WO2012065059A2 - Traitement de l'athérosclérose par des antagonistes de bmp-alk3 - Google Patents

Traitement de l'athérosclérose par des antagonistes de bmp-alk3 Download PDF

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WO2012065059A2
WO2012065059A2 PCT/US2011/060365 US2011060365W WO2012065059A2 WO 2012065059 A2 WO2012065059 A2 WO 2012065059A2 US 2011060365 W US2011060365 W US 2011060365W WO 2012065059 A2 WO2012065059 A2 WO 2012065059A2
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alk3
functional variant
bmp
treatment
atherosclerosis
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WO2012065059A3 (fr
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Paul B. Yu
Matthias Derwall
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The General Hospital Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • Atherosclerosis is the leading cause of illness and death in the United States and most developed countries. It is estimated that cardiovascular disease, primarily due to coronary artery disease and stroke, affects at least 22 million individuals in the United States, and causes nearly 900,000 deaths each year.
  • Atherosclerotic lesions in afflicted individuals are marked by fatty cholesterol deposits (atheroma), inflammation, cells, scar tissue, and vessel calcification.
  • Atherosclerosis is a polygenic complex disease of mammals characterized by the deposits or plaques of lipids and other blood derivatives in the arterial walls (aorta, coronary arteries, and carotid arteries). These plaques can be calcified to a greater or lesser extent according to the progression of the process.
  • vascular calcification has been found to be strongly associated with increased risk of cardiovascular events and mortality. They are also associated with the accumulation of fatty deposits consisting mainly of cholesterol esters in the arteries.
  • Cholesterol accumulates in the foam cells of the arterial wall, thereby narrowing the lumen and decreasing the flow of blood. This is accompanied by a thickening of the arterial wall, with hypertrophy of the smooth muscle, the appearance of foam cells and the accumulation of the fibrous tissue. Atherosclerosis can therefore result in very serious cardiovascular pathologies such as infarction, peripheral vascular disease, stroke, sudden death, cardiac decompensation, cerebral vascular accidents and the like. Often times, by the time that heart problems are detected, the underlying cause, atherosclerosis, is usually quite advanced, having progressed for decades.
  • Atherosclerosis remains the largest cause of morbidity and mortality in developed countries. Thus there continues to be an unmet need for additional effective therapies targeting pathways critical for the development and progression of atherosclerosis.
  • ALK3-Fc and/or other functional variants thereof can be used to inhibit, prevent, and/or treat the process of atherosclerosis and its associated vascular endothelial dysfunction
  • ALK3-Fc and/or other functional variants thereof can be
  • an ALK3-Fc and/or other functional variants thereof can be administered to individuals receiving intravascular stents or angioplasty who have had a history of aggressive restenosis with previous stent or angioplasty procedures.
  • the invention provides for a method of preventing or treating atherosclerosis in a mammalian subject in need thereof, the method comprises administering to the subject an ALK3-Fc or functional variant thereof in a
  • a method of preventing or treating thrombosis of an atherosclerosic plaque in a mammalian subject in need thereof comprises administering to the subject an ALK3-Fc or functional variant thereof in a
  • a method of preventing or treating vascular calcification in a mammalian subject in need thereof comprises
  • composition comprising an
  • ALK3-Fc or functional variant thereof and at least one agent that is used in the treatment of cardiovascular disease in combination in a pharmaceutically acceptable carrier.
  • agents used in the treatment of cardiovascular disease include but are not limited to anti-atherosclerosis drugs, anti-thrombosis drugs, anti-inflammatory drugs, ACE Inhibitors and cholesterol lowering drugs.
  • the subject is a human subject.
  • the ALK3-Fc or functional variant thereof or composition can be administered orally, by bolus injection or by parenteral injection.
  • the atherosclerosis is associated with a disease selected from the group consisting of thrombosis, coronary heart disease, high blood pressure, metabolic syndrome, dyslipidemia, myocardial infartion, stroke, critical limb ischemia, and angina.
  • Figure 1 shows the native amino acid sequence of human ALK3 precursor (SEQ.
  • ALK3 extracellular domain (residues 24-152) is underlined.
  • Figure 2 shows the native nucleotide sequence encoding human ALK3 precursor
  • Figure 3 shows the native amino acid sequence of the extracellular domain of human ALK3 (SEQ. ID. NO: 30).
  • Figure 4 shows the native nucleotide sequence encoding the extracellular domain of human ALK3 (SEQ. ID. NO: 31).
  • Figure 5 shows the native amino acid sequence of human IgGl Fc (constant) domain (SEQ. ID. NO: 32) from the human IgGl heavy chain. This sequence corresponds to residues 223-447 of the larger human IgGl heavy chain.
  • Figure 6 shows the native nucleotide sequence encoding human IgGl Fc domain
  • Figure 7 shows the amino acid sequence of leaderless hALK3(24-152)-hFc (SEQ.
  • Figure 8 shows the full amino acid sequence of hALK3(24-152)-hFc with TPA leader (SEQ. ID. NO: 35).
  • the human ALK3 extracellular domain is underlined, and the TGGG (SEQ ID NO: 1) linker sequence is in bold.
  • Figure 9 shows a nucleotide sequence encoding hALK3(24-152)-hFc with TPA leader (SEQ ID NOS 36 and 37, respectively, in order of appearance). The sequence encoding the human ALK3 extracellular domain is underlined.
  • Figure 10 shows the full amino acid sequence of hALK3(24-152)-mFc with TPA leader (SEQ. ID. NO: 38).
  • the human ALK3 extracellular domain is underlined, and the TGGG (SEQ ID NO: 1) linker sequence is in bold.
  • Figure 11 shows a nucleotide sequence encoding hALK3(24-152)-mFc with TPA leader (SEQ ID NOS 39 and 40, respectively, in order of appearance).
  • the sequence encoding the human ALK3 extracellular domain is underlined.
  • Figure 12A shows hematoxylin and eosin stained sections of the aortic root, valve, and aortic arch showing the presence of numerous fibrofatty plaques along the minor curvature of the aortic root and aortic arch in mutant (LDLr-/-) mice prone to
  • hypercholesterolemia a mouse model of atherosclerosis and athero -calcific vascular disease. These mