNA duplex of 10 to 50 nucléotides in length, which interfères with the expression of a target gene which is sufficiently complementary (e.g. at least 80% identity to the gene). In some embodiments, the siRNA molécule comprises a nucléotide sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to the nucléotide sequence of the target gene.
Additional RNAi molécules include short hairpin RNA (shRNA); also short interfering hairpin and microRNA (miRNA). The shRNA molécule contains sense and antisense sequences from a target gene connected by a loop. The shRNA is transported from the nucléus into the cytoplasm, and it is degraded along with the mRNA. Pol III or U6 promoters can be used to express RNAs for RNAi. Paddison et al. [Genes & Dev. (2002) 16:948-958, 2002] hâve used small RNA molécules folded into hairpins as a means to effect RNAi. Accordingly, such short hairpin RNA (shRNA) molécules are also advantageously used in the methods described herein. The length of the stem and loop of functional shRNAs varies; stem lengths can range anywhere from about 25 to about 30 nt. and loop size can range between 4 to about 25 nt without affecting silencing activity. While not wishing to be bound by any particular theory, it is believed that these shRNAs resemble the double stranded RNA (dsRNA) products of the DICER RNase and, in any event, hâve the same capacity for inhibiting expression of a spécifie gene. The shRNA can be expressed from a lentiviral vector. An miRNA is a single stranded RNA of about 10 to 70 nucléotides in length that are initially transcribed as pre-miRNA characterized by a “stem-loop” structure and which are subsequently processed into mature miRNA after further processing through the RISC.
Molécules that médiate RNAi, including without limitation siRNA, can be produced in vitro by chemical synthesis (Hohjoh, FEBS Lett 521:195-199. 2002). hydrolysis ot dsRNA (Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002), by in vitro transcription with T7 RNA polymerase (Donzeet et al., Nucleic Acids Res 30:e46, 2002; Yu et al., Proc Natl Acad Sci USA 99:6047-6052, 2002), and by hydrolysis of double-stranded RNA using a nuclease such as E. coli RNase III (Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002).
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According to another aspect, the disclosure provides polynucleotide antagonists încluding but not limited to, a decoy DNA, a double stranded DNA, a single-stranded DNA, a complexed DNA, an encapsulated DNA, a viral DNA, a plasmid DNA, a naked RNA, an encapsulated RNA, a viral RNA, a double stranded RNA, a molécule capable of generating RNA interférence, or combinations thereof.
In some embodiments, the polynucleotide antagonists of the disclosure are aptamers. Aptamers are nucleic acid molécules, încluding double stranded DNA and single stranded RNA molécules, which bind to and form tertiary structures that specifically bind to a target molécule, such as a GDFl l, GDF8, activin A, activin B, activin C, activin E. BMP6, BMP7. Nodal, ActRIlA, and ActRIIB polypeptide. The génération and lherapeutic use of aptamers
I are well established in the art. See, e.g., U.S. Pat.No. 5,475,096. Additional information on aptamers can be found in U.S. Patent Application Publication No. 20060148748. Nucleic acid aptamers are selected using methods known in the art, for example via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. SELEX is a method for the in vitro évolution of nucleic acid molécules with highly spécifie binding to target molécules as described in, e.g., U.S. Pat. Nos. 5,475,096, 5,580,737, 5,567,588, 5,707,796, 5,763,177, 6,011,577, and 6,699,843. Another screening method to identify aptamers is described in U.S. Pat. No. 5,270,163. The SELEX process is based on the capacity of nucleic acids for forming a variety of two- and three-dimensional structures, as well as the chemical versatility available within the nucléotide monomers to act as ligands (form spécifie binding pairs) with virtually any chemical compound, whether monomeric or polymeric, încluding other nucleic acid molécules and polypeptides. Molécules of any size or composition can serve as targets. The SELEX method involves sélection from a mixture of candidate oligonucleotides and step-wise itérations of binding, partitioning and amplification, using the same general sélection scheme, to achieve desired binding affïnity and selectivity. Starting from a mixture of nucleic acids, which can comprise a segment ofrandomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding; partitioning unbound nucleic acids from those nucleic acids which hâve bound specifically to target molécules; dissociating the nucleic acid-target complexes; amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand enriched mixture of nucleic acids. The steps of binding, partitioning, dissociating and amplifying are repeated through as many cycles as desired to yield highly spécifie high affïnity nucleic acid ligands to the target molécule.
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Typically, such binding molécules are separately administered to the animal [see, e.g., O'Connor (I99l) J. Neurochem. 56:560], but such binding molécules can also be expressed in vivo from polynucleotides taken up by a host cell and expressed in vivo. See, e.g., Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fia. (1988).
Any of the polynucleotide ActRII antagonists disclosed herein (e.g., a polynucleotide antagonist of one or more of GDFl l, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIlA, and/or ActRIIB) can be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., treat or prevent an anémia in a subject in need thereof and/or treat or prevent one or more complications of anémia including, for example, cutaneous ulcers). For example, an polynucleotide ActRII antagonist disclosed herein (e.g., a polynucleotide antagonist of one or more of GDFl 1, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIlA, and/or ActRIIB) can be used in combination with i) one or more additional polynucleotide ActRII antagonists disclosed herein, ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIlA and/or ActRIIB polypeptides), iii) one or more GDF Traps disclosed herein; iv) one or more ActRII antagonist antibodies disclosed herein (e.g., an antiGDFl 1 antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-antiBMP7 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody); v) one or more small molécule ActRII antagonists disclosed herein (e.g., a small molécule antagonist of one or more of GDFl 1, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIlA, and/or ActRIIB); vi) one or more follistatin polypeptides disclosed herein; and/or vii) one or more FLRG polypeptides disclosed herein.
F. Other Antagonists
In other aspects, an agent for use in accordance with the methods disclosed herein (e.g., methods of treating or preventing an anémia in an subject in need thereof and/or methods of treating or preventing one or more complications of anémia including, for example, cutaneous ulcers) is a follistatin polypeptide. The term follistatin polypeptide includes polypeptides comprising any naturally occurring polypeptide of follistatin as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that
-10319001 retain a useful activity, and further includes any functional monomer or multimer of follistatin. In certain embodiments, follistatin polypeptides of the disclosure bind to and/or inhibit activin activity, particularly activin A (e.g., activin-mediated activation of ActRIIA and/or ActRIIB SMAD 2/3 signaling). Variants of follistatin polypeptides that retain activin binding properties can be identified based on prevîous studies involving follistatin and activin interactions. For example, W02008/030367 discloses spécifie follistatin domains (FSDs) that are shown to be important for activin binding. As shown below in SEQ ID NOs: 18-20, the follistatin N-terminal domain (FSND SEQ ID NO: 18), FSD2 (SEQ ID NO: 20), and to a lesser extent FSD1 (SEQ ID NO: 19) represent exemplary domains within follistatin that are important for activin binding. In addition, methods for making and testing libraries of polypeptides are described above in the context of ActRIl polypeptides and such methods also pertain to making and testing variants of follistatin. Follistatin polypeptides include polypeptides derived from the sequence of any known follistatin having a sequence at least about 80% identical to the sequence of a follistatin polypeptide, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity. Examples of follistatin polypeptides include the mature follistatin polypeptide or shorter isoforms or other variants of the human follistatin precursor polypeptide (SEQ ID NO: 16) as described, for example, in W02005/025601.
The human follistatin precursor polypeptide isoform FST344 is as follows:
mvrarhqpgg Icllllllcq fmedrsaqag ncwlrqakng rcqvlyktel skeeccstgr Istswteedv ndntlfkwmi fnggapncip cketcenvdc 101 gpgkkcrmnk knkprcvcap dcsnitwkgp vcgldgktyr necallkarc 151 keqpelevqy qgrckktcrd vfcpgsstcv vdqtnnaycv tenriepepa 201 sseqylcgnd gvtyssachl rkatcllgrs iglayegkci kakscediqc 251 tggkkclwdf kvgrgrcslc delcpdsksd epvcasdnat yasecamkea 301 acssgvllev khsqscnsis edteeeeede dqdysfpiss ilew(SEQ ID NO: 16; NCB1 ReferenceNo. NP_037541.1 follistatin isoform FST344)
The signal peptide is underlined: also underlined above are the last 27 residues in which represent the C-terminal extension distinguishing this follistatin isoform from the shorter follistatin isoform FST317 shownbelow.
The human follistatin precursor polypeptide isoform FST317 is as follows:
MVRARHQPGG LCLLLLLLCQ FMEDRSAQAG NCWLRQAKNG RCQVLYKTEL
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SKEECCSTGR LSTSWTEEDV NDNTLFKWMI FNGGAPNCIP CKETCENVDC
101 GPGKKCRMNK KNKPRCVCAP DCSNITWKGP VCGLDGKTYR NECALLKARC
151 KEQPELEVQY QGRCKKTCRD VFCPGSSTCV VDQTNNAYCV TCNRICPEPA
201 SSEQYLCGND GVTYSSACHL RKATCLLGRS IGLAYEGKCI KAKSCEDIQC
251 TGGKKCLWDF KVGRGRC5LC DELCPDSKSD EPVCASDNAT YASECAMKEA
301 ACSSGVLLEV KHSGSCN(SEQ ID NO: 17; NCBI Reference No.
NP-006341.1)
The signal peptide is underlined.
The follistatin N-terminus domain (FSND) sequence is as follows:
GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWM
IFNGGAPNCIPCK (SEQ ID NO: 18; FSND)
The FSD1 and FSD2 sequences are as follows :
ETCENVDCGPGKKCRMNKKNKPRCV (SEQ ID NO: 19; FSD1) KTCRDVFCPGSSTCWDQTNNAYCVT (SEQ ID NO: 20; FSD2)
In other aspects, an agent for use in accordance with the methods disclosed herein (e.g., methods of treating or preventing an anémia in an subject in need thereof and/or methods of treating or preventing a complication of anémia including, for example, cutaneous ulcers) is a follistatin-like related gene (FLRG), also known as follistatin-related protein 3 (FSTL3). In some embodiments, the agent is used to treat a complication of anémia including, for example, cutaneous ulcers. In some embodiments, the agent is used to prevent a complication of anémia including, for example, cutaneous ulcers. The term FLRG polypeptide includes polypeptides comprising any naturally occurring polypeptide of FLRG as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. In certain embodiments, FLRG polypeptides of the disclosure bind to and/or inhibit activin activity, particuiarly activin A (e.g., activin-mediated activation of ActRIIA and/or ActRIIB SMAD 2/3 signaling). Variants of FLRG polypeptides that retain activin binding properties can be identified using routine methods to assay FLRG and activin interactions. See, e.g., US 6,537,966. In addition, methods for making and testing libraries of polypeptides are described above in the conlcxt of ActRIl polypeptides and such methods also pertain to making and testing variants of FLRG. FLRG polypeptides include polypeptides derived from the sequence of any known FLRG having a sequence at
-10519001 least about 80% identical to the sequence of an FLRG polypeptide, and optionally at least 85%, 90%, 95%, 97%, 99% or greater identity.
The human FLRG (follistatin-related protein 3 precursor) polypeptide is as follows:
MRPGAPGPLW PLPWGALAWA VGFVS5MGSG NPAPGGVCWL QQGQEATCSL
VLQTDVTRAE CCASGNIDTA WSNLTHPGNK INLLGFLGLV HCLPCKDSCD
101 GVECGPGKAC RMLGGRPRCE CAPDCSGLPA RLQVCGSDGA TYRDECELRA
151 ARCRGHPDLS VMYRGRCRKS CEHVVCPRPQ SCWDQTGSA HCVVCRAAPC
201 PVPSSPGQEL CGNNNVTYIS SCHMRQATCF LGRSIGVRHA GSCAGTPEEP
251 PGGESAEEEE NFV(SEQ ID NO:21; NCBI Référencé No. NP_005851.1)
The signal peptide is underlined.
In certain embodiments, functional variants or modified forms of the follistatin polypeptides and FLRG polypeptides include fusion proteins having at least a portion of the follistatin polypeptides or FLRG polypeptides and one or more fusion domains, such as, for example, domains that facilitate isolation, détection, stabilization or multimerization oi'the polypeptide. Suitable fusion domains are discussed in detail above with référencé to the ActRII polypeptides. In some embodiment, an antagonist agent of the disclosure is a fusion protein comprising an activin-binding portion of a follistatin polypeptide fused to an Fc domain. In another embodiment, an antagonist,agent of the disclosure is a fusion protein comprising an activin binding portion of an FLRG polypeptide fused to an Fc domain.
Any of the follistatin polypeptides disclosed herein may be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., treat or prevent an anémia in a subject in need thereof and/or treat or prevent one or more complications of anémia including, for example, cutaneous ulcers). For example, a follistatin polypeptide disclosed herein can be used in combination with i) one or more additional follistatin polypeptides disclosed herein. ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIIA and/or ActRIIB polypeptides), iii) one or more GDF Traps disclosed herein; iv) one or more ActRII antagonist antibodies disclosed herein (e.g, an anti-GDFl 1 antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activîn E antibody, an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-anti-BMP7 antibody, an antiActRIIA antibody, or an anti-ActRIIB antibody); v) one or more small molécule ActRII antagonists disclosed herein (e.g, a small molécule antagonist of one or more of GDFl 1,
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GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7. Nodal. ActRIIA, and/or ActRIIB); vi) one or more polynucleotide ActRII antagonists disclosed herein (e.g., a polynucleotide antagonist of one or more of GDFl l, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); and/or one or more FLRG polypeptides disclosed herein.
Similarly, any of the FLRG polypeptides disclosed herein may be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., treat or prevent an anémia in a subject in need thereof and/or treat or prevent one or more complications of anémia including, for example, cutaneous ulcers). For example, a FLRG polypeptide disclosed herein can be used in combination with i) one or more additional FLRG polypeptides disclosed herein, ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIIA and/or ActRIIB polypeptides), iii) one or more GDF Traps disclosed herein; iv) one or more ActRII antagonist antibodies disclosed herein (e.g, an antiGDFl l antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl I antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-antiBMP7 antibody, an anti-ActRIIA antibody, or an anti-ActRHB antibody); v) one or more small molécule ActRII antagonists disclosed herein (e.g., a small molécule antagonist of one or more of GDFl l, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6. BMP7, Nodal, ActRIIA, and/or ActRIIB); vi) one or more polynucleotide ActRII antagonists disclosed herein (e.g., a polynucleotide antagonist of one or more of GDFl l, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); and/or one or more follistatin polypeptides disclosed herein.
4. Screening Assays
In certain aspects, the présent disclosure relates to the use of the subject ActRII polypeptides (e.g., ActRIIA and ActRIIB polypeptides) andGDF Trap polypeptides to identify compounds (agents) which are agonist or antagonists of ActRIIB polypeptides. Compounds identified through this screening can be tested to assess their ability to modulalc red blood cell, hemoglobin, and/or réticulocyte levels as well as effect cutaneous ulcers. These compounds can be tested. for example, in animal models.
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There are numerous approaches to screening for therapeutic agents for increasing red blood cell or hemoglobin levels by targeting ActRII signaling (e.g., ActRlIA and/or ActRIIB SMAD 2/3 and/or SMAD 1/5/8 signaling). In certain embodiments, high-throughput screening of compounds can be carried out to identify agents that perturb ActRII-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 ActRII polypeptide or GDF Trap polypeptide to its binding partner, such as an ActRII ligand (e.g., activin A, activin B, activin AB, activin C, Nodal, GDF8, GDFl l or BMP7). Altematively, the assay can be used to identify compounds that enhance binding of an ActRII polypeptide or GDF Trap polypeptide to its binding partner such as an ActRII ligand. In a further embodiment, the compounds can be identified by their ability to interact with an ActRII polypeptide or GDF i
Trap 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. Altematively, the subject compounds may be naturally occurring biomolecules synthesized in 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 molécules, including peptidomimetics), or produced recombinantly. Test compounds contemplated by the present invention include non-peptidyl organic molécules, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molécules. In certain embodiments, the test agent is a small organic molécule having a molecular weight of less than about 2,000 Daltons.
The test compounds of the disclosure can be provided as single, discrète 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, aldéhydes, ethers and other classes of organic compounds. Présentation 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 derivatized with other compounds and hâve derivatizing groups that facilitate isolation of the compounds. Nonlimiting examples of derivatizing groups include biotin. fluorcscein. digoxygenin, green
-10819001 fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S transferase (GST), photoactivatible crosslinkers or any combinations thereof.
In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are désirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free Systems, such as may be derîved with purified or semî-purified proteins, are often preferred as “primary screens in that they can be generated to permit rapid development and relatively easy détection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity or bioavailability 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 in an alteration of binding affinity between an ActRII polypeptide or a GDF Trap polypeptide and ils binding partner (e.g., an ActRII ligand).
Merely to illustrate, in an exemplary screening assay of the présent disclosure, 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 (e.g., GDFl l). Détection and quantification of ActRIIB/ActRIIB 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 of the 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 ActRIIB ligand is added to a composition containing the ActRIIB polypeptide, and the formation of ActRIIB/ActRIIB ligand complex is quantitated in the absence of the test compound. It will be understood that, in general, the order in which the reactants may be admixed can be varied, and can be admixed simultaneously. Moreover, in place of purified proteins, cellular extracts and lysâtes may be used to render a suitable cell-free assay System.
Complex formation between an ActRII polypeptide or GDF Trap 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, detectably labeled proteins such as radiolabeled (e.g., 32P, 35S, l4C or 3H), fluorescently labeled (e.g., FITC), or
-10919001 enzymatically labeled ActRII polypeptide or GDF Trap polypeptide and/or its binding protein, by immunoassay, or by chromatographie détection.
In certain embodiments, the présent disclosure contemplâtes the use of fluorescence polarization assays and fluorescence résonance energy transfer (FRET) assays in measuring, either directly or îndirectly, the degree of interaction between an ActRII polypeptide of GDF Trap polypeptide and its binding protein. Further, other modes of détection, such as those based on optical waveguides (see, e.g., PCT Publication WO 96/26432 and U.S. Pat. No. 5,677,196), surface plasmon résonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodiments of the disclosure.
Moreover, the présent disclosure contemplâtes the use of an interaction trap assay, also known as the “two hybrid assay,” for identifying agents that disrupt or potentiate interaction between an ActRII polypeptide or GDF Trap polypeptide and its binding partner. See, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura e/ al. (1993) J Biol Chem 268:12046-12054; Bartel et a/.(1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). In a spécifie embodiment, the présent disclosure contemplâtes the use of reverse two hybrid Systems to identify compounds (e.g., small molécules or peptides) that dissociate interactions between an ActRII polypeptide or GDF Trap and its binding protein. See, e.g., Vidal and Legrain, (1999) Nucleic Acids Res 27.91929; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; and U.S. Pat. Nos. 5.525.490. 5,955,280; and 5,965,368.
In certain embodiments, the subject compounds are identified by their ability to interact with an ActRII polypeptide or GDF Trap polypeptide. The interaction between the compound and the ActRII polypeptide or GDF Trap polypeptide may be covalent or noncovalent. For example, such interaction can be identified at the protein level using in vitro biochemical methods, including photo-crosslinking, radiolabeled ligand binding, and affinity chromatography. See, e.g., Jakoby WB et ai. (1974) Methods in Enzymology 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 ActRII polypeptide of GDF Trap polypeptide. This may include a solid phase or fluid phase binding event. Altematively, the gene encoding an ActRII polypeptide or GDF Trap polypeptide can be transfected with a reporter System (e.g.. β-galactosidase, luciferase, 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
-11019001 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 electrophoresîs. The bound compounds may be detected usually using colorimétrie endpoints or fluorescence or surface plasmon résonance.
5. Exemplary Therapeutic Uses
In certain aspects, an ActRII antagonîst agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to increase red blood cell levels in a subject (e.g., a patient) in need thereof, particularly mammals such as rodents, primates, and humans. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to treat or prevent an anémia in a subject (e.g., a patient) in need thereof and/or one or more complications of anémia including, for example, an ulcer, particularly a cutaneous ulcer. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to treat an anémia in a subject (e.g., a patient) in need thereof and/or one or more complications of anémia including, for example, an ulcer, particularly a cutaneous ulcer. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to prevent an anémia in a subject (patient) in need thereof and/or one or more complications of anémia including, for example, an ulcer, particularly a cutaneous ulcer. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to treat or prevent an ulcer in a subject (e.g., a patient) having anémia, particularly mammals such as rodents, primates, and humans. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to treat or prevent an ulcer that is associated with anémia in a subject (e.g., a patient) in need thereof, particularly mammals such as rodents, primates, and humans. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to treat or prevent a cutaneous (e.g., skin) ulcer in a subject (e.g., a patient) having anémia, particularly mammals such as rodents, primates, and humans. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to treat or prevent a cutaneous ulcer associated with anémia in a subject (e.g., patient) in need thereof, particularly mammals such as rodents, primates, and humans. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to treat or prevent an ulcer (e.g., a cutaneous ulcer) in a subject (e.g., patient) having a hemolytic anémia, particularly mammals such as rodents, primates, and humans. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to treat or prevent an ulcer (e.g., a cutaneous ulcer) in a subject (e.g., patient) having a hemoglobinopathy anémia, particularly mammals such as rodents, primates, and humans. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to treat or prevent an ulcer (e.g., a cutaneous ulcer) in a subject (patient) having a thalassemia syndrome (e.g., β-thalassemia syndrome, β-thalassemia intermedia, etc.), particularly mammals such as rodents, primates, and humans. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure can be used to treat or prevent an ulcer (e.g., a cutaneous ulcer) in a subject (patient) having sickle-cell disease, particularly mammals such as rodents, primates, and humans. In some of the foregoing embodiments, the ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure are used to treat an ulcer (e.g., a cutaneous ulcer) in a subject (e.g., patient) having anémia (e.g., hemolytic anémia, hemoglobinopathy anémia, a thalassemia syndrome (e.g., β-thalassemîa syndrome, βthalassemia intermedia, etc.), sickle-cell disease, etc.). In some of the foregoing embodiments, the ActRII antagonist agent, or combination of ActRII antagonist agents, of the présent disclosure are used to prevent an ulcer (e.g., a cutaneous ulcer) in a subject (e.g., patient) having anémia (e.g., hemolytic anémia, hemoglobinopathy anémia, a thalassemia syndrome (e.g., β-thalassemia syndrome, β-thalassemia intermedia, etc.), sickle-cell disease. etc.). In some embodiments, the subject having anémia has sickle cell disease. In some embodiments, the subject having anémia has a thalassemia syndrome (e.g., β-thalassemia syndrome, β-thalassemia intermedia, etc.). In some embodiments, the subject having anémia has a cutaneous ulcer. In some embodiments, the cutaneous ulcer is a skin ulcer. In some embodiments, the ulcer occurs on legs or ankes.
As used herein, a therapeulic that “prevents” a disorder or condition refers to a compound that, in a statîstîcal 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. For example, using an ActRII antagonist of the disclosure to prevent an ulcer (e.g., cutaneous ulcer) in a subject having anémia refers to reducing the occurrence of ulcer in
-11219001 the subject or delays the onset or reduces the severity of ulcer in the subject relative to a subject having anémia who is not receiving an ActRII antagonist.
The term treating” as used herein inciudes amelioration or élimination of the condition once it has been established. In either case, prévention or treatment may be discemed in the diagnosîs provided by a physician or other health care provider and the intended resuit of administration of the therapeutic agent. In some embodiments, treating an ulcer refers to promoting wound healing of ulcer tissues.
In general, treatment or prévention of a disease or condition as described in the présent disclosure is achieved by administering one or more of the ActRII antagonists (e.g., an ActRIlA and/or ActRIIB antagonist) of the présent disclosure in an effective amount. An effective amount of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic resuit. A therapeutically effective amount of an agent of the présent disclosure may vary according to factors such as the disease state, âge, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic resuit.
Ulcers and Anémia i
An ulcer is a sore on the skin or mucous membrane which is accompanied by the disîntegration of tissue. Cutaneous (skin) ulcers can resuit in complété loss of epidermis and often portions of the dermis and even subcutaneous fat. Cutaneous ulcers are most common on the skin of the Iower extremities but do occur on other areas of the body. Typically, ulcers appear as open craters, often round, with layers of skin that hâve eroded, and such lésions are highly susceptible to infection. The skin around the ulcer may be red, swollen, and/or tender. In general, ulcers tend to heal more slowly that other types of skin injuries and are résistant to treatment.
Ulcers develop in stages. In stage l, the skin is red with soft underlying tissue. In the second stage, the redness of the skin becomes more pronounccd, swelling appears, and lhere may be some blisters and loss of outer skin layers. During the next stage, the skin may become necrotic down through the deep layer of the skin, and the fat beneath may become exposed. In the last two stages, the sore may cause a deeper loss of fat and necrosis of muscle - in serve cases, it can extend to destruction of the bone and cause sepsis. In view of
-i 1319001 staged progression of ulcer pathology, physicians hâve adopted grading Systems to classify ulcers. The Wagner Grading System classifies ulcers into 5 categories: i) a superficial ulcer is designated as Grade l ; ii) a ulcer deeper into subcutaneous tissue exposing soft tissue (but no abscess or osteomyelitis) is designated as Grade 2; iii) an ulcer with abscess formation and/or osteomyelitis is designated as Grade 3; iv) an ulcer having associated gangrené on part of a tissue or limb is designated as Grade 4; and v) an ulcer having extensive gangrené to a large area or entire limb is designated as Grade 5.
Ulcers, particularly cutaneous ulcers, occur as a complication of many anémias. In most patients, these ulcers occur in the legs or ankles, but may occur on other parts ofthc body. The relationship between anémia and ulcer formation is multitactorial, but it is generally expected that elevated hemolysis, oxidative stress, poor tissue oxygénation and vascular congestion may ail contribute to the formation of ulcers. Elevated hemolysis causes the release of free hemoglobin into the sérum, which causes oxidative damage and consumes nitric oxide that is needed to maintain proper vascular tone. Ulcers are associated with many hereditary and acquired anémias, including hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseô-phosphate dehydrogenase deficiency, sickle cell disease, thalassemia (both alpha and beta), paroxysmal noctumal hemoglobinuria. Sickle cell disease and the thalassemias are particularly noted for causing ulcers, probably because ail of the risk factors are présent in these diseases. Ulcers are associated with many hemolytic anémias, which describes an anémie condition that results from excessive destruction of red blood cells. Hemolytic anémias may resuit from infections, such as hepatitis, cytomégalovirus (CMV), Epstein-Barr virus (EBV). typhoid fever, E. coli (escherichia coli), mycoplasma pneumonia, or streptococcus, médications, such as penicillin, antimalaria médications, sulfa médications, or acetaminophen, cancers such as leukemia or lymphoma and solid tumors of various types, autoimmune disorders, such as systemic lupus erythematous (SLE, or lupus), rheumaloid arthritis, Wiskott-Aldrich syndrome, or ulcerative colitis, hypersplenism, and autoimmune hemolytic anémia, in which the body's immune System créâtes an antibody against its own blood cells. Microangiopathic hemolytic anémia and thrombotic thrombocytopénie purpura are also associated with anémia and ulcer formation.
In some embodiments, an ActRII antagonist agent, or combination of ActR.II antagonist agents, of the présent disclosure can be used to treat or prevent an ulcer (e.g., a cutaneous ulcer) in a subject (patient) having an anémia selected from: including hereditary spherocytosis, hereditary elliptocytosis, hereditary stomacytosis, glucoseô-phosphate
-11419001 dehydrogenase deficiency, a hemolytic anémia, a hemoglobinopathy anémia, sickle-cell disease, thalassemia (both alpha and beta), a β-thalassemia syndrome , β-thalassemia intermedia, paroxysmal nocturnal hemoglobinuria, microangiopathic hemolytic anémia, thrombotic thrombocytopénie purpra, an anémia associated with an infection (e.g., hepatitis, cytomégalovirus (CMV), Epstein-Barr virus (EBV), typhoid fever, E. coli (escherichia coli), mycoplasma pneumonia, or streptococcus), an anémia associated with administration of a médication (e.g., penicillin, antimalaria médications, sulfa médications, or acetaminophen), anémia associated with a cancer (e.g., leukemia, lymphoma, and solid tumors of varions types), and anémia associated with an autoimmune disorder (e.g., systemic lupus erythematous (SLE, or lupus), rheumatoid arthritis, Wiskott-Aldrich syndrome, or ulcerative colitis, hypersplenism, and autoimmune hemolytic anémia, in which the body’s immune System créâtes an antibody against its own blood cells). In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used to treat or prevent an ulcer (e.g., a cutaneous ulcer) in a subject (patient) having a hemolytic anémia. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used to treat or prevent an ulcer (e.g., a cutaneous ulcer) in a subject (patient) having a hemoglobinopathy anémia. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, ol the present disclosure can be used to treat or prevent an ulcer (e.g., a cutaneous ulcer) in a subject (patient) having a thalassemia syndrome. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used to treat or prevent an ulcer (e.g., a cutaneous ulcer) in a subject (patient) having a β-thalassemia syndrome. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used to treat or prevent an ulcer (e.g., a cutaneous ulcer) in a subject (patient) having β-thalassemia intermedia. In some embodiments, an ActRII antagonist agent, or combination of ActRII antagonist agents, ofthe present disclosure can be used to improve the Grade classification (e.g., the Wager Grading System) ofthe ulcer (e.g., a cutaneous ulcer) by at least one Grade (e.g., by at least one, two, three, four, or five Grades).
In certain embodiments, one or more ActRII antagonist agents ofthe disclosure (e.g., a GDF-ActRIl antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, e/c.)may be used in combination with supportive thérapies for ulcers. Conventional care of cutaneous ulcers involves debridement and cleansing of the wound followed by application of
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I occlusive dressing. See, e.g., Marti-Carvajal et\al (2012) The Cochrane Collaboration, Published by Wiley & Sons, Ltd. Additional interventions can generally be classified into two major treatment groups: pharmaceutical interventions (systemic and topical agents) and non-pharmaceutical interventions. Systemic pharmaceutical interventions include, for example, vascular drugs (e.g., pentoxifylline, isoxsuprine hydrochloride, and xanthinol nicotinate), antioxidant agents (e.g., L-camitine), EPO and EPO-stimulating agents, growth factors (e.g., Bosentan), minerais (e.g., zinc sulphate), agonists of HbF synthesis (e.g., arginine butyrate), and antibiotics. Topical pharmaceutical interventions include, for example, antibiotics, antiseptics, growth factors (e.g., GM-CSF, RGD peptide matrix, Solcoseryl®), steroids (e.g., cortisone), and pain relievers (e.g, opioids). Non-pharmaceutical interventions include, for example, reconstructijve surgery, cell therapy, laser therapy, and hyperbaric oxygen.
Ulcers and Sickle Cell Disease
Numerous genes contribute to classical sickle-cell disease (SCD; drepanocytosis; sickle cell anémia). Primarily, SCD is an inherited disorder caused by a mutation in the βglobin gene (a mutation of a glutamate to a valîne at codon 6). See, e.g., Kassim et al. (2013) Annu Rev Med, 64: 451-466. Sickle-cell anémia refers to the most common form of SCD, with a homozygous mutation in the allele (HbSS), affecting 60 to 70% of people with SCD.
Because of the mutation in the /Tglobin gene, abnormal hemoglobin molécules are produced with a hydrophobie motif that is exposed when it is in a deoxygenated state. See, e.g., Eaton et al. (1990) Adv Protein Chem, 40: 63-279; Steinberg, MH (1999) N Engl J Med 340(13): 1021-1030; and Ballas et al. (1992) Blood, 79(8) 2154-63. Once exposed, the chains of the separate hemoglobin molécules polymerize, which results in damage to the red blood cell membrane and cellular déhydration. The membrane damage is manifested, in part, by a redistribution of membrane lipids leading to the expression of phosphatidylserinc on the outer leaflet of the érythrocyte membrane. See, e.g., (2002) Blood 99(5): 1564-1571. Extemalized phosphatidylserîne promûtes adhesion to both macrophages and activated endothélial cells, which contributes to vascular (vaso) occlusion. Thus, at low oxygen states, the red cell’s hemoglobin précipitâtes into long crystals that cause it to elongate, niorphologically switching into a “sickled” red blood cell. Both génotype and the extent and degree of deoxygenation contribute to the severity of hemoglobin polymerization. It has been
-11619001 demonstrated that the presence of fêtai hemogltjjbin proportionally reduces the amount of pathological hemoglobin polymers and is protective from vaso-occlusive crises.
Most sickle-cell disease patients expérience painful épisodes call pain crises. A sickle-cell pain crisis refers to acute sickling-related pain that lasts for at least l hour (e.g., at least l, 2, 3, 4, 5, 6, or 10 hours) and optionally requires pain management therapy such as, e.g., administration of one or more narcotic and/or non-steroid anti-inflammatory agent. A pain crisis typically results in patient admission to a medical facility for pain management therapy. Acute pain in patients with SCD is generally ischémie in nature and can resuit from the occlusion of microvascular beds. Clinical data indicate that some patients with SCD hâve from three to ten épisodes of pain crisis per year. In many patients a pain crisis épisode will typically be resolved in about a week. In some|cases, sevcrc épisodes may persist for scvcral weeks or even months. SCD pain management often requires administration of one or more opioid analgésies (e.g. hydromorphone, meperidine, etc.), non-steroidal anti-inflammatory drugs (e.g., ketorolac tromethamine), and corticosteroids. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent pain crisis in a patient with SCD. In some embodiments, one or more ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to reduce the frequency of pain management (e.g., treatment with one or more narcotics, non-steroid anti-inflammatory drugs, and/or corticosteroids) in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to reduce the dosage amount of one or more pain management agents narcotics, non-steroid anti-inflammatory drugs, and/or corticosteroids) in a SCD patient.
Vaso-occlusive crises are one of the clinical hallmarks of SCD. See, e.g., Rees et al. (2010) Lancet, 376: 2018-2031. Hypoxia, acidosis, inflammatory stress, and endothélial cell activation promote the entrapment of rigid, polymerized sickled érythrocytes and leukocytes within small vessels.Sickled red blood cells obstruct capillaries and restrict bloodflow to the organ, leading to ischemia, pain, tissue necrosis,and damage to varions organs. This can cause vascular obstruction,leading to tissue ischemia. Although polymerizationand early membrane damage are initially réversible, repeated sicklingepisodes lead to irreversibly sickled érythrocytes, which can impact a variety of organsystems and lead to death. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in
-II719001 combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent vaso-occlusive crisis in a SCD patient. In some embodiments, one or more ActRIl antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent vasoocclusion in a SCD patient. In some embodiments, one or more ActRIl antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent a complication of vaso-occlusion in a SCD patient. In some embodiments, one or more ActRIl antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent vaso-occlusion pain in a SCD patient.
Like vaso-occlusive complications, hemolytic anémia leads to significant morbidity in SCD patients. See, e.g., Pakbaz et al. (2014) Hematol Oncol Clin N Am 28: 355-374; Rassi m et al. (2013) Annu Rev Med 64: 451-466. Multiple factors contribute to chronic anémia in SCD. As érythrocytes become deformed, antibodies are created to exposed antigens, which leads to increased destruction of érythrocytes, with an average lifespan of 17 days instead of 110 to 120 days. The release of hemoglobin during hemolysis înhibits nitric oxide signaling, leading to endothélial cell dysfunction and conlributing to a hypercoagulable state. Chronic hemolysis contributes to anémia along with an impaired érythrocyte compensatory mechanism caused by hormone and vitamin deficiencies. Progressive rénal disease is common in SCD, leading to decreaseid erythropoietin and thus impaired stimulation erythropoiesis. Folate and iron deficiency are common because of higher demand from érythrocyte production and increased urinary iron losses. Ail of these factors contribute to chronic anémia in SCD patients. In some embodiments, one or more ActRIl antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent anémia in a SCD patient. In some embodiments, one or more ActRIl antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating sickle cell disease, may be used to treat or prevent a complication of anémia in a SCD patient. In some embodiments, one or more ActRIl antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat anémia in a SCD patient. In some embodiments, one or more ActRIl antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat a complication of anémia in a SCD patient.
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In some embodiments, one or more ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive therapies for treating SCD, may be used to prevent anémia in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or 5 supportive therapies for treating SCD, may be used to prevent a complication of anémia in a SCD patient.
Acute anémia, which can be severe and potentially fatal, is associated with a 10% to 15% mortality rate, in SCD patients. In general, severe épisodes are precipitated by three main causes: splenic séquestration crises, aplastic crises, or hyperhemolytic crises. See, e.g., 10 Ballas étal. (2010) Am J Hematol, 85: 6-13.
Splenic séquestration crises occur as a resuit of érythrocyte vaso-occlusîon within the spleen, where a poohng of érythrocytes causes its rapid enlargement. As such, there is a decrease in circulating hemoglobin (e.g., decreasing by 2 g/dL) and effective circulating volume, which may lead to hypovolémie shock. In some embodiments, one or more ActRII 15 antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive therapies for treating SCD, may be used to treat or prevent splenic séquestration crises m a SCD patient. In some embodiments, one or more ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive therapies for treating SCD, may be used to treat or prevent splenic séquestration of red blood cells in a 20 SCD patient. In some embodiments, one or more ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive therapies for treating SCD, may be used to treat or prevent splenomegaly in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive therapies for treating SCD, may be used to treat splenomegaly 25 in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive therapies for treating SCD, may be used to prevent splenomegaly in a SCD patient.
Aplastic crises arise when erythropoiesis is impaired. Becauseof the constant overproduction of erythrocytes, an aplasticcrisis can rapidiy resuit in severe anémia. 30 Infections, such asparvovirus B19, streptococci, Salmonella, and Epsteîn-Barrvirus, are common causes for the transient arrest of erythropoiesis.CircuIating erythrocytes and réticulocytes areboth decreased during aplastic crises.In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents
-11919001 and/or supportive thérapies for treating SCD, may be used to treat or prevent aplastic crises in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent aplastic anémia in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat aplastic crises in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to prevent aplastic crises in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat aplastic anémia in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to prevent aplastic anémia in a SCD patient.
Hyperhemolysis occurswhen there is a sudden exacerbation of anémia with reticulocytosis, without evidence of splenic sequeslration.Hyperhemolysiscrises hâve been documented in patients with multiple transfusions or in patients receiving intravenous immunoglobulin therapy. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent hyperhemolytic crises in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent hyperhemolytic anémia in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies ior treating SCD, may be used to treat hyperhemolytic crises in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to prevent hyperhemolytic crises in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat hyperhemolytic anémia in a SCD patient. In some embodiments, one or more ActRII antagonist agents of the disclosure, optionally in combination with one or
-12019001 more agents and/or supportive thérapies for treating SCD, may be used to prevent hyperhemolytic anémia in a SCD patient.
In certain aspects, ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent a cardiac complication of SCD. Typically, chronic anémia in SCD causes a compensatory increased cardiac output. This, in tum, leads to cardiomegaly and left ventricular hypertrophy with left ventricular dysfunction. See, e.g., Adebayo et al. (2002) Niger J Med, 11: 145-152; Sachdev et al. (2007) J Am Coll Cardiol, 49: 472-279; and Zilberman et al. (2007) Am J Hematol 82: 433-438. Acute myocardial infarction can occur, even without coronary artery disease, and is thus underdiagnosed in SCD-See. e.g., Pannu et al. (2008) Crit Pathw Cardio, 7: 133-138. Cardiac arrhythmias and congestive heart failure hâve also been linked to prématuré death in SCD patients..See, e.g., Fitzhugh et al. (2010) Am J Hematol 85: 36-40. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent one or more cardiac complications of SCD including, e.g., increased cardiac output, cardiomegaly, cardiomyopathy, left ventricular hypertrophy, acute myocardial infarction, arrhythmia, and congestive heart failure. In some embodiments, ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat one or more cardiac complications of SCD. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to prevent one or more cardiac complications of SCD.
In certain aspects, ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent a pulmonary complication of SCD. SCD frequently results in both acute and chronic pulmonary complications. See, e.g., Rucknagel, DL (2001) Pediatr Pathol MOI Med, 20: 137-154; Haynes et al. (1986) Am J Med 80: 833-840. Acute complications may include infection, pulmonary emboli from thrombi, bone marrow infarction, and fai emboli. Pulmonary dysfunction may occur because of local pain irom rib and sternal infarctions, leading to hypoventilation and atelectasis with hypoxemia.Chronic complications include sickle cell chronic lung disease and pulmonary hypertension. Acute chest syndrome (ACS) is unique to people with sickle disease and is defined by a new pulmonary infiltrate involving at least 1 complété lung segment, chest pain, and température above 38.5°C along with tachypnea, wheeze, or cough. See, e.g, Vichinsky et al. (2000) N Engl J Med, 342:
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1855-1865. Development of pulmonary infarction, fat embolism, and infections may ail contribute to ACS. Infection is a major cause of morbidity and mortality in ACS patients.
Pulmonary hypertension is currently a major cause of morbidity and mortality in SCD.See, e.g., De Castro et al. (2008) Am J Hematol, 83: 19-25; Gladwin et al. (2004) N Engl J Med 350: 886-895. Pulmonary hypertension has been documented in 32% of adults with SCD and is related to vaso-occlusive crises and hemolysis. See, e.g., Machado et al. (2010) Chest, 137(6 supple): 30S-38S. Cell-free hemoglobin from hemolysis is thought to decrease nitric oxide, a pulmonary vasodilator, contributing to vaso-occlusion. See, e.g., Wood et al. (2008) Free Radie Biol Med 44: 1506-1528. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent one or more pulmonary complications of SCD încluding, e.g., fat or bone marrow emboli, pulmonary edema, sicklecell lung disease, pulmonary hypertension, thromboemboli, and Acute chest syndrome. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat one or more pulmonary complications of SCD. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to prevent one or more pulmonary complications of SCD.
In certain aspects, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent a hepatic complication of SCD. Liver pathology is common in SCD, with hepatomegaly being observed in -90% of autopsy cases. See, e.g., Bauer et al. (1980) Am J med 69: 833-837; Mills et al. (1988) Arch Pathol Lab Med 112: 290-294. The effects of sickle cell anémia on the liver include intrasinusoidal sickling with proximal sinusoïdal dilation, Kupffer cell hyperplasia with erythrophagocytosis, and hemosiderosis. Focal necrosis, regenerative nodules, and cirrhosis hâve also been described in postmortem examinations. Vaso-occlusion can iead to sinusoïdal obstruction and ischemia, resulting in acute sickle hepatic crises.Similar to splenic séquestration, érythrocytes can be sequestered within the liver, leading to acute anémia. See, e.g., Lee et al. (1996) Postgrad Med J 72: 487488. Hepatic séquestration can also lead to intrahepatic cholestasis. See, e.g., Shao ei al. (1995) Am J Gastroenterol 90: 2045-2050.Ischemia within hépatocytes from sickling episodesalso leads to ballooning of érythrocytes and intracanalicularcholestasis. Some thérapies used for treating SCD also contribute to liver pathology.For example, frequent
-12219001 transfusions lead to increased iron depositionwithin Kupffer cells (which may lead to iron overload) and increase the risk of infection withblood-bome disease such as viral hepatitis. In some embodiments, ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent one or more hepatic complications of SCD including, e.g., hepatic failure, hepatomegaly, hepatic séquestration, intrahepatic cholestasis, cholelithiasis, and iron overload.
In certain aspects, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent a splenic complication of SCD. Splenic séquestration, as previously discussed, occurs as a resuit of vaso-occlusion of érythrocytes within the spleen. Acute exacerbations resuit in splenomegaly and occasionally splenic infarction. More commonly, subclinical splenic séquestration may lead to the graduai loss of splenic function, leading to fimctional hyposplenia and asplénia. This, in tum, can lead to an increased susceptibility to sepsis as a resuit of encapsulated bacteria. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent one or more splenic complications of SCD including, e.g., acute or chronic splenic séquestration, splenomegaly, hyposplenia, asplénia, and splenic infarction. In some embodiments, ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat one or more splenic complications of SCD. In some embodiments, ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to prevent one or more splenic complications of SCD.
In certain aspects, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent a rénal complication of SCD. Approximately twelve percent of people with SCD develop rénal failure. See, e.g., Powars et al. (2205) Medicine 84: 363-376; Scheinman, JI (2009) Nat Clin Pract Nephrol 5: 78-88.Vaso-occlusion within the vasa recta capillaries leads tomicrothrombotic infarction and extravasation of erythrocytesinto the rénal medulla.Blood becomes more viscousin the rénal medulla because of low oxygen tension, low pH,and high osmolality and, if severe, can contrîbute to ischemia,infarction, and papillary necrosis.Repeated glomerularischemia leads to glomerulosclerosis.Clinical consequencesof ischémie damage include hematuria, proteinuria.decreased concentrating
-I2319001 abilîty, rénal tubular acidosis, abnormalproximal tubular function, acute and chronic rénal failure, and urinary tract infections. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent one or more rénal complications of SCD including, e.g., acute and/or chronic rénal failure, pyelonephritis, and rénal medullary carcinoma. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat one or more rénal complications of SCD. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to prevent one or more rénal complications of SCD.
In certain aspects, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD. may be used to treat or prevent a bone and/or joint complication of SCD. Bone and joint complications are a common complication in SCD patients. See, e.g., Hernigou et al. (1991) J Bone Join Surg Am, 73; 81-92. Pain from the small bones in the hands and feet, dactylitîs, occurs frequently in infants with SCD. Long-term conséquences of vaso-occlusion within bone marrow include infarcts, necrosis, and ultimately degenerative changes. Because of hyposplenia, bacterial infections are more common in SCD. Infarcted bone and bone marrow are common sites of infection, leading to osteomyelitis and septic arthritis. Osteonecrosis, or avascular necrosis, occurs after infarction with bone and bone marrow. Infarctions are most common within long bones such as the humérus, tibia, and fémur. Chronic weight bearing causes stress on abnormal fémoral heads and leàds to progressive joint destruction and arthritis.In some embodiments, ActRII antagonist agents of the disclosure. optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent one or more bone and/or joint complications of SCD including, e.g., infarction, necrosis, osteomyelitis, septic artliritis, osteonecrosis, and osteopenia. In some embodiments, ActRII antagonist agents ot the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat one or more bone and/or joint complications complications of SCD. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to prevent one or more bone and/or joint complications complications of SCD.
-I2419001
In certain aspects, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent a neurological complication of SCD. Approximately 25 percent of individuals with SCD are affected by neurological injury. See, e.g., Ohene-Frempong et al. (1998) Blood, 91: 288-294; Verduzco étal. (2009) Blood 114: 5117-5125. The injuries may be acute or chronic. Cerebrovascular accidents are most conimon in adults, but dépend on the génotype. A person with HbSS has the highest cerebrovascular risk, with a 24 percent likelihood of having a clinical stroke by the âge of 45.Ischémie strokes are more common in children under 9 years of âge, whereas hémorrhagie strokes are more common in adults. Ischémie strokes occur because of the occlusion of large intracranial arteries, leading to ischemia. The ischemia is secondary to occlusion of smaller vessels by rigid érythrocytes, exacerbated by chronic anémia, a hypercoagulable state, and flow-related hémodynamie
I injury to the arterial endothélium, further increasîng the likelihood of érythrocyte adhesion. In contrast, hemorrhagic strokes may occur in intraventricular, intraparenchymakand subarachnoid spaces. See, e.g., Anson, et al. (1991) J Neurosurg. 75: 552-558. Intraventricular hemorrhage may be associated with rupture of anterior cérébral artery aneurysms or direct extension of intraparenchymalhemorrhage into the latéral or third ventricle. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent one or more neurological complications of SCD inclduing, e.g., aneurysm, ischémie stroke, intraparenchymal hemorrhage, subarachnoid hemorrhage, and intraventricular hemorrhage. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat one or morejneurological complications complications of SCD. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to prevent one or more neurological complications complications of SCD.
In certain aspects, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent an ophthalmic complication of SCD. Eye complications in SCD mainly affect the retina. See, e.g., Downes et al. (2005) Opthalmology, 112: 1869-1875; Fadugbagbe et al. (2010) Ann Trop Paediatr 30: 19-26. As a resuit of vaso-occlusive crises, peripheral retinal ischemia occurs. New blood vessels (sea fan formations) form mostly near
I
-12519001 arteriovenous crossings and are known as proliférative sickle retinopathy.These new vessels can bleed easily, causing traction retinal detachments and ultimately blindness.Nonproliferative retinal changes are also more common in SCD. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent one or more ophthalmîc complications of SCD including, e.g., peripheral retinal ischemia, proliférative sickle retinopathy, vitreous hemorrhage, retinal detachment, and non-proliferative retinal changes. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat one or more ophthalmîc complications complications of SCD. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may bc used to prevent one or more ophthalmîc complications complications of SCD.
In certain aspects, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent a cutaneous (skin) complication of SCD. One of the common cutaneous complications of SCD is the manifestation of ulcers. See, e.g., Keast et al. (2004) Ostomy Wound Manage., 50(10): 64-70; Trent et al. (2004) Adv Skin Wound Care, 17(8): 410-416; J.R. Eckman (1996) Hematol Oncol Clin North Am., 10(6): 1333-1344; and Chung et al. (1996) Advances in Wound Care, 9(5): 46-50. While the mechanism for ulcer development in SCD patients has not been fully elucidated, it is believed to be a multifactorial process that is influenced by various aspects of SCD including, for example, vascular obstruction, increased venous and capillary pressure, abnormal blood rheology, tissue hypoxia, and increased susceptibility to bacterial invasion causcd by venous stasis. increased venous pressure, or both . The rate of ulcer healing has been found to be three to 16 limes slower that the rate in patients with SCD. Ulcers may persîst for months to years and there is a high incidence of reoccurrence in SCD patients. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat or prevent one or more cutaneous complication of SCD including, e.g., ulcers. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating SCD, may be used to treat one or more cutaneous complications complications of SCD, e.g., ulcers. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for
-12619001 treating SCD, may be used to prevent one or more cutaneous complications complications of SCD, e.g., ulcers.
In certain aspects, ActRII antagonist agents of the disclosure may be administered to a subject in need thereof în combination with one or more additional agents (e.g., hydroxyurea, an EPO antagonist, EPO, an opioid analgésie, a non-steroidal anti-inflammatory drug, a corticosteroids, an iron-chelating agent) or supportive thérapies (e.g., red blood cell transfusion) for treating sickle-cell disease or one or more complications of sickle-cell disease (e.g., cutaneous complications such as cutaneous ulcers).
The mainstay of treatment for the majority of SCD patients is supportive. Current treatment options for patients with sickle cell disease include antibiotics, pain management, intravenous fluids, blood transfusion, surgery, and compounds such as hydroxyurea.
Hydroxyurea (e.g. Droxia®)is an approved drug for treating Sickle Cell Disease. Hydroxyurea is an S-phase cytotoxic drug and is used for long-term therapy. It is believed to increase the levels of hemoglobin F which prevents formation of S-polymers and red cell sickling. It is also believed to increase NO production. A multi-center trial of hydroxyurea in adults with Sickle Cell Disease showed that hydroxyurea reduced the incidence of painful épisodes by nearly half. However, presently hydroxyurea is used only in patients who suffer severe complications of SCD and who are capable of following the daily dosage régimes. The general belief is that hydroxyurea therapy is effective only if given in a structured environment with a high potential for compliance. Unfortunately, many SCD patients are refractory to hydroxyurea. In some embodiments, the methods of the présent disclosure relate to treating sickle-cell disease in a subject in need thereof by administering a combination of an ActRII antagonist of the disclosure and hydroxyurea. In some embodiments, the methods of the présent disclosure relate to treating or preventing one or more complications (e.g., cutaneous complications such as cutaneous ulcers) of sickle-cell disease in a subject in need thereof by administering a combination of an ActRII antagonist of the disclosure and hydroxyurea.
Regular red blood cell transfusions are also a common therapy for SCD patients. However, several issues make them unsuitable for long-term use. Aithough regular transfusions hâve been shown to prevent stroke, ACS, and vaso-occlusive pain crises, they do not prevent the development of siient infarcts or the progression of moyamoya disease, a disorder of the cérébral circulation în which certain arteries are constricted and the compensatory collateral vessels are prone to hemorrhage. See, e.g., Bishop et al. (2011) Blood Cells, Molécules & Disease, 47: 125-128; DeBaun et al. (2012) Blood, 119: 478719001
4596. Furthennore, SCD patients may develop iron overload as a conséquence of red blood cell transfusion, which is associated with its own morbidity. Regular red blood cell transfusion requires exposure to varions donor units of blood and hence a higher risk of alloimmunization. Difficulties with vascular access, availability of and compliance with iron chélation, and the high cost are some of the reasons why regular transfusions are not an optimal option for universal therapy. Wayne étal. (2000) Blood, 96: 2369-2372. In some embodiments, the methods of the présent disclosure relate to treating sickle-cell disease in a subject in need thereof by administering a combination of an ActRII antagonist of the disclosure and one or more blood cell transfusions. In some embodiments, the methods of the présent disclosure relate to treating or preventing one or more complication of sickle-cell disease in a subject in need thereof by administering a combination of an ActRII antagonist of the disclosure and one or more red blood cell transfusions. In some embodiments, treatment with one or more ActRII antagonists of the disclosure is effective at decreasing the transfusion requirement in a SCD patient, e.g., reduces the frequency and/or amount of blood transfusion required to effectively treat SCD or one or more complications of SCD.
In certain embodiments, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRll antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, c7c.)may be used in combination with supportive thérapies for SCD. Such thérapies include transfusion with either red blood cells or whole blood to treat anémia. In SCD patients, normal mechanisms for iron homeostasis are overwhelmed by repeated transfusions, eventually leading to toxic and potentially fatal accumulation of iron in vital tissues such as heart. liver, and endocrine glands. Thus, supportive thérapies for SCD patients also include treatment with one or more iron-chelating molécules to promote iron excrétion in the urine and/or stool and thereby prevent, or reverse, tîssue iron overload. Effective iron-chelating agents should be able to selectively bind and neutralize ferrie iron, the oxidized form of nontransferrin bound iron which iikely accounts for most iron toxicity through catalytic production of hydroxyl radicals and oxidation products. See, e.g., Esposito et al. (2003) Blood 102:2670-2677. These agents are structurally diverse, but ail possess oxygen or nitrogen donor atoms able to form neutralizing octahedral coordination complexes with individual iron atoms in stoichiometries of 1:1 (hexadentate agents), 2:1 (tridentate), or 3:1 (bidentate). Kalinowski et al. (2005) Pharmacol Rev 57:547-583. In general, effective ironchelating agents also are relatively low molecular weight (e.g., less than 700 daltons), with solubility in both water and lipids to enabie access to affected tissues. Spécifie examples of iron-chelating molécules include deferoxamine (also known as desferrioxamine B,
-12819001 desferoxamine B, DFO-B, DFOA, DFB, or Desferal®), a hexadentate agent of bacterial origin requiring daily parentéral administration, and the orally active synthetic agents deferiprone (also known as Ferriprox®) (bidentate) and deferasîrox (also known as bishydroxyphenyl-triazole, 1CL670, or Exjade®) (tridentate). Combination therapy consisting of same-day administration of two iron-chelating agents shows promise in patients unresponsive to chélation monotherapy and also in overcoming issues of poor patient compliance with dereroxamine alone. Cao et al. (2011) Pediatr Rep 3(2):e 17; and Galanello et al. (2010) Ann NY Acad Sci 1202:79-86.
Ineffective Erythropoiesis and Ulcers
In certain aspects, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies, may be used to treat or prevent an ineffective erythropoiesis in a subject in need thereof. Originally distinguished from aplastic anémia, hemorrhage, or peripheral hemolysis on the basis of ferrokinetic studîes (Ricketts et al., 1978, Clin Nucl Med 3:159-164), ineffective erythropoiesis describes a diverse group of anémias in which production of mature RBCs is less than would be expected given the number of erythroid precursors (erythroblasts) present in the bone marrow (Tanno et al., 2010, Adv Hematol 2010:358283). In such anémias, tissue hypoxia persists despite elevated erythropoietin levels due to ineffective production of mature RBCs. A vicious cycle eventually develops in which elevated erythropoietin levels drive massive expansion of erythroblasts, potentially leading to splenomegaly (spleen enlargement) due to extramedullary erythropoiesis (Aizawa et al, 2003, Am J Hematol 74:68-72), erythroblastinduced bone pathology (Di Matteo et al., 2008, J Biol Regul Homeost Agents 22:211-216), and tissue iron overload, even in the absence of therapeutic RBC transfusions (Pippard et al., 1979, Lancet 2:819-821). Thus, by boosting erythropoietic effectiveness, an ActRII antagonist of the disclosure may break the aforementioned cycle and may alleviate not only the underlying anémia but also the associated complications of elevated erythropoietin levels, splenomegaly, bone pathology, and tissue iron overload. ActRII antagonists can treat ineffective erythropoiesis, including anémia and elevated EPO levels, as well as complications such as splenomegaly, erythroblast-induced bone pathology, and iron overload, cutaneous ulcers, and their attendant pathologies. With splenomegaly, such pathologies include thoracic or abdominal pain and réticuloendothélial hyperplasia. Extramedullary hematopoiesis can occur not only in the spleen but potentially in other tissues in the form of
-12919001 extramedullary hematopoietic pseudotumors (Musallam et al., 2012, Cold Spring Harb Perspect Med 2:a013482). With erythroblast-induced bone pathology, attendant pathologies include low bone minerai density, osteoporosis, and bone pain (Haidar i., 2011, Bone 48:425-432). With iron overload, attendant pathologies include hepcidin suppression and hyperabsorption of dietary iron (Musallam et al., 2012, Blood Rev 26(Suppl 1):S 16-S19), multiple endocrinopathies and lîver fibrosis/cirrhosis (Galanello et al., 2010, Orphanet J Rare Dis 5:11), and iron-overload cardiomyopathy (Lekawanvijit et al., 2009, Can J Cardiol 25:213-218).
The most common causes of ineffective erythropoiesis are the thalassemia syndromes, hereditary hemoglobinopathîes in which imbalances in the production of intact alpha- and beta-hemoglobin chains lead to increased apoptosis during erythroblast maturation (Schrier, 2002, Cuir Opin Hematol 9:123-126). Thalassemias are collectively among the most frequent genetic disorders worldwide, with changing épidémiologie patterns predicted to contribute to a growing public health problem in both the U.S. and globally (Vichinsky, 2005, Ann NY Acad Sci 1054:18-24). Thalassemia syndromes are named according to their severity. Thus, ct-thalassemias include α-thalassemia minor (also known as a-thalassemia trait; two affected α-globin genes), hemoglobinjH disease (three affected a-globin genes), and α-thalassemia major (also known as hydrops fetalis; four aflected α-globîn genes). βThalassemias include β-thalassemia minor (also known as β-thalassemia trait; one affected βglobin gene), β-thalassemia intermedia (two affected β-globin genes), hemoglobin E thalassemia (two affected β-globin genes), and β-thalassemia major (also known as Cooley’s anémia; two affected β-globin genes resulting in a complété absence of β-globin protein). βThalassemia impacts multiple organs, is associated with considérable morbidity and mortality, and currently requires life-long care. Although life expectancy in patients with β-thalassemia has increased in rccent years due to use of regular blood transfusions in combination with iron chélation, iron overload resulting both from transfusions and from excessive gastrointestinal absorption of iron can cause sei-ious complications such as heart disease, thrombosis, hypogonadism, hypothyroidism, diabètes, osteoporosis, and osteopenîa (Rund et al, 2005, N Engl J Med 353:1135-1146). As demonstrated herein with a mouse model of βthalassemia, an ActRIIa antagonist, optionally combined with an EPO receptor activator, can be used to treat thalassemia syndromes. Furthermore, data disclosed herein demonstrates that a GDF Trap polypeptide can be used to promote positive effects on red blood cell parameters
-13019001 (e.g., increased levels of sérum hemoglobin) as well as treat complications of thalassemia (e.g., cutaneous ulcers) in human thalassemia patients.
In certain aspects, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating an ineffective erythropoîesis disorder, such as a thalassemia syndrome, may be used to treat or prevent a cutaneous (skin) complication of ineffective erythropoîesis. A common cutaneous complication of ineffective erythropoîesis, particularly thalassemia, is the manifestation of ulcers. While the mechanism for ulcer development in thalassemia patients has not been fully elucidated, it is believed to be a multifactorial process thaï is inlluenced by various aspects of thalassemia including, for example, and tissue hypoxia. In some embodiments, ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating ineffective erythropoîesis (e.g., thalassemia), may be used to treat or prevent one or more cutaneous complication of ineffective erythropoîesis (e.g., thalassemia) including, e.g., ulcers. In some embodiments, ActRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating ineffective erythropoîesis (e.g., thalassemia), may be used to treat one or more cutaneous complications complications of ineffective erythropoîesis (e.g., thalassemia) including, e.g., ulcers. In some embodiments, ^ctRII antagonist agents ofthe disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating ineffective erythropoîesis (e.g., thalassemia), may be used to prevent one or more cutaneous complications complications of ineffective erythropoîesis (e.g., thalassemia) including, e.g., ulcers. In some embodiments, ActRII antagonist agents of the disclosure, optionally m combination with one or more agents and/or supportive thérapies ior treating β-thalassemia (e.g., β-thalassemia intermedia), may be used to treat one or more cutaneous complications complications of β-thalassemia (e.g., β-thalassemia intermedia) including, e.g., ulcers. In some embodiments, ActRII antagonist agents of the disclosure, optionally in combination with one or more agents and/or supportive thérapies for treating β-thalassemia (e.g., βthalassemia intermedia), may be used to prevent one or more cutaneous complications complications of β-thalassemia (e.g., β-thalasseinia intermedia) including, e.g., ulcers.
Other Anémia Indications
-13119001
ActRII antagonist of the disclosure, optionally combined with one or more supportive thérapies, can be used for treating disorders of ineffective erythropoiesis besides thalassemia syndromes. Such disorders include siderblastic anémia (inherited or acquired); dyserythropoietic anémia (Types I and II); sickle cell anémia; hereditary spherocytosis; pyruvate kinase deficiency; megaloblastic anémias, potentially caused by conditions such as folate deficiency (due to congénital diseases, decreased intake, or increased requirements), cobalamin deficiency (due to congénital diseases, pemicious anémia, impaired absorption, pancreatic insuffleiency, or decreased intake), certain drugs, or unexplained causes (congénital dyserythropoietic anema, refractoryl megaloblastic anémia, or erylhroleukemia); myelophthisic anémias, including myelofibrosis (myeloid metaplasia) and myelophthisis; congénital erythropoietic porphyria; and lead poisoning.
As shown herein, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator and one or more additional supportive thérapies, may be used to increase red blood cell, hemoglobin, or réticulocyte levels in healthy individuals and selected patient populations. Examples of appropriate patient populations include those with undesirably low red blood cell or hemoglobin levels, such as patients having an anémia, sickle-cell patients, 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 resuit in substantial blood loss. In some embodiments, a patient with adéquate red blood cell levels is treated with one or more ActRII antagonist agents to increase red blood cell levels, and then blood is drawn and stored for later use in transfusions.
One or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator and/or other one or more additional supportive thérapies, may be used to increase red blood cell levels, hemoglobin levels, and/or hematocrit levels in a patient having an anémia (e.g., a sickle-cell patient, a thalassemia patient, etc.). When observing hemoglobin and/or hematocrit^ levels in humans, a level of iess than normal for the appropriate âge and gender category may be indicative of anémia, although indîvidual variations are taken into account. For example, a hemoglobin level from 10-12.5 g/dl, and typically about 11.0 g/dl is considered to be within the normal range in health adults, although, in tenns of therapy, a lower target level may cause fewer cardiovascular side effects.
-13219001
See, e.g., Jacobs et al. (2000) Nephrol Dial Transplant 15, 15-19. Alternatively, hematocrit levels (percentage of the volume of a blood sample occupied by the cells) can be used as a measure for anémia. Hematocrit levels for healthy individuals range from about 41-51 % for adult males and from 35-45% for adult females. In certain embodiments, a patient may be treated with a dosing regimen intended to restore the patient to a target level of red blood cells, hemoglobin, and/or hematocrit. As hemoglobin and hematocrit levels vary from person to person, optimally, the target hemoglobin and/or hematocrit level can be individualized for each patient.
Anémia is frequently observed in patients having a tissue injury, an infection, and/or a chronic disease, particularly cancer. In some subjects, anémia is distinguished by low erythropoietin levels and/or an inadéquate response to erythropoietin in the bone marrow. See, e.g., Adamson, 2008, Harrison’s Principles of Internai Medicine, 17th ed.; McGraw Hill, New York, pp 628-634. Potential causes of anémia include, for example, blood-loss, nutritional déficits (e.g. reduced dietary intake of protein), médication reaction, various problems associated with the bone marrow, and many diseases. More particularly, anémia has been associated with a variety of disorders and conditions that include, for example, bone marrow transplantation; solid tumors (e.g., breast cancer, lung cancer, and colon cancer); tumors of the lymphatic System (e.g., chronic lymphocyte leukemia, non-Hodgkin’s lymphoma, and Hodgkin’s lymphoma); tumors of the hematopoietic System (e.g., leukemia, a myelodysplastic syndrome and multiple myeloma); radiation thcrapy; chemothcrapy (e.g.. platinum containing regimens); inflammatory and autoimmune diseases, including, but not limited to, rheumatoid arthritis, other inflammatory arthritides, systemic lupus erythematosis (SLE), acute or chronic skin diseases (e.g., psoriasis), inflammatory bowel disease (e.g, Crohn's disease and ulcerative colitis); acute or chronic rénal disease or failure, including idiopathic or congénital conditions; acute or chronic liver disease; acute or chronic bleeding; situations where transfusion of red blood cells is not possible duc to patient allô- or autoantibodies and/or for religious reasons (e.g, some Jehovah's Witnesses); infections (e.g., malaria and osteomyelitis); hemoglobinopathies including, for example, sickle cell disease (anémia), a thalassemias; drug use or abuse (e.g., alcohol misuse); pédiatrie patients with anémia from any cause to avoid transfusion; and elderly patients or patients with underlying cardiopulmonary disease with anémia who cannot receive transfusions due to concerns about circulatory overload. See, e.g., Adamson (2008) Harrison’s Principles of Internai Medicine, 17th ed.; McGraw Hill, New York, pp 628-634. In some embodiments, one or more ActRII
-13319001 antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, may be used to treat or prevent anémia associated with one or more of the disorders or conditions disclosed herein. In some embodiments, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, may be used to treat anémia associated with one or more of the disorders or conditions disclosed herein. In some embodiments, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, may be used to prevent anémia associated with one or more of the disorders or conditions disclosed herein.
Many factors can contribute to cancer-related anémia. Some are associated with the disease process itself and the génération of inflammatory cytokines such as interleukin-l, interferon-gamma, and tumor necrosis factor. Bron et al. (2001) Semin Oncol 28(Suppl 8):Ιό. Among its effects, inflammation induces the key iron-regulatory peptide hepcidin, thereby inhibiting iron export from macrophages and generally limiting iron availability for erythropoiesis. See, e.g., Ganz (2007) J Am Soc Nephrol 18:394-400. Blood loss through various routes can also contribute to cancer-related anémia. The prevalence of anémia due to cancer progression varies with cancer type, ranging from 5% in prostate cancer up to 90% in multiple myeloma. Cancer-related anémia has profound conséquences for patients, including fatigue and reduced quality of life, reduced treatment efficacy, and increased mortality. In some embodiments, one or more ActRII antagonist agents of the disclosure (e.g., a GDFActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, may be used to treat or prevent a cancer-related anémia. In some embodiments, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, may be used to treat a cancer-related anémia. In some embodiments, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, may be used to prevent a cancer-related anémia. '
A hypoproliferative anémia can resuit from primary dysfunction or failure of the bone marrow. Hypoproliferative anémias include: anémia of chronic disease, anémia of kidney
-13419001 disease, anémia associated with hypometabolic states, and anémia associated with cancer. In each of these types, endogenous erythropoietin levels are inappropriately low for the degree of anémia observed. Other hypoproliferative anémias include: early-stage iron-deficient anémia, and anémia caused by damage to the bone marrow. In these types, endogenous erythropoietin levels are appropriatelyelevated for the degree of anémia observed. Prominent examples would be myelosuppression caused by cancer and/or chemotherapeutic drugs or cancer radiation therapy. A broad review of clinical trials found that mild anémia can occur in 100% of patients after chemotherapy, while more severe anémia can occur in up to 80% of such patients. See, e.g., Groopman et al. (1999) J Natl Cancer Inst 91:1616-1634. Myelosuppressive drugs include, for example: 1) alkylating agents such as nitrogen mustards (e.g., melphalan) and nitrosoureas (e.g., streptozocin); 2) antimetabolites such as folie acid antagonists (e.g, methotrexate), purine analogs (e.g., thioguanine), and pyrimidine analogs (e.g., gemcitabine); 3) cytotoxic antibiotics such as anthracyclines (e.g., doxorubicin); 4) kinase inhibitors (e.g., gefitinib); 5) mitotic inhibitors such as taxanes (e.g, paclitaxel) and vinca alkaloids (e.g., vinorelbine); 6) monoclonal antibodies (e.g, rituximab); and 7) topoisomerase inhibitors (e.g, topotecan and etoposide). In addition, conditions resulting in a hypometabolic rate can produce a mild-to-moderate hypoproliferative anémia. Among such conditions are endocrine deficiency states. For example, anémia can occur in Addison’s disease, hypothyroidism, hyperparathyroîdism, or males who are castrated or treated with estrogen. In some embodiments, one or more ActRIl antagonist agents oflhc disclosure (e.g. a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor actîvator, may be used to treat or prevent a hyperproliferative anémia.
Chronic kidney disease is sometimes associated with hypoproliferative anémia, and the degree of the anémia varies in severity with the level of rénal impairment. Such anémia is primarily due to inadéquate production of erythropoietin and reduced survival of red blood cells. Chronic kidney disease usually proceeds gradually over a period of years or décades to end-stage (Stage-5) disease, at which point dialysis or kidney transplantation is required for patient survival. Anémia often develops early in this process and worsens as disease progresses. The clinical conséquences of anémia of kidney disease are well-documented and include development of left ventricular hypertrophy, impaired cognitive fonction, reduced quality of life, and altered immune fonction. See, e.g., Levin et al. (1999) Am J Kidney Dis 27:347-354; Nissenson (1992) Am J Kidney Dis 20(Suppl 1):21-24; Revicki et al. (1995) Am
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J Kidney Dis 25:548-554; Gafter et al., (1994) Kidney Int 45:224-231. In some embodiments, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc ), optionally combined with an EPO receptor activator, may be used to treat or prevent anémia associated with acute or chronic rénal disease or failure. In some embodiments, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, may be used to treat anémia associated with acute or chronic rénal disease or failure. In some embodiments, one or more ActRII antagonist agents of the disclosure (e.g, a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, may be used to prevent anémia associated with acute or chronic rénal disease or failure.
Anémia resulting from acute blood loss of sufficient volume, such as from trauma or postpartum hemorrhage, is known as acute post-hemorrhagic anémia. Acute blood loss initially causes hypovolemia without anémia since there is proportional déplétion of RBCs along with other blood constituents. However, hypovolemia will rapidly trigger physiologie mechanisms that shift fluid from the extravascular to the vascular compartment, which results in hemodilution and anémia. If chronic, blood loss gradually depletes body iron stores and eventually leads to iron deficiency. In some embodiments, one or more ActRII antagonist agents of the disclosure (e.g, a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), may be used to treat anémia resulting from acute blood loss.
Iron-deficiency anémia is the final stage in a graded progression of increasing iron deficiency which includes négative iron balance and iron-deficicnt erythropoîesis as intermediate stages. Iron deficiency can resuit from increased iron demand, decreased iron intake, or increased iron loss, as exemplified in conditions such as pregnancy, inadéquate diet, intestinal malabsorption, acute or chronic inflammation, and acute or chronic blood loss. With mild-to-moderate anémia of this type, the bone marrow remains hypoproliferative, and RBC morphology is largely normal; however, even mild anémia can rcsult in some microcytic hypochromie RBCs, and the transition to severe iron-deficient anémia is accompanied by hyperproliferation of the bone marrow and increasingly prévalent microcytic and hypochromie RBCs. See, e.g., Adamson (2008) Harrison’s Principles of Internai Medicine, 17th ed.; McGraw Hill, New York, pp 628-634. Appropriate therapy for irondeficiency anémia dépends on its cause and severity, with oral iron préparations, parentéral
-13619001 iron formulations, and RBC transfusion as major conventional options. In some embodiments, one or more ActRII antagonist agents of the disclosure (e.g.. a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, may be used to treat a chronic iron-deftciency.
Myelodysplastic syndrome (MDS) is a diverse collection of hematological conditions characterized by ineffective production of myeloid blood cells and risk of transformation to acute mylogenous leukemia. In MDS patients, blood stem cells do not mature into healthy red blood cells, white blood cells, or platelets. MDS disorders include, for example, refractory anémia, refractory anémia with ringed sideroblasts, refractory anémia with excess blasts, refractory anémia with excess blasts in transformation, refractory cytopenia with multilineage dysplasia, and myelodysplastic syndrome associated with an isolated 5q chromosome abnormality. As these disorders manifest as irréversible defects in both quantity and quality of hematopoietic cells, most MDS patients are afflicted with chronic anémia. Therefore, MDS patients eventually require blood transfusions and/or treatment with growth factors (e.g., erythropoietin or G-CSF) to increase red blood cell levels. However, many MDS patients develop side-effect due to frequency of such therapies. For example, patients who receive frequent red blood cell transfusion can hâve tissue and organ damage from the buildup of extra iron. Accordingly, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, may be used to treat patients having MDS. In certain embodiments, patients suffering from MDS may be treated using one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally in combination with an EPO receptor activator. In other embodiments, patient suffering from MDS may be treated using a combination of one or more ActRII antagonist agents ofthe disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.) and one or more additional therapeutic agents for treating MDS including, for example, thalidomide, lenalidomide, azacitadine, decitabine, erythropoietins, deferoxamine, antithymocyte globulin, and filgrastrim (G-CSF).
As used herein, “in combination with” or “conjoint administration” refers to any form of administration such that the second therapy is still effective in the body (e.g., the two agents or compounds are simultaneously effective in the patient, which may include synergîstic effects of the two agents or compounds). Effectiveness may not correlate to measurable concentration of the agent in blood, sérum, or plasma. For example, the different therapeutic agents or compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially, and on different schedules. Thus, an îndividual who receives such treatment can benefit from a combined effect of different thérapies. One or more GDFl l and/or activin B antagonist agents (optionally further antagonists of one or more of GDF8, activin A, activin C, activin E, and BMP6) of the disclosure can be administered concurrently with, prîor to, or subséquent to, one or more other additional agents or supportive thérapies. In general, each therapeutic agent will be administered at a dose and/or on a time schedule determined for thaï particular agent. The particular combination to employ in a regimen will take into account compatibility of the antagonist of the présent disclosure with the therapy and/or the desired therapeutic eifect to be achieved.
In certain embodiments, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc. )may be used in combination with hepcidin or a hepcidin agonist for treating sickle-cell disease, particularly sickle-cell disease complications associated with iron overload. A circulating polypeptide produced mainly in the liver, hepcidin is considered a master regulator of iron metabolism by virtue of its ability to induce the dégradation of ferroportin, an iron-export protein localized on absorptive enterocytes, hépatocytes, and macrophages. Broadly speaking, hepcidin reduces availability of extracellular iron, so hepcidin agonists may be bénéficiai in the treatment of sickle-cell disease , particularly sickle-cell disease complications associated with iron overload.
One or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), optionally combined with an EPO receptor activator, would also be appropriate for treating anémias of disordered RBC maturation, which are characterized in part by undersized (microcytic), oversized (macrocytic), misshapen, or abnormally colored (hypochromie) RBCs.
In certain embodiments, the présent disclosure provides methods of treating or preventing anémia in an îndividual in need thereof by administering to the îndividual a therapeutically effective amount of one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, e/c.)and a EPO receptor activator. In certain embodiments, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide.
-13819001 an ActRIIB polypeptide, a GDF Trap, etc.) may be used in combination with EPO receptor activators to reduce the required dose of these activators in patients that are susceptible to adverse effects of EPO. These methods may be used for therapeutic and prophylactic treatments of a patient. |
One or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.) of the disclosure may be used in combination with EPO receptor activators to achieve an increase in red blood cells, particularly at lower dose ranges. This may be bénéficiai in reducing the known off-target effects and risks associated with high doses of EPO receptor activators. The primary adverse effects of EPO include, for example, an excessive increase in the hematocrît or hemoglobin levels and polycythemia. Elevated hematocrit levels can lead to hypertension (more particularly aggravation of hypertension) and vascular thrombosîs. Other adverse effects of EPO which hâve been reported, some of which relate to hypertension, are headaches, influenza-like syndrome, obstruction of shunts, myocardial infarctions and cérébral convulsions due to thrombosîs, hypertensive encephalopathy, and red cell blood cell aplasia. See, e.g., Singibarti (1994) J. Clin Investig 72(suppl 6), S36-S43; Horl et al. (2000) Nephrol Dial Transplant I5(suppl 4), 51-56; Delanty et al. (I997) Neurology 49, 686-689; and Bunn (2002) N Engl J Med 346(7), 522-523).
Provided that antagonists of the présent disclosure act by a different mechanism thaï EPO, these antagonists may be useful for increasing red blood cell and hemoglobin levels in patients that do not respond well to EPO. For example, an ActRII antagonist of the présent disclosure may be bénéficiai for a patient in which administration of a normal to increased (>300 lU/kg/week) dose of EPO does not resuit in the increase of hemoglobin level up to the target level. Patients with an inadéquate EPO response arc found for ail types of anémia, but higher numbers of non-responders hâve been observed particularly frequentfy in patients with cancers and patients with end-stage rénal disease. An inadéquate response to EPO can be either constitutive (observed upon the first treatment with EPO) or acquired (observed upon repeated treatment with EPO).
In certain embodiments, the présent disclosure provides methods for managing a patient that has been treated with, or is a candidate to be treated with, one or more one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.) by measuring one or more hématologie parameters in the patient. The hématologie parameters may be used to evaluate
-I3919001 appropriate dosîng for a patient who is a candidate to be treated with the antagonist ot the présent disclosure to monitor the hématologie parameters during treatment, to evaluate whether to adjust the dosage during treatment with one or more antagonist of the disclosure, and/or to evaluate an appropriate maintenance dose of one or more antagonists of the disclosure. If one or more of the hématologie parameters are oulside the normal level, dosîng with one or more ActRIl antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, e/c.)may be reduced, delayed or terminated.
Hématologie parameters that may be measured in accordance with the methods provided herein include, for example, red blood cell levels, blood pressure, iron stores, and other agents found in bodily fluids that correlate with increased red blood cell levels, using art recognized methods. Such parameters may be determined using a blood sample from a patient. Increases in red blood cell levels, hemoglobin levels, and/or hematocrit levels may cause increases in blood pressure.
In one embodiment, if one or more hématologie parameters are outside the normal range or on the high side of normal in a patient who is a candidate to be treated with one oi more ActRIl antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), then onset of administration of the one or more antagonists of the disclosure may be delayed until the hématologie parameters hâve returned to a normal or acceptable level either naturally or via therapeutic intervention. For example, if a candidate patient is hypertensive or pre-hypertensive, then the patient may be treated with a blood pressure lowering agent în order to reduce the patient’s blood pressure. Any blood pressure lowering agent appropriate for the individual patient’s condition may be used including, for example, diuretîcs, adrenergic inhibitors (including alpha blockers and beta blockers), vasodilators, calcium channel blockers, angiotensin-converting enzyme (ACE) inhibitors, or angiotensin II receptor blockers. Blood pressure may alternatively be treated using a diet and exercise regimen. Similarly, if a candidate patient has iron stores that are iower than normal, or on the low side of normal, then the patient may be treated with an appropriate regimen of diet and/or iron suppléments until the patient’s iron stores hâve 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 one or more antagonists of the disclosure may be delayed until the levels hâve returned to a normal or acceptable level.
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In certain embodiments, if one or more hématologie parameters are outside the normal range or on the high side of normal in a patient who is a candidate to be treated with one or more ActRII antagonist agents of the disclosure (e.g, a GDF-ActRII antagonist, an
ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.), then the onset of administration may not be delayed. However, the dosage amount or frequency of dosing of the one or more antagonists of the disclosure may be set at an amount that would reduce the risk of an unacceptable increase in the hématologie parameters arising upon administration ol the one or more antagonists of the disclosure. Alternalively, a therapeutic regimen may be developed for the patient that combines one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, e/c.)with a therapeutic agent that addresses the undesirable level of the hématologie parameter. For example, if the patient has elevated blood pressure, then a therapeutic regimen may be designed involving administration of onc or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.) and a blood pressure lowering agent. For a patient having lower than desired iron stores, a therapeutic regimen may be developed involving one or more ActRII antagonist agents of the disclosure (e.g, a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, e/c.)and iron supplémentation.
In one embodiment, baseline parameter(s) for one or more hématologie parameters may be established for a patient who is a candidate to be treated with one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.) and an appropriate dosing regimen established for that patient based on the baseline value(s). Altematively, established baseline parameters based on a patient’s medical history could be used to inform an appropriate antagonist dosing regimen for a patient. For example, if a healthjJ patient has an established baseline blood pressure reading that is above the defrned normal range it may not be necessary to bring the patient’s blood pressure into the range that is considered normal for the general population prior to treatment with the one or more antagonist of the disclosure. A patient s baseline values for one or more hématologie parameters prior to treatment with one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, eic.)may also be used as the relevant comparative
-14119001 values for monitoring any changes to the hématologie parameters during treatment with the one or more antagonists of the disclosure.
In certain embodiments, one or more hématologie parameters are measured in patients who are being treated with one or more ActRII antagonist agents of the disclosure (e.g.. a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.). The hématologie parameters may be used to monitor the patient during treatment and permit adjustment or termination of the dosing with the one or more antagonist of the disclosure or additional dosing with another therapeutic agent. For example, if administration of one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.) results in an increase in blood pressure, red blood cell level, or hemoglobin level, or a réduction in iron stores, then the dose of the one or more antagonist of the disclosure may be reduced in amount or frequency in order to decrease the effects of the one or more antagonist ofthe disclosure on the one or more hématologie parameters. If administration of one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.) results in a change in one or more hématologie parameters that is adverse to the patient, then the dosing of the one or more antagonist of the disclosure may be terminated either temporarily, until the hématologie parameter(s) retum to an acceptable level, or permanently. Similarly, if one or more hématologie parameters are not brought within an acceptable range after reducing the dose or frequency of administration of the one or more antagonist of the disclosure, then the dosing may be terminated. As an alternative, or in addition to, reducing or terminating the dosing with the one or more antagonist of the disclosure, the patient may be dosed with an additional therapeutic agent that addresses the undesirable level in the hématologie parameter(s), such as, for example, a blood pressure lowering agent or an iron supplément. For example, if a patient being treated with one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF d rap. etc.)e has elevated blood pressure, then dosing with the one or more antagonist of the disclosure may continue at the same level and a blood pressure lowering agent is added to the treatment regimen, dosing with the one or more antagonist of the disclosure may be reduce (e.g., in amount and/or frequency) and a blood pressure lowering agent is added to the treatment regimen, or dosing with the one or more antagonist of the disclosure may be terminated and the patient may be treated with a blood pressure lowering agent.
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6. Pharmaceutical Compositions
In certain aspects, one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, e/c.)can be administered alone or as a component of a pharmaceutical formulation (also referred to as a therapeutic composition or pharmaceutical composition). A pharmaceutical formation refers to a préparation which is in such form as to permit the biological activity of an active ingrédient (e.g., an agent of the présent disclosure) contained therein to be effective and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The subject ActRII antagonist agents may be formulated for administration in any convenient way for use in human or veterinary medicine. For example, one or more ActRII antagonist agents of the présent disclosure may be formulated with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier refers to an ingrédient in a pharmaceutical formulation, other than an active ingrédient, which is generally nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, and/or preservative. In general, pharmaceutical formulations for use in the présent disclosure are in a pyrogen-free. physiologicallyacceptable form when administered to a subject. Therapeutically uselul agents other than those described herein, which may optionally be included in the formulation as described above, may be administered in combination with the subject ActRII antagonist agents in the methods ofthe présent disclosure.
In certain aspects, the disclosure provides a method of using a pharmaceutical compostion comprising an ActRII antagonist and a pharmacetucally acceptable carrier to treat or prevent treat or prevent an anémia in a subject in need thereof and/or treat or prevent one or more complication of anémia including, for example, cutaneous ulcers. In some embodiments, the disclosure provides methods of using a pharmaceutical composition comprising an ActRII antagonist, or combination of ActRII antagonists, and a pharmaceutically acceptable carrier to treat an anémia in a subject in need thereof and/or treat one or more complications of anémia including, for example, cutaneous ulcers in a subject having anémia. In some embodiments, the disclosure provides methods of using a pharmaceutical composition comprising an ActRII antagonist, or combination of ActRII antagonists, and a pharmaceutically acceptable carrier to prevent an anémia in a subject in
-14319001 need thereof and/or prevent one or more complications of anémia including, for example, cutaneous ulcers in a subject having anémia. In some of the foregoing embodiments, the pharmaceutical compositions comprising an ActRII antagonist, or combination of ActRII antagonists, and a pharmaceutically acceptable carrier of the présent disclosure are used to treat an ulcer (e.g., a cutaneous ulcer) in a subject (e.g., patient) having anémia (e.g., hemolytic anémia, hemoglobinopathy anémia, a thalassemia syndrome (e.g., β-thalassemia syndrome, β-thalassemia intermedia, etc.), sickle-cell disease, etc.). In some of the foregoing embodiments, the pharmaceutical compositions comprising an ActRII antagonist, or combination of ActRII antagonists, and a pharmaceutically acceptable carrier of the présent disclosure are used to prevent an ulcer (e.g., a cutaneous ulcer) in a subject (e.g., patient) having anémia (e.g., hemolytic anémia, hemoglobinopathy anémia, a thalassemia syndrome (e.g., β-thalassemia syndrome, β-thalassemia intermedia, etc.), sickle-cell disease, etc.). In some embodiments, the subject having anémia has sickle cell disease. In some embodiments, the subject having anémia has a thalassemia syndrome (e.g., β-thalassemia syndrome, βthalassemia intermedia, etc.). In some embodiments, the subject having anémia has a cutaneous ulcer. in some embodiments, the cutaneous ulcer is a skin ulcer. In some embodiments, the ulcer occurs on legs or ankes.
In certain embodiments, the ActRII antagonist agents or the pharmaceutical compositions of the disclosure will be administered parenterally [e.g., by intravenous (I.V.) injection, intraarterial injection, intraosseous injection, intramuscular injection, intrathecal injection, subcutaneous injection, or intradermal injection]. Pharmaceutical compositions suitable for parentéral administration may comprise one or more ActRII antagonist agents of the disclosure in combination with one or more pharmaceutically acceptable stérile isotonie aqueous or nonaqueous solutions, dispersions, suspensions or émulsions, or stérile powders which may be reconstituted into stérile injectable solutions or dispersions just prior to use. Injectable solutions or dispersions may contain antioxidants, buffers, bacteriostats, suspending agents, thickening agents, or solutés which render the formulation isotonie with the blood of the intended récipient. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical formulations of the présent disclosure include water, éthanol, polyols (e.g, glycerol, propylene glycol, polyethylene glycol, etc.), vegetable oils (e.g., olive oil), injectable organic esters (e.g, ethyl oleate), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials (e.g, lecithin), by the maintenance of the required particle size in the case of dispersions, and by
-14419001 the use of surfactants. In certain embodiments, the ActRII antagonist agents or the pharmaceutical compositions of the disclosure will be administeredsubcutaneously (e.g., subcutaneous injection). In certain embodiments, the ActRII antagonist agents or the pharmaceutical compositions of the disclosure will be admînîstered topically.
In some embodiments, a therapeutic method of the présent disclosure includes administering the pharmaceutical composition of the présent disclosure systemically, or locally, from an implant or device. Further, the pharmaceutical composition of the présent disclosure may be encapsulated or înjected in a form for delivery to a target tissue site (e.g., bone marrow or muscle). In certain embodiments, the pharmaceutical compositions of the présent disclosure may include a matrix capable of delivering one or more of the agents of the présent disclosure to a target tissue site (e.g., bone marrow or muscle), 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 one or more agents of the présent disclosure. Such matrices may be formed of materials presently in use for other implanted medical applications.
The choice of matrix material may be based on one or more of: biocompatibility, biodegradability, mechanical properties, cosmetic appearance. and interface properties. The particular application of the subject compositions will defîne the approprîate formulation. Potential matrices for the compositions may be biodégradable and chemically defmed calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, and polyanhydrides. Other potential materials are biodégradable and biologically well-defined including, for example, bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defmed including, for example, sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material including, for example, polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition (e.g., calciumaluminate-phosphate) and processing to alter one or more of pore size, particle size, particle shape, and biodegradability.
In certain embodiments, the pharmaceutical compositions of the présent disclosure can be administered topically. “Topical application” or “topically” means contact of the pharmaceutical composition with body surfaces including, for example, the skin, wound sites, ulcer sites, and mucous membranes. The topical pharmaceutical compositions can hâve
-14519001 various application forms and typically comprise a drug-containing layer, which is adapted to be placed near to or in direct contact with the tissue upon topically administering the composition. Pharmaceutical compositions suitable for topical administration may comprise one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIlA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc.) fonnulatcd as a liquid, a gel. a cream, a lotion, an ointment, a foam, a paste, a putty, a semi-solid, or a solid. Pharmaceutical compositions in the liquid, gel, cream, lotion, ointment, foam, paste, or putty form can be applied by spreading, spraying, smearing, dabbing or rolling the composition on the target tissue. The pharmaceutical compositions also may be împregnated into stérile dressings, transdermal patches, plasters, and bandages. Pharmaceutical ompositions of the putty, semi-solid or solid forms may be déformable. They may be elastic or non-elastic (e.g., flexible or rigid). In certain aspects, the pharmaceutical composition forms part of a composite and can include fibers, particulates, or multiple layers with the same or different compositions.
Topical compositions in the liquid form may include pharmaceutically acceptable solutions, émulsions, microemulsions, and suspensions. In addition to the active ingredient(s), the liquid dosage form may contain an inert diluent commonly used in the art including, for example, water or other solvent, a solubilizing agent and/or emulsifier [e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, or l,3-butylene glycol, an oil (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oil), glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fatty acid ester of sorbitan, and mixtures thereof].
Topical gel, cream, lotion, ointment, semi-solid or solid compositions may include one or more thickening agents, such as a polysaccharide, synthetic polymer or protein-based polymer. In one embodiment of the invention, the gelling agent herein is one that is suitably nontoxic and gîves the desired viscosity. The thickening agents may include polymers, copolymers, and monomers of: vinylpyrrolidones, methacrylamides, acrylamides Nvinylimidazoles, carboxy vinyls, vinyl esters, vinyl ethers, silicones, polyelhylencoxides, polyethyleneglycols, vinylalcohols, sodium acrylates, acrylates, maleic acids, NNdimethylacrylamides, diacetone acrylamides, acrylamides, acryloyl morpholme, pluromc, collagens, polyacrylamides, polyacrylates, polyvinyl aicohols, polyvinylenes, polyvinyl silicates, polyacrylates substituted with a sugar (e.g., sucrose, glucose, glucosamines, galactose, trehalose, mannose, or lactose), acylamidopropane sulfonic acids,
-14619001 tetramethoxyorthosilicates, methyltrimethoxyorthosilicates, tetraalkoxyorthosilicates, trialkoxyorthosilicates, glycols, propylene glycol, glycérine, polysaccharides, alginates, dextrans, cyclodextrin, celluloses, modified celluloses, oxidized celluloses, chitosans, chitins, guars, carrageenans, hyaluronic acids, inulin, starches, modified starches, agarose, metliylcelluloses, plant gums, hylaronans, hydrogels, gelatins, glycosaminoglycans, carboxymethyl celluloses, hydroxyethyl celluloses, hydroxy propyl methyl celluloses, pectins, low-methoxy pectins, cross-linked dextrans, starch-acrylonitrile graft copolymers, starch sodium polyacrylate, hydroxyethyl méthacrylates, hydroxyl ethyl acrylates, polyvinylene, polyethylvinylethers, polymethyl méthacrylates, polystyrènes, polyuréthanes, polyalkanoates, polylactic acids, polylactates, poly(3-hydroxybutyrate), sulfonated hydrogels, AMPS (2acrylamido-2-methyl-l-propanesulfonic acid), SEM (sulfoethylmethacrylate), SPM (sulfopropyl méthacrylate), SPA (sulfopropyl acrylate), N,N-dîmethyl-N-methacryloxyethylN-(3-sulfopropyl)ammonium betaine, methacryllic acid amidopropyl-dimethyl ammonium sulfobetaine, SPl (itaconic acid-bis(l-propyl sulfonizacid-3) ester di-potassium sait), itaconic acids, AMBC (3-acrylamido-3-methylbutanoic acid), beta-carboxyethyl acrylate (acrylic acid dimers), and maleic anhydride-methylvinyl ether polymers, dérivatives thereof. salts thcrcol. acids thereof, and combinations thereof.ln certain embodiments, pharmaceutical compositions of present disclosure can be administered orally, for example, in the form of capsules, cachets, pills, tablets, lozenges (usîng a flavored basis such as sucrose and acacia or tragacanth), powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid émulsion, or an élixir or syrup, or pastille (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and/or a moulh wash, each containing a predetermined amount of an ActRII antagonist agent of the present disclosure and optionally one or more other active ingrédients. An ActRII antagonist agent of the present disclosure and optionally one or more other active ingrédients may also be administered as a bolus, electuary, or paste.
In solid dosage forms for oral administration (e.g.. capsules, tablets, pills. dragées, powders, and granules), one or more ActRII antagonist agents ofthe present disclosure may be mixed with one or more pharmaceutically acceptable carriers including, for example, sodium citrate, dicalcium phosphate, a filler or extender (e.g., a starch, lactose, sucrose, glucose, mannitol, and silicic acid), a binder (e.g. carboxymethylcellulose, an alginate, gelatin, polyvinyl pyrrolidone, sucrose, and acacia), a humectant (e.g., glycerol), a disintegrating agent (e.g., agar-agar, calcium carbonate, potato or tapioca starch. alginic acid, a silicate, and
-I4j719001 sodium carbonate), a solution retarding agent (e.g. paraffin), an absorption accelerator (e.g. a quaternary ammonium compound), a wetting agent (e.g., cetyl alcohol and glycerol monostearate), an absorbent (e.g., kaolin and bentonite clay), a lubricant (e.g., a talc, calcium stéarate, magnésium stéarate, solid polyethylene glycols, sodium lauryl sulfate), a coloring agent, and mixtures thereof. In the case of capsules, tablets, and pills, the pharmaceutical formulation (composition) may also comprise a bufferîng agent. Solid compositions of a similar type may also be employed as fillers in soft and hard-lilied gclalin capsules using one or more excipients including, e.g., lactose or a milk sugar as well as a high molecular-weight polyethylene glycol.
Liquid dosage forms for oral administration of the pharmaceutical composition of the disclosure may include pharmaceutically acceptable émulsions, microemulsions, solutions, suspensions, syrups, and élixirs. In addition to the active ingredient(s), the liquid dosage form may contain an inert diluent commonly used in the art including, for example, water or other solvent, a solubilîzing agent and/or emulsifier [e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyiene glycol, or l ,3butylene glycol, an oil (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oil), glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fatty acid ester of sorbitan, and mixtures thereof], Besides inert diluents, the oral formulation can also include an adjuvant including, for example, a wetting agent, an emulsifying and suspending agent, a sweetening agent, a flavoring agent, a coloring agent, a perfuming agent, a preservative agent, and combinations thereof.
Suspensions, in addition to the active ActRII antagonist agents, may contain suspending agents including, for example, an ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, a sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite. agar-agar, tragacanth, and combinations thereof.
Prévention of the action and/or growth of microorganisms may be ensured by the inclusion of varions antibacterial and antifungal agents including, for example, paraben, chlorobutanol, and phénol sorbic acid.
In certain embodiments, it may be désirable to include an isotonie agent including, for example, a sugar or sodium chloride into the pharmaceutical compositions. In addition, prolonged absorption of an injectable pharmaceutical form may be brought about by the
-14819001 inclusion of an agent that delay absorption including, for example, aluminum monostearate and gelatin.
It is understood that the dosage regimen will be determîned by the attending physician considering various factors which modify the action of the one or more oi the agents ot the present disclosure. The various factors include^ but are not limited to, the patient’s red blood cell count, hemoglobin level, the desired targetïed blood cell count, the patients âge, the patient’s sex, the patient’s diet, the severity of any disease that may be contributing to a depressed red blood cell level, the time of administration, and other clinical factors. The addition of other known active agents to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of one or more of red blood cell levels, hemoglobin levels, réticulocyte levels, and other indicators of the hematopoietic process.
In certain embodiments, the present disclosure also provides gene therapy for the in vivo production of one or more of the ActRII antagonist agents of the present disclosure. Such therapy would achieve its therapeutic effect by introduction of the agent sequences into cells or tissues having one or more of the disorders as listed above. Delivery ol the agent sequences can be achieved, for example, by using a recombinant expression vector such as a chimeric virus or a colloïdal dispersion System.I In some embodiments, therapeutic delivery of one or more of agent sequences ofthe disclosure is the use oftargeted liposomes.
Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or an RNA virus (e.g., a retrovirus). The retroviral vector may be a dérivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. Ail of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. Retroviral vectors can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein. In some embodiments, targeting is accomplished by using an antibody. fhose of skill in the art will recognîze that spécifie polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target spécifie delivery of the retroviral vector containing one or more of the agents of the present disclosure.
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Altematively, tissue culture cells can be directiy transfected with plasmids encoding the retroviral structural genes (gag, pol, and env), by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
Another targeted delivery system for onl or more of the agents of the présent
I disclosure is a colloïdal dispersion system. Colloïdal dispersion Systems include, for example, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based Systems including oil-in-water émulsions, micelles, mixed micelles, and liposomes. In certain embodiments, the colloïdal system of this disclosure is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated wîthin the aqueous interior and be delivered to cells in a biologically active form. See, e.g., Fraley, et al. (1981) Trends Biochem. Sci., 6:77. Methods for efficient gene transfer using a liposome vehicle are known in the art. See, e.g., Mannino, et al. (1988) Biotechniques, 6:682, 1988.
The composition of the liposome is usually a combination of phospholipîds, which may include a steroid (e.g.cholestérol). The physical characteristics of liposomes dépend on pH, ionic strength, and the presence of divalent; cations. Other phospholipîds or other lipids may also be used including, for example a phosphatidyl compound (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, a sphingolipid. a cerebroside, and a ganglioside), egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. The targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art.
EXEMPLIFICATION
The invention now being generally described, it will be more readily understood by référencé to the following examples, which are included merely for purposes of illustration ot certain embodiments and embodiments of the présent invention, and are not intended to limit the invention.
Example l : ActRIIa-Fc Fusion Proteins
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Applicant constructed a soluble ActRIIA fusion protein that has the extracellular domain of human ActRIIa fused to a human or mouse Fc domain with a minimal linker in between. The constructs are referred to as ActRIIA-hFc and ActRIIA-mFc, respectively.
ActRIIA-hFc is shown below as purified from CHO cell lines (Fc portion underlined) (SEQ ID NO:22):
ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEI VKQGCWLDDINCYDRTDCVEKKDSPEVTFCCCEGNMCNEKFSYFPEMEVTQPTSNP VTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAK.TKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKVSN KALPVPIEKTISKAKGQPREPOVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQOGNVFSCSVMHEALHNHYTOK SLSLSPGK
The ActRIIA-hFc and ActRIIA-mFc proteins were expressed in CHO cell lines.
Three different leader sequences were considered:
(i) Honey bee mellilin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO:23) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ
ID NO:24) (iii) Native: MGAAAKLAFAVFLISC^SGA (SEQ ID NO:25).
The selected form employs the TPA leader and has the following unprocessed amino acid sequence:
MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTG VEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFC CCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:26)
This polypeptide is encoded by the following nucleic acid sequence:
ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGC AGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGGAGTGT CTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACC GTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATT TCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTA TGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGC
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TGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCGGAGATGGAAGÎCA CACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCA CACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTAC AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGTCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCÀGCCCCGAGAACCACAGGTGTACACCC TGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGG TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT CCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAATGAGAATTC (SEQ ID NO:27)
Both ActRIIA-liFc and ActRIIA-mFc were remarkably amenable to recombinant expression. As shown in Figures 3A and 3B, the protein was purified as a single, welldefined peak of protein. N-terminal sequencing revealed a single sequence ot ILGRSl·. IQE (SEQ ID NO:34). Purification could be achievéd by a sériés of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification could be completed with viral filtration and buffer exchange. The ActRIIA-hFc protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS PAGE.
ActRIIA-hFc and ActRIIA-mFc showed a high affmity for ligands, particularly activin A. GDF-l l or activin A were immobilîzed on a Biacore™ CM5 chip using standard amine coupling procedure. ActRIIA-hFc and ActRIIA-mFc proteins were loaded onto the System, and binding was measured. ActRIIA-hFc bound to activin with a dissociation constant (KD) of 5xl0‘l2, and bound to GDFl l with a KD of 9.96x10‘9. Sec Figures 4A and 4B. ActRIIA-mFc behaved sîmilarly.
The ActRIIA-hFc was very stable in pharmacokinetic studies. Rats were dosed with 1 mg/kg, 3 mg/kg or 10 mg/kg of ActRIIA-hFc protein and plasma levels of the protein were measured at 24, 48, 72, 144 and 168 hours. In a separate study, rats were dosed at 1 mg/kg,
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ΙΟ mg/kg or 30 mg/kg. In rats, ActRIIA-hFc had an 11-14 day sérum half-life and circulating levels of the drug were quite high alter two weeks (l l pg/ml, 110 pg/ml or 304 pg/ml for initial administrations of l mg/kg, 10 mg/kg or 30 mg/kg, respectively.) In cynomolgus monkeys, the plasma half-life was substantially greater than 14 days and circulating levels of the drug were 25 pg/ml, 304 pg/ml or 1440 pg/ml for initial administrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.
Example 2: Characterization of an ActRIIA-hFc Protein
ActRIIA-hFc fusion protein was expressed in stably transfccted CI IO-DUKX B11 cells from a pAID4 vector (SV40 ori/enhancer, CMV promoter), using a tissue plasminogen leader sequence of SEQ ID NO:24. The protein, purified as described above in Example 1, had a sequence of SEQ ID NO:22. The Fc portion is a human IgGl Fc sequence, as shown in SEQ ID NO:22. Protein analysis reveals that the ActRIIA-hFc fusion protein is formed as a homodimer with disulfide bonding.
Example 3. ActRIIA-hFc Increases Red Blood Cell Levels in Non-Human Primates
The study employed four groups of five male and five female cynomolgus monkeys each, with three per sex per group scheduled for termination on Day 29, and two per sex per group scheduled for termination on Day 57. Each animal was administered the vehicle (Group I) or ActRIIA-Fc at doses of 1, 10, or 3Q mg/kg (Groups 2, 3 and 4, respectively) via intravenous (IV) injection on Days 1,8, 15 and'22. The dose volume was maintained at 3 mL/kg. Various measures of red blood cell levels were assessed two days prior to the first administration and at days 15, 29 and 57 (for the remaining two animais) after the first administration.
The ActRIIA-hFc caused statistically signifïcant increases in mean red blood cell parameters [red blood cell count (RBC, hemoglobin (HGB), and hematocrit (HCT)] for males and females, at ail dose levels and time points throughout the study, with accompanying élévations in absolute and relative réticulocyte counts (ARTC; RTC). See Figures 5-8.
Statistical significance was calculated for each treatment group relative to the mean for the treatment group at baseline. .
Notably, the increases in red blood cell counts and hemoglobin levels are roughly équivalent in magnitude to effects reported with erythropoietin. The onset of these effects is more rapid with ActRIIA-Fc than with erythropoietin.
Similar results were observed with rats and mice.
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Example 4: ActRIIA-hFc Increases Red Blood Ceil Levels and Markers of Bone Formation in Human Patients
The ActRIIA-hFc fusion protein described in Example l was administered to human patients in a randomized, double-blind, placebo-controlled study that was conducted to evaluate, primarily, the safety ofthe protein in healthy, postmenopausal women. Forty-eight subjects were randomized in cohorts of 6 to receive either a single dose of ActRIIA-hFc or placebo (5 active:l placebo). Dose levels ranged from 0.01 to 3.0 mg/kg intravenously (IV) and 0.03 to O.l mg/kg subcutaneously (SC). Ail subjects were followed for 120 days. In addition to pharmacokinetic (PK) analyses, the biologie activity of ActRIIA-hFc was also assessed by measurement of biochemical markers of bone formation and résorption as well as FSH levels.
To look for potential changes, hemoglobin and RBC numbers were examined in detail for ail subjects over the course of the study and compared to the baseline levels. Platelet counts were compared over the same time as the control. There were no clinically significant changes from the baseline values over time for the platelet counts.
PK analysis of ActRIIA-hFc revealed a linear profile with dose, and a mean half-life of approximately 25-32 days. The area-under-curve (AUC) for ActRIIA-hFc was hnearly related to dose, and the absorption after SC dosing was essentially complété. See Figures 9 and 10. These data indicate that SC is a désirable approach to dosing because it provides équivalent bioavailability and serum-half life for the drug while avoiding the spike in sérum concentrations of drug associated with the first few days of IV dosing. See Figure 10. ActRIIA-hFc caused arapid, sustained dose-dependent increase in sérum levels of bonespecific alkaline phosphatase (BAP), which is a marker for anabolic bone growth, and a dosedependent decrease in C-terminal type 1 collagen telopeptide and tartrate-resistant acid phosphatase 5b levels, which are markers for bone résorption. Other markers, such as P1NP showed inconclusive results. BAP levels showed near saturating effects at the highest dosage of drug, indicating that half-maximal effects on this anabolic bone biomarker could be achieved at a dosage of 0.3 mg/kg, with increases ranging up to 3 mg/kg. Calculated as a relationship of pharmacodynamie effect to AUC for drug, the EC50 was 51,465 (day ng/ml). See Figure 11. These bone biomarker changes [were sustained for approximately 120 days at the highest dose levels tested. There was also q dose-dependent decrease in sérum FSH levels consistent with inhibition of activin.
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Overall, there was a very small non-drug related réduction in hemoglobin over the first week of the study probably related to study phlebotomy in the 0.01 and 0.03 mg/kg groups whether given IV or SC. The 0.1 mg/kg SC and IV hemoglobin results were stable or showed modest increases by Day 8-15. At the 0.3 mg/kg IV dose level there was a clear increase in HGB levels seen as early as Day 2 and often peaking at Day 15-29 that was not seen in the placebo-treated subjects. At the 1.0 mg/kg IV dose and the 3.0 mg/kg IV dose, mean increases in hemoglobin of greater than 1 g/dl were observed in response to the single dose, with corresponding increases in RBC counts and hematocrit. These hématologie parameters peaked at about 60 days after the dose and substantial decrease by day 120. This indicates that dosing for the purpose of increasing red blood cell levels may be more effective if donc at intervals less than 120 days (i.e., prior to retum to baseline), with dosing intervals of 90 days or less or 60 days or less may be desjrable. For a summary of hematological changes, see Figures 12-15.
Overall, ActRIIA-hFc showed a dose-dependent effect on red blood cell counts and réticulocyte counts.
Example 5: Treatment of an Anémie Patient with ActRIIA-hFc
A clinical study was designed to treat patients with multiple doses of ActRIIA-hFc, at 30 dose levels of 0.1 mg/kg, 0.3 mg/kg, and 1.0 mg/kg, with dosing to occur every thirty days. Normal healthy patients in the trial exhibited an increase in hemoglobin and hematocrit that is consistent with the increases seen in the Phase I clinical trial reported in Example 4, except that in some instances, the hemoglobin (Hg) and hematocrit (FIct) are elevated beyond the normal range. An anémie patient with hemoglobin levels of approximately 7.5 g/dL also received two doses at the 1 mg/kg level, resulting in a hemoglobin level of approximately 10.5 g/dL after two months. The patient’s anémia was a microcytic anémia, thought to be caused by chronic iron deficiency.
ActRIIA-Fc fusion proteins hâve been further demonstrated to be effective in increasing red blood cell levels in varions models of anémia including, for example, chemotherapy-induced anémia and anémia associated with chronic kidney disease. See, e.g., U.S. Patent Application Publication No. 2010/0028331.
I
Example 6: Alternative ActRIIA-Fc Proteins
A variety of ActRIIA variants that may be used according to the methods described herein are described in the International Patent Application published as W02006/012627
-15519001 (see e.g., ρρ. 59-60), incorporated herein by référencé in its entirety. An alternative construct may hâve a délétion of the C-terminal tail (the final 15 amino acids of the extracellular domain of ActRIIA. The sequence for such a construct is presented below (Fc portion underlined) (SEQ ID NO:28): ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQG CWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMTGGGTHTÇPPÇPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPVPIEKTISKAKGOPRE PQVYTLPPSREEMTKNOVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Example 7: Génération of ActRIIB-Fc fusion proteins
Applicant constructed a soluble ActRIIB fusion protein that has the extracellular domain of human ActRIIB fused to a human or mouse Fc domain with a minimal linker (three glycine amino acids) in between. The constructs are referred to as ActRIIB-hFc and ActRIIB-mFc, respectively.
ActRIIB-hFc is shown below as purified from CHO cell lines (SEQ ID NO:29) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKK GCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTFILPEAGGPEVTYEPPPT APTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PVPIEKTISKAKGQPREPOVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQOGNVFSCSVMHEALHNHY I QKSLS LSPGK
The ActRIIB-hFc and ActRIIB-mFc proteins were expressed in CHO cell lines.
Three different leader sequences were considered:
(i) Honey bee mellitin (HBML): MK.FLVNVALVFMVVYISYIYA (SEQ ID NO.2j) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO:24) (iii) Native: MGAAAKLAFAVFLISCSSGA (SEQ ID NO:30).
The selected form employs the TPA leader and has the following unprocessed amino acid sequence (SEQ ID NO: 31): i
MDAMKRGLCCVLLLCGAVFVSPGASGRcjEAETREClYYNANWELERTNQSGLERCE GEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCE
-I5619001
GNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHODWLNGKEYKCKVSNKALPVPIEKTISKAKGOPREPOVYÎLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOQ GNVFSCSVMHEALHNHYTQKSLSLSPGK
This polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO:32): A TGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCT GTGTGGAGCA GTCTTCGTTT CGCCCGGCGC CTCTGGGCGT GGGGAGGCTG AGACACGGGA GTGCATCTAC TACAACGCCA ACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCG CTGCGAAGGC GAGCAGGACA AGCGGCTGCA CTGCTACGCC TCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGA AGGGCTGCTG GCTAGATGAC TTCAACTGCT ACGATAGGCA GGAGTGTGTG GCCACTGAGG AGAACCCCCA GGTGTACTTC TGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTC ATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCC ACCCCCGACA GCCCCCACCG GTGGTGGAAC TCACACATGC CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAG TCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACT CCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT AAATGA
N-terminal sequencing of the CHO-cell produced material revealed a major polypeptide sequence of-GRGEAE (SEQ ID NO:33). Notably, other constructs reported in the literature begin with an -SGR... sequence.
Purification could be achieved by a sériés of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q
-15719001 sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification could be completed with virai filtration and buffer exchange.
I
ActRIIB-Fc fusion proteins were also expressed in HEK293 cells and COS cells. Although material from ail cell lines and reasonable culture conditions provided protein with muscle-building activity in vivo, variability in potency was observed perhaps relating to cell line sélection and/or culture conditions.
Applîcant generated a sériés of mutations in the extracellular domain of ActRIIB and produced these mutant proteins as soluble fusion proteins between extracellular ActRIIB and an Fc domain. The background ActRIIB-Fc fusion has the sequence of SEQ ID NO:29.
Various mutations, including N- and C-terminal truncations, were introduced into the background ActRIIB-Fc protein. Based on the data presented in Example l, it is expected that these constructs, if expressed with a TPA leader, will lack the N-terminal serine. Mutations were generated in ActRIIB extracellular domain by PCR mutagenesis. After PCR, fragments were purified through a Qiagen column, digested with Sfol and Agel and gel purified. These fragments were ligated into expression vector pAID4 (see W02006/012627) such that upon ligatîon it created fusion chimera with human IgGl. Upon transformation into E. coli DFI5 alpha, colonies were picked and DNAs were isolated. For murine constructs (mFc), a murine IgG2a was substituted for the human IgGl. Seqeunces of ail mutants were verified.
Ali of the mutants were produced in HEK.293T cells by transient transfection. In summary, in a 500ml spinner, HEK293T cells were set up at 6xlO5 cells/ml in Freestyle (Invitrogen) media in 250ml volume and grown ovemight. Next day, these cells were trcaled with DNAiPEI (l:l) complex at 0.5 ug/ml final DNA concentration. After 4 hrs, 250 ml media was added and cells were grown for 7 days. Conditioned media was harvested by spinning down the cells and concentrated.
Mutants were purified using a variety of techniques, including, for example, a protein A column and eluted with low pH (3.0) glycine buffer. After neutralization, these were dialyzed against PBS.
Mutants were also produced in CHO cells by similar methodology.
Mutants were tested in binding assays and/or bioassays described in WO 2008/097541 and WO 2006/012627 incorporated by reference herein. In some instances, assays were performed with conditioned medium rather than purified proteins. Additional variations of ActRIIB are described in U.S. Patent No. 7.842,663.
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Example 8: ActRIIB-Fc Stimulâtes Erythropoiesis in Non-Human Primates
Cynomolgus monkeys were aliocated into seven groups (6/sex/group) and administered ActRIIB(20-l34)-hFc as a subcutaneous injection at dosages of 0.6, 3, or 15 mg/kg every 2 weeks or every 4 weeks over a 9 month period. The control group (6/sex/group) received the vehicle at the same dose volume (0.5 ml/kg/dose) as ActRIIB(20134)-hFc -treated animais. Animais were monitored for changes in general clinical pathology parameters (e.g., hematology, clinical chemîstry, coagulation, and urinalysis). Hematology, coagulation, and clinical chemîstry parameters (including iron parameters, lipase, and amylase) were evaluated twice prior to initiation of dosing and on Days 59, 143, 199, 227, and on Days 267 (for groups dosed every 4 weeks) or 281 (for groups dosed every 2 weeks). The évaluations on Days 267/281 occured 2 weeks after the final dose was administered.
Administration of ActRIIB(20-134)-hFc resulted in non-adverse, dose-related changes to hematology parameters in male and female monkeys. These changes included increased red blood cell count, réticulocyte count and red cell distribution width and decreased mean corpuscular volume, mean corpuscular hemoglobin, and platelet count. In males, RBC count was increased at ail dose levels and the magnitude of increase was generally comparable whether ActRIIB(20-134)-hFc was administered every 2 weeks or every 4 weeks. Mean RBC count was increased at ail time points between Days 59 and 267/281 (except RBC count was not increased in Group 2 males [0.6 mg/kg every 2 weeks] on Day 281 ). In females, RBC count was increased at > 3 mg/kg every 2 weeks and the changes occurred between Days 143 and 281 ; at 15 mg/kg every 4 weeks mean RBC count was increased between Days 59 and 267.
These effects are consistent with a positive effect of ActRIlB(20-l 34)-hFc on stimulating erythropoiesis. I
Example 9: Génération of a GDF Trap
Applicant constructed a GDF Trap as follows. A polypeptide having a modified extracellular domain of ActRIIB (amino acids 20-134 of SEQ ID NO:1 with an L79D substitîon) with greatly reduced activin A binding relative to GDF11 and/or myostatin (as a conséquence of a leucine-to-aspartate substitution at position 79 in SEQ ID NO: 1 ) was fused to a human or mouse Fc domain with a minimal linker (three glycine amino acids) in between. The constructs are referred to as ActRIIB(L79D 20-134)-hFc and ActRIIB(L79D 20-134)mFc. respectively. Alternative forms with a glutamate rather than an aspartate at position 79
-15919001 performed similarly (L79E). Alternative forms with an alanine rather than a valine at position 226 with respect to SEQ ID NO:36, below were also generated and performed equivalently in ail respects tested. The aspartate at position 79 (relative to SEQ ID NO: 1, or position 60 relative to SEQ ID NO:36) is indicated with double underlining below. The valine at position 226 relative to SEQ ID NO:36 is also indicated by double underlining below.
The GDF Trap ActRIIB(L79D 20-134)-hFc is shown below as purified from CHO cell Unes (SEQ ID NO:36).
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKK GCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPT APTGGGTHTCPPCPAPELLGGPSVFLFPPK.PKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKAL PyPIEKTISKAKGQPREPOVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTQKSLS LSPGK
The ActRIIB-derived portion of the GDF Trap has an amino acid sequence set forth below (SEQ ID NO: 37), and that portion could be used as a monomer or as a non-Fc fusion protein as a monomer, dimer or greater order complex. GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKK GCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPT APT (SEQ ID NO: 37)
The GDF Trap protein was expressed in CHO cell lines. Thrce different leader sequences were considered:
(i) Honey bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO:23) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO:24) (iii) Native: MTAPWVALALLWGSLCAGS (SEQ ID NO:30).
The selected form employs the TPA leader and has the lollowing unproccsscd amino acid sequence:
MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECrYYNANWELERTNQSGLERCE GEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCE GNFCNF.RFTT-TT PFAGGPFVTYF.PPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEAKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV ltvlhodwlngkeykckvsnkalpvpiektiskakgqprepovytlppsreemtkno
J
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VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQO GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:38)
This polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO:39): A TGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCT GTGTGGAGCA GTCTTCGTTT CGCCCGGCGC CTCTGGGCGT GGGGAGGCTG AGACACGGGA GTGCATCTAC TACAACGCCA ACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCG CTGCGAAGGC GAGCAGGACA AGCGGCTGCA CTGCTACGCC TCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGA AGGGCTGCTG GGACGATGAC TTCAACTGCT ACGATAGGCA GGAGTGTGTG GCCACTGAGG AGAACCCCCA GGTGTACTTC TGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTC ATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCC ACCCCCGACA GCCCCCACCG GTGGTGGAAC TCACACATGC CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAG TCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACT CCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCC'l CTCCC T G TC TCCGGG T AAATGA
Purification could be achieved by a sériés of column chromatography steps, încluding, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification could be completed with viral filtration and buffer exchange. In an example of a purification scheme, the cell culture medium is passed over a protein A column, washed in 150 mM Tris/NaCl (pH 8.0), then washed in 50 mM Tris/NaCl (pH 8.0) and eluted with O.l M glycine, pH 3.0. The low pH eluate is kept at room température for 30 minutes as a viral clearance step. The eluate is
-I6l19001 then neutralized and passed over a Q sepharose ion exchange column and washed in 50 mM Tris pH 8.0, 50 mM NaCl, 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 50 mM Tris pH 8.0, l.l M ammonium sulfate and passed over a phenyl sepharose column, washed, and eluted in 50 mM i
Tris pH 8.0 with ammonium sulfate between 150 and 300 mM. The eluate is dialyzed and filtered for use.
Additional GDF Traps (ActRIIB-Fc fusion proteins modified so as to reduce the ratio of activin A binding relative to myostatin or GDFl l binding) are described in WO 2008/097541 and WO 2006/012627, incorporated by reference herein.
Example 10: Bioassay for GDF-11 and Activin-Mediated Signaling
An A-204 reporter gene assay was used to evaluate the effects of ActRIIB-Fc proteins and GDF Traps on signaling by GDF-11 and activin A. Cell line: human rhabdomyosarcoma
I (derived from muscle). Reporter vector: pGL3(CAGA)12 (described in Dennler et al, 1998, i
EMBO 17: 3091-3100). The CAGA12 motif is présent in TGF-Beta responsive genes (e.g, PAI-1 gene), so this vector is of general use for factors signaling through SMAD2 and 3.
Day 1 : Split A-204 cells into 48-well plate.
Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 or pGL3(CAGA) 12(10 ug)+ pRLCMV (1 pg) and Fugene.
Day 3: Add factors (diluted into medium+ 0.1 % BSA). Inhibitors need to be preincubated with factors for 1 hr before adding to cells. Six hrs later, cells were rinsed with PBS, and lysed.
This is followed by a luciferase assay. In the absence of any inhibitors, Activin A showed 10-fold stimulation of reporter gene expression and an ED50 - 2 ng/ml. GDF-11:16 fold stimulation, ED50: - 1.5 ng/ml.
ActRIIB(20-134) is a potent inhibitor of activin A, GDF-8, and GDF-11 activity in this assay. As described below ActRIIB variants were also tested in this assay.
Example 11: ActRIIB-Fc Variants. Cell-Based Activity
Activity of ActRIIB-Fc proteins and GDF Traps was tested in a cell based assay, as described above. Results are summarized in the table below. Some variants were tested in different C-terminal truncation constructs. As discussed above, truncations of five or fifteen
-16219001 amino acids caused réduction in activity. The GDF Traps (L79D and L79E variants) showed substantial loss of activin A inhibition while retaining almost wild-type inhibition of GDF-l l.
Soluble ActRIIB-Fc binding to GDF11 and Activin A:
ActRIIB-Fc Variations Portion of ActRIIB (corresponds to amino acids ofSEQ ID NO:l) GDF1I Inhibition Activity Activin Inhibition Activity
R64 20-134 +4--1- | | |
(approx. 10'8 M K[) (approx. 10’8 M K|)
A64 20-134 + +
(approx. 10'6 M K|) (approx. 10'6 M Kj)
R64 20-129 +4-4- +H-+
R64 K74A 20-134 ++++ i n i
R64 A24N 20-134 -F-H- ; | |
R64 A24N 20-119 -H- -H-
R64 A24N K74A 20-119 + 1
R64 L79P 20-134 + +
R64 L79P K.74A 20-134 + +
R.64 L79D 20-134 4—1—F +
R64 L79E 20-134 | | 1 “F
R64K 20-134 | | | +++
R64K 20-129 1 | | 4—1—h
R64P129S P130A 20-134 1 1 H—h4- 4—1—I-
R64N 20-134 + +
+ Poor activity (roughly lxlO‘É Kj) ++ Moderate activity (roughly IxlO' Kj) g
+-H- Good (wild-type) activity (roughly 1x10’ K[) ++++ Greater than wild-type activity
Several variants hâve been assessed for sérum half-life in rats. ActRlIB(20-134)-Fc has a sérum half-life of approximately 70 hours. ActRIIB(A24N 20-134)-Fc has a sérum half-life of approximately 100-150 hours. The A24N variant has activity in the cell-based assay (above) and in vivo assays (below) that is équivalent to the wild-type molécule. Coupled with the longer half-life, this means that over time an A24N variant will give greater effect per unit of protein than the wild-type molécule. The A24N variant, and any of the other variants tested above, may be combined with the GDF Trap molécules, such as the L79D or L79E variants.
Example 12: GDF-11 and Activin A Binding.
Binding of certain ActRIIB-Fc proteins and GDF Traps to ligands was tested in a Biacorc™ assay.
The ActRIIB-Fc variants or wild-type p rotein were captured onto the System using an anti-hFc antibody. Ligands were injected and flowed over the captured receptor proteins. Results are summarized in the tables, below.
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Ligand-binding specifïcity IIB variants.
GDF11
Protein Kon (1/Ms) Koff(l/s) KD (M)
ActRIIB(20-l34)-hFc 1.34e-6 1.13e-4 8.42e-11
ActRIIB(A24N 20-l34)-hFc 1.21e-6 6.35e-5 5.19e-l1
ActRIIB(L79D 20-134)-hFc 6.7e-5 4.39e-4 6.55e-10
ActRIIB(L79E 20-l34)-hFc 3.8e-5 2.74e-4 7.16e-10
ActRIIB(R64K 20-l34)-hFc 6.77e-5 2.41e-5 3.56e-ll
GDF8
Protein Kon (1/Ms) | Koff(l/s) KD (M)
ActRIIB(20-l34)-hFc 3.69e-5 3.45e-5 9.35e-l 1
ActRIIB(A24N 20-l34)-hFc
ActRlIB(L79D 20-l34)-hFc 3.85e-5 8.3e-4 2.15e-9
ActRIIB(L79E 20-l34)-hFc 3.74e-5 9e-4 2.41e-9
ActRIIB(R64K 20-l34)-hFc 2.25e-5 4.71e-5 2.1e-10
ActRIIB(R64K 20-l29)-hFc 9.74e-4 2.09e-4 2.15e-9
1,67e-9
ActRIIB(P129S. P130R 20- !34)-hFc 1.08e-5 1.8e-4
ActRlIB(K74A 20-l34)-hFc 2.8e-5 2.03e-5 7.18e-l 1
Activin A
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Protein Kon (1/Ms) Koff(l/s) KD (M)
ActRIIB(20-134)-hFc 5.94e6 1.59e-4 2.68e-11
ActRIIB(A24N 20-134)-hFc 3.34e6 3.46e-4 1.04e-10
ActRIIB(L79D 20-134)-hFc Low binding
ActRIIB(L79E 20-134)-hFc Low binding
ActRIIB(R64K 20-134)-hFc 6.82e6 3.25e-4 4.76e-l1
ActRIIB(R64K 20-129)-hFc 7.46e6 6.28e-4 8.41e-l 1
ActRII B(PI29S, P130R 20- 134)-hFc 5.02e6 4.17e-4 8.31e-l 1
These data obtained from a cell-free assay confirm the cell based assay data, demonstrating that the A24N variant retains ligand-binding activity that is similar to that of the ActRlIB(20-l34)-hFc molécule, and that the L79D or L79E molécule retains myostatin 5 and GDFl 1 binding but shows markedly decreased (non-quantifiable) binding to activin A.
Other variants hâve been generated and tested, as reported in W02006/012627 (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, K.74Y, K.74F, K.74I (and presumably other hydrophobie substitutions at K.74, such as K74L), and D80I, cause a 10 decrease in the ratio of activin A (ActA) binding to GDFl 1 binding, relative to the wild-type
K.74 molécule. A table of data with respect to these variants is reproduced below:
Soluble ActRIIB-Fc variants binding to GDF11 and Activin A (BiaCore assay)
ActRIIB ActA GDF11
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WT (64A) KD=1.8e-7M (+) KD= 2.6e-7M (+)
WT (64R) na KD= 8.6e-8M (+++)
+15tail KD -2.6 e-8M (+++) | KD= 1.9e-8M (il)
E37A * *
R40A - -
D54A - *
K55A -H- *
R56A * *
K74A KD=4.35e-9M +++++ KD=5.3e-9M 1 1 1 1 1
K74Y * —
K74F * —
K741 *
W78A * *
L79A + *
D80K * 1 *
D80R * *
D80A * *
D80F *
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D80G * *
D80M * *
D80N * *
D80I * —
F82A ++
* No observed binding
- < l/5 WT binding
- - l/2 WT binding + WT
Ή- < 2x increased binding
4-H- ~5x increased binding ++++ ~!0x increased binding i i ( i i ~ 40x increased binding
Example 13: A GDF Trap Increases Red Blood Cell Levels in vivo
Twelve-week-old male C57BL/6NTac mice were assigned to one of two treatment groups (N=10). Mice were dosed with either vehicle or with a variant ActRIIB polypeptide (“GDF Trap) [ActRIIB(L79D 20-134)-hFc] by subcutaneous injection (SC) at 10 mg/kg twice per week for 4 weeks. At study termination, whole blood was collected by cardiac puncture into EDTA containing tubes and analyzed for cell distribution using an HM2 hematology analyzer (Abaxis, Inc).
I
Group Désignation I
Group N Mice Injection Dose (mg/kg) Route Frequency
1 10 C57BL/6 PBS 0 SC Twice/week
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2 10 C57BL/6 GDF Trap [ActRIIB(L79D 20-134)-hFc] 10 SC Twice/week
Treatment with the GDF Trap did not hâve a statisticaily significant effect on the number of white blood cells (WBC) compared to the vehicle controls. Red blood cell (RBC) numbers were increased in the treated group relative to the controls (see table below), Both 5 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 animais, indicating an increase in the pool of immature red blood cells. Therefore, treatment with the GDF Trap leads to increases in red blood cells, with no distinguishable effects on white blood cell populations.
Hematology Results
RBC 10,2/L HGB (g/dL) HCT (%) RDWc (%)
PBS 10.7 ±0.1 14.8 ±0.6 44.8 ±0.4 17.0 ± 0.1
GDF Trap 12.4 ± 0.4** 17.0 ± 0.7* 48.8 ± 1.8* 18.4 ± 0.2**
*=p<0.05, **= p<0.01
Example 14: A GDF Trap is Superior to ActRIIB-Fc for Increasing Red Blood Cell l.cvclsw vivo.
!5 Nineteen-week-old male C57BL/6NTac mice were randomly assigned to one of three groups. Mice were dosed with vehicle (10 mM Tris Buffered Saline, TBS), wild-type ActRIIB(20-134)-mFc, or GDF trap ActRIIB(L79D 20-134)-hFc by subeutaneous injection twice per week for three weeks. Blood was collected cheek bleed at baseline and after three
-16919001 weeks of dosing and analyzed for cell distribution using a hematology analyzer (HM2, Abaxis, Inc.)
Treatment with ActRIIB-Fc or the GDF trap did not hâve a significant effect on white blood cell (WBC) numbers compared to vehicle controls. The red blood cell count (RBC), hematocrit (HCT), and hemoglobin levels were ail elevated in mice treated with GDF Trap compared to either the controls or the wild-type construct (see table below), Therefore, in a direct comparison, the GDF trap promûtes increases in red blood cells to a si g nificantly greater extent than a wild-type ActRIIB-Fc protein. In fact, in this experîment, the wild-type ActRIIB-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 Résulte after three weeks of dosing
RBC (10l2/ml) HCT % HGB g/dL
TBS 11.06 ± 0.46 46.78 ± l.9 15.7 ± 0.7
ActRIIB-mFc ll.64± 0.09 49.03 ± 0.3 16.5 ± 1.5
GDF Trap I3.l9± 0.2** 53.04 ±0.8** 18.4 ± 0.3**
**=p<0.0l
Example 15: Génération of a GDF Trap with Truncated ActRIIB Extracellular Domain As described in Example 9, a GDF Trap referred to as ActRIIB(L79D 20-l34)-hFc was generated by N-terminal fusion of TPA leader to the ActRIIB extracellular domain (residues 20-134 in SEQ ID NO:l) containing a leucine-to-aspartate substitution (at residue 79 in SEQ ID NO: l ) and C-terminal fusion of human Fc domain with minimal linker (three glycine residues) (Figure 16). A nucléotide sequence corresponding to this fusion protein is shown in Figures 17A and 17B.
A GDF Trap with truncated ActRIIB extracellular domain, referred to as ActRIIB(L79D 25-l 31 )-hFc, was generated by N-terminal fusion of TPA leader to truncated
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I extracellular domain (residues 25-13l in SEQ ID NO;l) containing a leucine-to-aspartate substitution (at residue 79 in SEQ ID NO:l) and C-terminal fusion of human Fc domain with minimal linker (three glycine residues) (Figure 18). A nucléotide sequence corresponding to this fusion protein is shown in Figures 19A and 19B.
Example 16; Sélective Ligand Binding by GDF Trap with Double-Truncated ActRIIB Extracelluar Domain
The affmity of GDF Traps and other ActRIIB-hFc proteins for several ligands was evaluated in vitro with a Biacore™ instrument. Results are summarized in the table below. Kd values were obtained by steady-state affinity fit due to the very rapid association and dissociation of the complex, which prevented accurate détermination of kon and kOff.
Ligand Selectivity of ActRIIB-hFc Variants:
Fusion Construct Activin A (Kde-11) Activin B (Kd c-11) GDFl 1 (Kd e-11)
ActRIIB(L79 20-134)-hFc ! 16 1.2 3.6
ActRIIB(L79D 20-l34)-hFc 1'350.0 78.8 12.3
ActRlIB(L79 25-131)-hFc 1.8 1.2 3.1
ActRIIB(L79D 25-13 l)-hFc 2290.0 62.1 7.4
The GDF Trap with a truncated extracellular domain, ActRIIB(L79D 25-l3l)-hFc, equaled or surpassed the ligand selectivity displayed by the longer variant, ActRHB(L79D 20-l34)-hFc, with pronounced loss of activin A binding, partial loss of activin B binding, and nearly full rétention of GDFl l binding comparèd to ActRIIB-hFc counterparts lacking the L79D substitution. Note that truncation alone (without L79D substitution) did not alter selectivity among the ligands displayed here [compare ActRIIB(L79 25-l3l)-hFc with ActRIIB(L79 20-l34)-hFc].
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Example 17: Génération of ActRIIB(L79D 25-131)-hFc with Alternative Nucléotide Sequences
To generale ActRIIB(L79D 25-13l)-hFc, the human ActRIIB extracellular domain with an aspartate substitution at native position 79 (SEQ ID NO: 1) and with N-terminal and 5 C-terminal truncations (residues 25-131 in SEQ ID NO: 1) was fused N-terminally with a TPA leader sequence instead of the native ActRIIB leader and C-terminally with a human Fc domain via a minimal linker (three glycine residues) (Figure 18). One nucléotide sequence encoding this fusion protein is shown in Figure 19 (SEQ ID NO: 42), and an alternative nucléotide sequence encoding exactly the samei fusion protein is shown in Figures 22A and 10 22B (SEQ ID NO: 46). This protein was expressed and purified using the methodology described in Example 9.
Example 18: GDF Trap with a Truncated ActRIIB Extracellular Domain Increases Prolifération of Ervthroid Progenitors in Mice
ActRIIB(L79D 25-13 l)-hFc was evaluated to determine its effect on prolifération of erythroid progenitors. Male C57BL/6 mice (8 weeks old) were treated with ActRIIB(L79D 25-131)-hFc (10 mg/kg, s.c.; n = 6) or vehicle (JTBS; n = 6) on Days 1 and 4, then eulhanized on Day 8 for collection of spleens, tibias, fémurs, and blood. Cells of the spleen and bone marrow were isolated, diluted in Iscove’s modified Dulbecco’s medium containing 5% fêtai bovine sérum, suspended in specialized methylcellulose-based medium, and cultured for either 2 or 12 days to assess levels of clonogenîc progenitors at the colony-forming uniterythroid (CFU-E) and burst forming unit-erythroid (BFU-E) stages, respectively. Methylcellulose-based medium for BFU-E détermination (MethoCult M3434, Stem Cell Technologies) included recombinant murine stem cell factor, interleukin-3, and interleukin-6, which were not présent in methylcellulose medium for CFU-E détermination (MethoCult M3334, Stem Cell Technologies), while both media contained erythropoietin, among other constituées. For both BFU-E and CFU-E, the number of 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 ActRlIB(L79D 25-131 )-hFc had twice the number of CFU-E colonies as did corresponding cultures from control mice (P < 0.05),
-17219001 whereas the number of BFU-E colonies did not differ 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 spleenderived cultures were accompanied by highly significant (P < 0.001 ) changes in red blood cell level (l l .6% increase), hemoglobin concentration ( 12% increase), and hematocrit level (l l.6% increase) at euthanasia in mice treated with ActRIIB(L79D 25-l3l)-hFc compared to controls. These results indicate that in vivo administration of a GDF Trap with truncated ActRIIB extracellular domain can stimulate prolifération of erythroid progenitors as part of its overall effect to increase red blood cell levels.
GDF Trap fusion proteins hâve been fuijther demonstrated to be effective în increasing red blood cell levels in varions models of anémia including, for example, chemotherapyinduced anémia, nephrectomy-induced anémia, and in a blood loss anémia. See, e.g., International Patent Application Publication No.WO 2010/019261.
Example 19: GDF Trap with Truncated ActRIIB Extracellular Domain Increases Levels of Red Blood Cells in Non-Human Primates
Two GDF Traps, ActRHB(L79D 20-134)-hFc and ActRlIB(L79D 25-131)-hFc, were evaluated for their ability to stimulate red blood cell production in cynomolgus monkey. Monkeys were treated subcutaneously with GDF Trap (10 mg/kg; n = 4 males/4 females), or vehicle (n = 2 males/2 females) on Days 1 and 8. Blood samplcs were collectcd on Days I (pretreatment baseline), 3, 8, 15, 29, and 44, and were analyzed for red blood cell levels (Figure 24), hematocrit (Figure 25), hemoglobin levels (Figure 26), and réticulocyte levels (Figure 27). Vehicle-treated monkeys exhibited decreased levels of red blood cells, hematocrit, and hemoglobin at ail post-treatment time points, an expected effect of repeated blood sampling. In contrast, treatment with ActRIIB(L79D 20-134)-hFc or ActRlIB(L79D 25-131 )-hFc increased these parameters by the first post-treatment time point (Day 3) and maîntained them at substantially elevated levels for the duration of the study (Figures 24-26). Importantly, réticulocyte levels in monkeys treated with ActRIIB(L79D 20-134)-hFc or
I
ActRIIB(L79D 25-131)-hFc were substantially increased at Days 8, 15, and 29 compared to vehicle (Figure 27). This resuit demonstrates that GDF Trap treatment increased production of red blood cell precursors, resulting in elevated red blood cell levels.
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Taken together, these data demonstrate that truncated GDF Traps, as well as a lulllength variants, can be used as sélective antagoniste of GDFl l and potentially related ligands to increase red blood cell formation in vivo.
Example 20: GDF Trap Derived from ActRIIB5
Others hâve reported an altemate, soluble form of ActRIIB (designated ActRIIB5), in which exon 4, including the ActRIIB transmembrane domain, has been replaced by a different C-terminal sequence. See, e.g., WO 2007/053775.
The sequence of native human ActRIIB5 without its leader is as follows. GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVK KGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEGPWAST TIPSGGPEATAAAGDQGSGALWLCLEGPAHE (SEQ ID NO:49)
An leucine-to-aspartate substitution, or other acidic substitutions, may be performed at native position 79 (underlined) as described to construct the variant ActRIIB5(L79D), which has the following sequence:
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDK.RLHCYASWRNSSGTIELVK. KGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEGPWAST TIPSGGPEATAAAGDQGSGALWLCLEGPAHE (SEQ ID NO:50)
This variant may bc connected to human Fc (double underline) with a TGGG linker (single underline) to generate a human ActRIlB5(L79D)-hFc fusion protein with the following sequence: GRGEAETRECIYYNANWELERTNQSGLERCEGEQDK.RLHCYASWRNSSGTIELVK KGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTIILPEAGGPEGPWAST TIPSGGPEATAAAGDQGSGALWLCLEGPAHETGGGTIITCPPCPAPELLGGPSVFL FPPKPKDTLMiSRTPEVTCVVVDVSHF.DPEVKFNWYVDGVEVHNAKTKPREEOYN STYRVVSVI.TVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPREPQVYTLP
-17419001 psreemtknovsltclvkgfypsdiavewîesngopennykttppvldsdgsffly
I
SKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLSLSPGK (SEQ ID NO:51).
This construct may be expressed in CHO cells.
Example 21 : Effects in Mice of Combined Treatment with EPO and a GDF Trap with a Truncated ActRIIB Extracellular Domain
EPO induces formation of red blood cells by increasing the prolifération of erythroid precursors, whereas GDF Traps could potentially affect formation of red blood cells in ways that complément or enhance EPO’s effects. Therefore, Applicants investigated the effect of combined treatment with EPO and ActRIIB(L7pD 25-13 l)-hFc on erythropoietîc parameters. Male C57BL/6 mice (9 weeks old) were gîven a single i.p. injection of recombinant human EPO alone (epoetin alfa, 1800 units/kg), ActRIIB(L79D 25-l3l)-hFc alone (10 mg/kg), both EPO and ActRIIB(L79D 25-l3l)-hFc, or vehicle (Tris-buffered saline). Mice were euthanîzed 72 h after dosing for collection of blood, spleens, and fémurs.
Spleens and fémurs were processed to obtain erythroid precursor cells for flow cytométrie analysis. After removal, the spleen was minced in Iscove’s modified Dulbecco’s medium containing 5% fêtai bovine sérum and mechanically dissociated by pushing through a 70-pm cell strainer with the plunger from a stérile l -mL syringe. Fémurs were cleaned of any residual muscle or connective tissue and ends were trimmed to permit collection of marrow by flushing the remaining shaft with Iscove’s modified Dulbecco’s medium containing 5% fêtai bovine sérum through a 2l-gauge needle connected to a 3-mL syringe. Cell suspensions were centrifuged (2000 rpm for 10 min) and the cell pellets resuspended in PBS containing 5% fêtai bovine sérum. Cells ( l O6) from each tissue were incubated with anti-mouse IgG to block nonspecific binding, then incubated with fluorescently labeled antibodies against mouse cell-surface markers CD7I (transferrin receptor) and Terl 19 (an antigen associated with cell-surface glycophorin A), washed, and analyzed by flow cytrometry. Dead cells in the samples were excluded from analysis by counterstaining with propidium iodide. Erythroid différentiation in spleen or bone marrow was assessed by the degree of CD7l labeling, which decreases over the course of différentiation, and Terl 19 labeling, which is increased during terminal erythroid différentiation beginning with the proerythroblast stage (Socolovsky et al., 2001, Blood 98:3261-3273; Ying et al., 2006, Blood 108:123-133). Thus, flow cytometry was used to détermine the number of proerythroblasts
-17519001 (CD7lh,ghTerll9low), basophilie erythroblasts (CD7lhlghTerl I9hlgh), polychromatophilic + orthochromatophilic erythroblasts (CD7linedTerl I9hlgh), and late orthochromatophilic erythroblasts + réticulocytes (CD7llowTerl 19h,gh), as described.
Combined treatment with EPO and ActRIIB(L79D 25-l3l)-hFc led to a surprisingly vigorous increase in red blood cells. In the 72-h time frame of this experiment, neither EPO nor ActRIIB(L79D 25-l3l)-hFc alone increased hematocrit significantly compared to vehicle, whereas combined treatment with the two agents led to a nearly 25% increase in hematocrit that was unexpectedly synergistic, i.e., greater than the sum of their separate effects (Figure 28). Synergy of this type is generally considered evidence that individual agents are acting through different cellular mechanisms. Similar results were also observed for hemoglobin concentrations (Figure 29) and red blood cell concentrations (Figure 30), each of which was also increased synergistically by combined treatment.
Analysis of erythroid precursor levels revealed a more complex pattern. In the mouse, the spleen is considered the primary organ responsible for inducible (“stress”) erythropoîesis. Flow cytométrie analysis of splenic tissue at 72 h revealed that EPO markedly altered the erythropoietic precursor profile compared to vehicle, increasing the number of basophilie erythroblasts by more than 170% at the expense of late precursors (late orthochromatophilic erythroblasts + réticulocytes), which decreased by more than one third (Figure 31). Importantly, combined treatment increased basophilie erythroblasts significantly compared to vehicle, but to a lesser extent than EPO alone, while supporting undiminished maturation of late-stage precursors (Figure 31). Thus, combined treatment with EPO and ActRIIB(L79D 25-13 l)-hFc increased erythropoîesis through a balanced enhancement of precursor prolifération and maturation. In contrast to spleen, the precursor cell profile in bone marrow after combined treatment did not differ appreciably from that after EPO alone. Applicants predict from the splenic precursor profile that combined treatment would lead to increased reticulocyte levels and would be accompanied by sustained élévation of mature red blood cell levels, if the experiment were extended beyond 72 h.
Taken together, these findings demonstrate that a GDF Trap with a truncated ActRIIB extracellular domain can be administered in combination with EPO to synergistically increase red blood cell formation in vivo. Acting through a complementary but undefined mechanism,
I a GDF trap can moderate the strong proliférative effect of an EPO receptor aelivator alone and still permit target levels of red blood cells to be attained with lower doses of an EPO
-17619001 receptor activator, thereby avoiding potential adverse effects or other problems associated with higher levels of EPO receptor activation.
Example 22: Effect of GDF Trap with a Truncated ActRIIB Extracellular Domain on RBC Levels and Morphology in a Mouse Model of β-Thalassemia
In thalassemia syndromes, which represent the most common causes of ineffective erythropoiesis, imbalances in the expression of a- and β-globin chains resuit in anémia due to increased apoptosis during erythroblast maturation. RBC transfusion is currently a key maintenance therapy in thalassemia but over time causes potentially léthal iron accumulation in certain tissues (Tanno et al, 2010, Adv Hematol 2010:358283). For example, heart disease associated with iron overload can account for 50% of mortality in patients with thalassemia major (Borgna-Pignatti et al, 2005, Ann NY Acad Sci 1054:40-47). Importantly, endogenous EPO levels are typically elevated and contribute to disease etiology in thalassemia syndromes as well as other disorders of ineffective erythropoiesis; therefore, therapeutic use of recombinant EPO may be inappropriate. Thus, there is the need for alternative thérapies for thalassemia and other disorders of ineffective erythropoiesis that would increase RBC levels without the iron overload that accompanies chronic transfusions.
Applicants investigated the effect of ActRIIB(L79D 25-131 )-mlc on RBC formation in a mouse model of β-thalassemia întennedia in which the entire coding région of the βmajor globin coding gene has been deleted. Mice homozygous for this Hbbih~' allele exhibit a hypochromie, mîcocytic anémia with inclusion bodies in a hîgh proportion of circulating RBCs (Skow et al, 1983, Cell 1043:1043-1052). In a preliminary experiment, Hbb'1’ βthalassemic mice (C57BL/6J-/7&ôi/Ji;,/J) at 2-5 months of âge were randomly assigned to receive ActRlIB(L79D 25-131)-mFc (10 mg/kg) or vehicle (Tris-buffercd saline) by subeutaneous injection twice-weekly. Wildtype littermates dosed with vehicle served as additional controls. Blood samples (100 μΐ) were collected by cheek bleed before the onset of dosing and at regular intervals thereafter for CBC analysis. Characterization of hématologie parameters at baseline confirmed that Hbb'1' β-thalassemic mice were severely anémie (Figures 32A-C), and treatment of Hbb\' mice with ActRIIB(L79D 25-131 )-mFc for 4 weeks increased RBC number markedly compared with vehicle-treatcd Hbb'1' mice, thereby reducing the anémia observed in this model by half (Figure 33). Treatment-associated increases in hematocrit and hemoglobin concentration were also seen. Importantly, treatment
-17719001 of Hbb'1'mice with ActRIIB(L79D 25-131)-mFc also resulted in improved RBC morphology and reduced hemolysis and erythrocytic débris compared to vehicle-treated Hbb'1' mice (Figure 34), thus indicating a fondamental improvement in erythropoiesis. Hence, a GDF Trap polypeptide with truncated ActRIIB extracellular domain can provide therapeutic benefit for anémia in a murine model of β-thalassemia by increasing both RBC number and morphology. By promoting erythroblast maturation while reducing anémia, GDF Trap polypeptides can treat ineffective erythropoiesis. Unlike transfusions, which are inherently a source of exogenous iron, a GDF Trap polypeptide can raise RBC levels by promoting use of endogenous iron stores via erythropoiesis, thereby avoiding iron overloading and its négative conséquences.
Example 23: Effect of a GDF Trap with Truncated ActRIIB Extracellular Domain on EPO Levels. Splenomegaly. Bone Density. and Iron Overload in a Mouse Model of β-Thalassemia
Hypoxia associated with ineffective erythropoiesis causes elevated EPO levels that can drive massive expansion of erythroblasts both within and outside the bonc marrow, leading to splenomegaly (spleen enlargement), erythroblast-induced bone pathology, and tissue iron overload. even in the absence of therapeutic RBC transfusions. Untreated iron overload leads to tissue iron déposition, multiple organ dysfunction, and prématuré mortality (Borgna-Pignatti et ai, 2005, Ann NY Acad Scj 1054:40-47; Borgna-Pignatti et al., 2011, Expert Rev Hematol 4:353-366), most often due to cardiomyopathy in severe forms of thalassemia (Lekawanvijit et al., 2009, Can J Cardîol 25:213-218). By increasing erythropoietic effectiveness, a GDF Trap polypeptide may alleviate not only the underlying anémia and elevated EPO levels but also the associated complications of splenomegaly, bone pathology, and iron overload.
Applicants investigated effects of a GDF Trap polypeptide on these parameters in the same mouse model of β-thalassemia intermedia studied in Example 21. Hbb'1' β-thalassemic mice (C57BU6J-Hbbd3,hiï) at 3 months of âge were randomly assigned to receive ActRIlB(L79D 25-13 l)-mFc (1 mg/kg, n = 7) or vehicle (Tris-buffered saline, n = 7) by subcutaneous injection twice weekly for 2 monTis. Wildtype littermates dosed with vehicle (n = 13) served as additional controls. Blood simples (100 μΐ) were collected at study termination for CBC analysis. At study termination, bone minerai density was determined by dual energy x-ray absorptiometry (DEXA), sérum EPO levels were determined by ELISA,
-17819001 reactive oxygen species (ROS) were quantitated with 2',7'-dichlorodihydrofluorescein diacetate and flow cytometry (Suragani et al., 2012, Blood 119:5276-5284), and hepcidin mRNA levels were determined by quantitative polymerase chain reaction.
This GDF Trap polypeptide exerted multiple hématologie effects consistent with alleviation of ineffective erythropoiesis. Treatment of Hbb'1' mice with ActRIIB(L79D 25131)-mFc for 2 months increased RBC counts by 25% compared with vehicle-dosed Hbb'1' mice (Figure 35). In Hb^' mice, ActRIIB(L79D 25-13 l)-mFc treatment also increased hemoglobin concentration and hematocrit significantly at 2 months compared to vehicle controls. These changes were accompanied by reduced levels of circulating réticulocytes (31.3 ± 2.3% vs. 44.8 ± 5.0% for Hbb'1''mice tréated with ActRIlB(L79D 25-131)-mFc or vehicle, respectively), which is consistent with alleviation of anémia. As in Example 21, treatment of Hbb'^ mice with ActRIIB(L79D 25-131)-mFc resulted in improved RBC morphology and reduced erythrocytic débris compared to vehicle-dosed Hbb ' mice. Compared to healthy indîviduals, patients with thalassemia exhibit an increased rate of RBC
IS destruction and elevated sérum levels of bilirubin, which is a product of heme catabolism and marker of hemolysis (Orten, 1971, Ann Clin Lab Sci 1:113-124). In Hbb1 mice, treatment with ActRIIB(L79D 25-13 l)-mFc reduced sérum bilirubin levels at 2 months by nearly half compared to vehicle (Figure 36), thereby providing evidence that ActRIIB(L79D 25-131)mFc can unexpectedly improve the structural/functional integrity ol mature RBCs as it promûtes RBC formation. Importantly, treatment of Hbb'1' mice with ActRIlB(L79D 25131)-mFc reduced sérum EPO levels at 2 montas by more than 60% compared to vehicle in the same model (Figure 37). Since elevated EPO levels are a hallmark of ineffective erythropoiesis in β-thalassemia, the réduction of such levels here is strong évidence that ActRIIB(L79D 25-13 l)-mFc alleviates ineffective erythropoiesis itseli, not just the anemia it causes, in this murine model of thalassemia.
This GDF Trap polypeptide also produced bénéficiai changes in endpoints representing major complications of ineffective erythropoiesis. In thalassemia patients, both splenomegaly and bone détérioration are caused by EPO-stimulated erythroid hyperplasia and extramedullary erythropoiesis. In Hbb'1' mice, treatment with ActRIIB(L79D 25-131 )-mFc for 2 months reduced spleen weight significantly compared to vehicle (Figures 38A and 38B) and fuily restored bone minerai density to wildtype values (Figure 39). Iron homeostasis was also improved significantly by treatment with this GDF Trap polypeptide. Sérum iron consists of both unbound (free) iron and iron bound to apotransferin (forming transfenn), a
-17919001 specialized protein for transporting elemental iiion in the circulation. Sérum iron constitutes a relatively small and labile component of total body iron, whereas sérum levels of ferritin, another form of iron storage found mainly intracellularly, represent a larger and less labile component. A third measure of iron load is transferin saturation, the degree to which the iron binding capacity of transferin is occupied. In Hbb^'mice, ActRIIB(L79D 25-l3l)-mFc treatment for 2 months reduced each of these indicators of iron overload significantly compared to vehicle (Figures 40A-C). In addition to its effects on these diverse parameters of iron homeostasis, ActRIIB(L79D 25-l3l)-mFc normalized tissue iron overload in Hbb'1' mice as determined by histochemical analysis in spleen, liver, and kidney (Figure 41). Moreover, this GDF Trap polypeptide exerted a bénéficiai effect on expression of hepcidin, a hepatic protein considered to be the master regulator of iron homeostasis (Gantz, 2011, Blood 117:4425-4433), whose levels vary inversely with dietary iron uptake. Treatment with ActRIIB(L79D 25-13 l)-mFc reversed the abnormally low expression of hepcidin in liver of Hbb'1' mice (Figure 42). Finally, another study with similar design was performed to détermine the effect of this GDF Trap on reactive oxygen species (ROS), which are thought to médiate many of the toxic effects of iron overload (Rund et al., 2005, N Engl J Med 353:1135-1146). In 3-month-old Hbb'!'mice, treatment with ActRIIB(L79D 25-l31)-mFc at 1 mg/kg twice weekly for 2 months nearly normalized ROS levels (Figure 43) and would therefore be predicted to greatly reduce the tissue damage mediated by ROS in thalassemia and other diseases characterized by ineffective erythropoiesis.
Together, the above findings demonstrate that GDF Trap polypeptides can treal ineffective erythropoiesis, încluding anémia and elevated EPO levels, as well as complications such as splenomegaly, erythroblast-induced bone pathology, and iron overload, and their attendant pathologies. With splenomegaly, such pathologies include thoracic or abdominal pain and réticuloendothélial hyperplasia. Extramedullary hematopoiesis can occur not only in the spleen but potentially in other tissues in the form of extramedullary hematopoietic pseudotumors (Musallam et al., 2012, Cold Spring I larb Perspect Med 2:a013482). With erythroblast-induced bone pathology, attendant pathologies include low bone minerai density, osteoporosis, and bone pain (Haidar et al., 2011, Bone 48:425-432). With iron overload, attendant pathologies include hepcidin suppression and hyperabsorption of dietary iron (Musallam et al., 2012, Blood Rev 26(Suppl 1):S16-S19), multiple endocrinopathies and liver fibrosis/cirrhosis (Galanello et al., 2010, Orphanet J Rare Dis 5:11), and iron-overload cardiomyopathy (Lekawanvijit et al., 2009, Can J Cardiol 25:213-18019001
218). In contrast to existing thérapies for ineffective erythropoiesis. GDl· T rap polypeptides such as ActRIIB(L79D 25-l 3 l)-mFc are able to reduce iron overloading in murine models while concurrently increasing RBC levels. This novel capability distinguishes GDF Trap polypeptides from blood transfusions, which inherently burden the body with exogenous iron in the course of treating anémia and do so without alleviating the underlying condition of ineffective erythropoiesis.
Example 24: GDF Trap Increases Hemoglobin Levels and Substantially Résolves a Cutaneous Ulcer in a Thalassemia Patient.
A clinical study was designed to treat thalassemia patients (β-thalassemia intermedia and major patients) with multiple does of ActRIIB(L79D 25-l3l)-hFc. The study comprised both non-transfusion dépendent patients (< 4 units/8 weeks, hemoglobin < 10 g/dL) and transfusion (blood) dépendent patients (> 4 units/ 8 weeks confirmed over 6 months). Patients were divided into one of four treatment groups: i) administration of 0.2 mg/kg ActRIlB(L79D 25-131 )-liFc by subcutaneous injection every three weeks; ii) administration of 0.4 mg/kg ActRIIB(L79D 25-13 l)-hFc by subcutaneous injection every three weeks; iii) administration of 0.6 mg/kg ActRIIB(L79D 25-13l)-hFc by subcutaneous injection every three weeks; and iv) administration of 0.8 mg/kg ActRII B(L79D 25-131 )-hFc by subcutaneous injection every three weeks. Over the course of three months of treatment, patients were observed to hâve significant, dose-dependent increases in hemoglobin levels. Furthermore, ActRIIB(L79D 25-13 l)-hFc treatment was effective at decreasing transfusion dependency, Le., ail transfusion dépendent patients experienced a >50% réduction in transfusion burden during the course of the study.
One patient with a baseline hemoglobin level of approximately 9.2 g/dL received 4 doses of ActRIIB(L79D 25-131 )-hFc at the 0.4 mg/kg level, resulting in a hemoglobin level of approximately 10.6 g/dL after three months of treatment. The patient’s thalassemia was βthalassemia intermedia, and the patient was non-transfusion dépendent. For approximately three years prior to this study, this patient had been afflicted with récurrent skin ulcers in the lower limbs. Such ulcers are common cutaneous complications of thalassemia. See, e.g.. Rassi et al. (2008) Pédiatrie Annals 37(5): 322-328. Prior to ActRIIB(L79D 25-13l)-hFc treatment, this patient was diagnosed with a leg ulcer. Ulcer healing was observed two weeks after administration of the first ActRIIB(L79D 25-13 l)-hFc dose. After six weeks of
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ActRHB(L79D 25-l3l)-hFc treatment, the leg ulcer was determined to be substantially resolved. A second non-transfusion dépendent patient began the study with a leg ulcer. The leg ulcer was substantially resolved after treatment with several doses of ActRllB(L79D 25131 )-hFc at 1.25 mg/kg. In addition, a transfusion-dependent patient began the study with an ulcer on the left ankle. After five doses of ActRIIB(L79D 25-13 l)-hFc at 1.0 mg/kg the ulcer was substantially resolved and remained so for the duration of the study. Accordingly, ActRIIB(L79D 25-13l)-hFc can be used to effectively treat ulcers that manifest in nontransfusion and transfusion dépendent thalassemia patients.
Accordingly, these data demonstrate that ActRIIB(L79D 25-131)-hFc treatment is effective in increasing hemoglobin levels and can be used to reduced transfusion dependency in human thalassemia patients. In addition to tfte positive effects on the anémia aspects of the disease, the signifïcant improvement in healîng of the leg ulcers indicates that ActRIIB(L79D 25-13l)-hFc can be used to effectively treat other non-anemia complications of thalassemia, which is consistent with the data from the mouse model of β-thalassemia described above.
Example 25: GDF Trap Increases Red Blood Cell Levels and Improves Red Blood Cell Morphology in Sickle-Cell Disease Model
Applicants investigated the effect of ActRIIB(L79D 25-131)-mFc on red blood cell (RBC) formation in a mouse model of sickle-cell disease (SCD) in which the mouse hemoglobin genes (α/α and β/β) hâve been replaced with the human sickle hemoglobin genes (α/α, γ/γ, and β /β ). Mice homozygous for the human β allele exhibit the major features (e.g., sever hemolytic anémia, irreversibly sickled red cells, vascular (vaso) occlusion, and inulti-organ pathology) found in humans with SCD. See, e.g., Wu et al., (2006) Blood, 108(4): 1183-1188; Ryan et al. (1997) Science 278: 873-876.
SCD mice (β5/β5) at 3 months ofage were randomly assigned to reçoive ActRIlB(L79D 25-13 l)-mFc (1 mg/kg) or vehicle [Tris-buffered saline (TBS)J by subcutaneous injections twice weekly. Non-symptomatic compound hétérozygote (β/β5) litermates dosed with vehicle served as additional controls (Wt animais). At baseline, SCD mice had reduced RBC levels (-28%, P<0.01 ) and hemoglobin levels (-14.5%, P<0.05) and increased réticulocyte levels (+50%, P<0.001) compared to the compound hétérozygote mice. demonstrating that the SCD mice were severel}| anémie.
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Following one month of treatment, subjects were assessed for changes in varions red blood cell parameters. Treatment of SCD mice|with ActRIIB(L79D 25-l31)-mFc for 4 weeks increased RBC levels markedly (+15.2%, p<0.01) compared to vehicle-treated SCD mice, thereby reducing the anémia observed in this model (Figures 44 and 45). ActRIIB(L79D 25-131)-mFc treatment-associated increases in hematocrit and hemoglobin concentrations were also observed (Figure 45) as well as significant decreases in mean corpuscular volume, RDC distribution width, réticulocyte numbers, and reactive oxygen species (Figure 46), which is ail consistent with improved red blood cell half-life. Surprisingly, treatment of SCD mice with ActRIIB(L79D 25-131 )-mFc for 6 weeks resulted in a substantial decrease in phosphatidylserine (PS) exposure in peripheral blood cells (-14%, P=0.08), as determined by scramblase enzyme assay and anncxin-V assay. indicaling a trcnd toward improved membrane phospholipid asymmetry compared to vehicle-treated subjects.
Following three months of treatment, subjects were observed to hâve improvements in additional blood chemistry parameters. In particular, treatment of SCD mice with ActRIIB(L79D 25-131 )-mFc for 12 weeks significantly decreased bilirubin (total) levels (17.0%, p<0.05), blood urea nitrogen levels (-19.2%, p<0.05), and cell free hemoglobin (30.7%, p = 0.06) compared to vehicle-treated SCD mice. These data indicate that GDF Traptreated subjects hâve decreased levels of red blood cell hemolysis in comparision to vehicletreated subjects, which is consistent with the observed increase of red blood cell levels observed as early as one month following the start of ActRIIB(L79D 25-131 )-mFc therapy. Annexîn-V assays demonstrated a significant decrease in phosphatidylserine (PS) exposure in peripheral blood cells (-13.4%, p = 0.06) after three months of therapy in comparison to vehicle-treated subjects. Moreover, blood smears performed aller three months of treatment showed fewer irreversibly sickle-formed red blood cells in ActRllB(L79D 25-131)-mFctreated mice (-66.5%, p< 0.0001; enumerated from approximately 2000 cells per group) in comparision to mice treated with vehicle alone. These data indicate a qualitative improvemenl in red blood cell morphology following ActRIlB(L79D 25-lol)-mFc treatment, which is consistent with the scramblase enzyme assay and annexin-V assay data obtained after one and three months of ActRIIB(L79D 25-131 )-mFc treatment. Furthermore, treatment of SCD mice with the GDF Trap for three months also resulted in a significant decrease in spleen weight (-20.5%, p<0.05) in comparision to vehicle-treated SCD mice. These data indicate that ActRIIB(L79D 25-131 )-mFc may be useful in the treatment of other complications associated with SCD including, for example, splenic séquestration of red blood cells, which can resuit in splenic séquestration crisis and/or spenomegaly.
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Together, these data indicate that a GDF Trap comprising a truncated ActRIIB extracellular domain can provide various therapeutic benefits in a murine model of SCD. In addition to increasing RBC levels and improving various blood parameters, the data demonstrate improvement in RBC morphology. This observed improvement in RBC morphology indicates that GDF Trap treatment: may be used to treat or prevent various other complications of SCD (e.g., complications arising from vaso-occlusion) in addition to anémia. This îs further supported by the observed decrease in spleen size in ActRIIB(L79D 25-131)mFc-treated subjects.
Accordingly, the data presented herein suggest that GDF Trap polypeptides can be used to treat a variety of complications of sickle-cell disease. Unlike red blood cell transfusions, which are inherently a source of exogenous iron, a GDF Trap polypeptide can raise RBC levels by promoting use of endogenous iron stores via erythropoiesis and thus avoid iron overloading and its négative conséquences.
As observed in thalassemia patients, skin ulcers are one of the most common cutaneous complications of sickle-cell disease. See, e.g., Keast et al. (2004) Ostomy Wound Manage., 50(10): 64-70; Trent et al. (2004) Adv Skin Wound Care, 17(8): 410-416; and J.R. Eckman (1996) Hematol Oncol Clin North Am., 10(6): 1333-1344. The underlying mechanism for ulcer formation in anémie patients has not been completely defined. However, it is believed that multiple complications oi anémia contribute to ulcer development including, for example, ischemia, decreased nitric oxide bioavailability, vascular obstruction (particularly in the case of sickle-cell anémia and thalassemia), thrombosis, high levels of circulating réticulocytes, and hypoxia. Id. As discussed above, the instant disclosure demonstrates that ActRIIB(L79D 25-131 )-Fc treatment alleviates many of these sickle-cell disease associated conditions. Accordingly, the data disclosed herein suggests that, as was observed in thalassemia patients described above, ActRIl antagonists may be used in the treatment and prévention of ulcers in patients tliat hâve sickle-cell disease.
INCORPORATION BY REFERENCE
Ail publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
While spécifie embodiments of the subject matter hâve been discussed, the above spécification is illustrative and not restrictive. Many variations will become apparent to those i
-18|419001 skilled in the art upon review ofthis spécification and the daims below. The full scope ofthe invention should be determined by reference to the daims, along with their fiill scope of équivalents, and the spécification, along with such variations,

Claims (30)

  1. We claim:
    l. An ActRIl antagonist for use in the treatment or prévention of a cutaneous ulcer in a subject that has anémia.
  2. 2.
    The ActRIl antagonist of claim 1, wherein the subject has a one or more of the diseases or conditions selected from the group consisting of: a hemolytic anémia, a hemoglobinopathy anémia, sickle-cell disease, a thalassemia syndrome, a β-thalassemia syndrome, and β-thalassemia intermedia.
  3. 3. The ActRIl antagonist of claim 1 or 2, wherein the ActRIl antagonist is an ActRIlA polypeptide.
  4. 4. The ActRIl antagonist of claim 3, wherein the ActRIIA polypeptide is selected from:
    a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 10;
    b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:H;
    c) a polypeptide comprising the amino acid sequence of SEQ ID NO:22 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:22;
    d) a polypeptide comprising the amino acid sequence of SEQ ID NO:28 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%. 90%. 95%. 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO;28; and
    e) a polypeptide comprising an amino acid sequence that is identical to amino acids 30-110 of SEQ ID NO:9 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of amino acids 30-110 of SEQ ID NO:9.
  5. 5. The ActRIl antagonist of claim 1 or 2, wherein the ActRIl antagonist is an ActRIIB polypeptide.
  6. 6. The ActRIl antagonist of claim 5, wherein the ActRIIB polypeptide is selected from:
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    a) a polypeptide comprising an amino acid sequence that is identical to amino acids
    29-109 of SEQ ID NO: 1 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of amino acids 29-109 of SEQ ID NO:1;
    b) a polypeptide comprising an amino acid sequence that is identical to amino acids 25-131 of SEQ ID NO: 1 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of amino acids 25-131 of SEQ ID NO:1;
    c) a polypeptide comprising the amino acid sequence of SEQ ID NO;2 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2;
    d) a polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a polypeptide comprising an amino acid sequence that is at least 80%. 85%. 90%. 95%.
    96%, 97%, 98%, or 99% identical to the amino acid sequence oi'SEQ ID NO:3; and i
    e) a polypeptide comprising the amino acid sequence of SEQ ID NO:29 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:29.
  7. 7. The ActRII antagonist of claim 1 or 2, wherein the ActRII antagonist is a GDF Trap polypeptide.
  8. 8. The ActRII antagonist of claim 7, wherein the GDF Trap polypeptide is selected from:
    a) a polypeptide comprising an amino acid sequence that is identical to amino acids 29-109 of SEQ ID NO:1 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of amino acids 29-109 of SEQ ID NO:1;
    b) a polypeptide comprising an amino acid sequence that is identical to amino acids 25-131 of SEQ ID NO: 1 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of amino acids 25-131 of SEQ ID NO:1;
    c) a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2;
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    d) a polypeptide comprising the amino acid sequence ofSEQ ID NO:3 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO:3;
    e) a polypeptide comprising the amino acid sequence of SEQ ID NO:36 or a polypeptide comprising an amino acid séquence that is al least 80%, 85%, 90%. 95%. 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO:36;
    f) a polypeptide comprising the amino acid sequence of SEQ ID NO:37 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:27;
    g) a polypeptide comprising the amino acid sequence ofSEQ ID NO:4l or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4l ;
    I
    h) a polypeptide comprising the amino acid sequence of SEQ ID NO:44 or a polypeptide comprising an amino acid séquence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thé amino acid sequence of SEQ ID NO:44; and
    i) a polypeptide comprising the amino acid sequence of SEQ ID NO:45 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:45; and wherein the GDF Trap polypeptide optionally comprises an acidic amino acid at position 79 with respect to SEQ ID NO: 1.
  9. 9. The ActRII antagonist of claim 8, wherein the GDF Trap polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 44.
  10. 10. The ActRII antagonist of claim 9, wherein the GDF Trap polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence ofSEQ ID NO: 44.
  11. 11. The ActRII antagonist of claim 8, wherein the GDF Trap polypeptide comprises the amino acid sequence of SEQ ID NO: 44.
  12. 12. The ActRII antagonist of any one of claims 3-8, wherein the polypeptide is a fusion protein comprising, in addition to an ActRIIA, ActRIIB, or GDF Trap polypeptide domain, one or more heterologous polypeptide domains that enhance one or more of: in vivo half-life, in vitro half-life, administration, tissue localization or distribution, formation of protein complexes, and purification.
    -18-8I
    I
  13. 13. The ActRII antagonist of claim 12, wherein the fusion protein comprises a heterologous polypeptide domain selected from: an immunoglobulin Fc domain and a sérum albumin.
  14. 14. The ActRII antagonist of claim 13, wherein the immunoglobulin Fc domain is an IgG l Fc domain and/or comprises an amino acid sequence selected from SEQ ID NO: 15 and 64.
  15. 15. The ActRII antagonist of claim 13 or 14, wherein the fusion protein further comprises a linker domain positioned between the ActRIIA, ActRIIB, or GDF Trap polypeptide domain and the immunoglobulin Fc domain.
  16. 16. The ActRII antagonist of claim 15, wherein the linker domain is a TGGG linker.
  17. 17. The ActRII antagonist of any one of daims 12-16, wherein the polypeptide is an ActRIIA-Fc fusion protein that comprises a polypeptide selected from:
    a) a polypeptide comprising the amino acid sequence of SEQ ID NO:22 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:22; and
    b) a polypeptide comprising the amino acid sequence of SEQ ID NO:28 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:28.
  18. 18. The ActRII antagonist of any one of daims 12-16, wherein the polypeptide is an ActRIIB-Fc fusion protein comprising a polypeptide that comprises the amino acid sequence of SEQ ID NO:29 or that comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:29.
    I
  19. 19. The ActRII antagonist of any one of daims 12-16, wherein the polypeptide is a GDF Trap polypeptide-Fc fusion protein that comprises a polypeptide selected from:
    a) a polypeptide comprising the amino acid sequence of SEQ ID NO:36 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:36; and
    b) a polypeptide comprising the amino acid sequence of SEQ ID NO:41 or a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:41.
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  20. 20. The ActRII antagonist of claim 19, wherein the GDF Trap polypeptide-Fc fusion protein comprises an acidic amino acid at position 79 with respect to SEQ ID NO:l.
  21. 21. The ActRII antagonist of any one of daims 3-20, wherein the polypeptide comprises one or more amino acid modifications selected from: a glycosylated amino acid, a PEGylated amino acid, a famesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a iipid moiety, and an amino acid conjugated to an organic derivatizing agent.
  22. 22. The ActRII antagonist of any one of daims 3-21, wherein the polypeptide binds to one or more proteins selected from the group consisting of GDFl l, GDF8, activin, and activin A.
  23. 23. The ActRII antagonist of daim l or 2, wherein the ActRII antagonist is:
    a) an anti-GDFI I antibody,
    b) an anti-GDF8 antibody,
    c) a multi-specific antibody that binds to at least GDFl I ;
    d) a multi-specific antibody that binds t<|> GDFl l and GDF8; or
    d) a multi-specific antibody that binds to GDFl l and any one or more of activin A, activin B, activin C, activin E, BMP7, Nodal, ActRIIA, and ActRIIB.
  24. 24. The ActRII antagonist of any one of claim l or 2, wherein the ActRII antagonist binds to any one or more of activin A, activin B, activin AB, activin C and/or activin E.
  25. 25. The ActRII antagonist of any one of claim l or 2, wherein the ActRII antagonist binds to activin A and to activin B.
  26. 26. The ActRII antagonist of any one of daims 23-25, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.
  27. 27. The ActRII antagonist of any one of clajms 23-26, wherein the antibody is a singlechain antibody, an F(ab’)2 fragment, a single chain diabody, a tandem single chain Fv fragment, a tandem single chain diabody, or a fusion protein comprising a single chain diabody and at least a portion of an immunoglobulin heavy chain constant région.
  28. 28. The ActRII antagonist of any one of daims l-27, wherein the method further comprises administering one or more supportive therapy for ulcers, anémia, sickle-cell disease, and/or a thalassemia syndrome.
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  29. 29. The ActRIl antagonist of claim 28, wherein the supportive therapy is one or more of the supportive thérapies selected from the group consîsting of: transfusion with red blood cells, administration of an iron-chelating agent or multiple iron-chelating agents, administering an EPO receptor activator, administration of hydroxyurea, and administration 5 ofhepcidin.
  30. 30. The ActRIl antagonist of any one of daims 1-25, wherein the ActRIl antagonist is administered topically.
OA1201600471 2014-06-13 2015-06-12 Methods and compositions for treating ulcers OA19001A (en)

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US62/045,808 2014-09-04

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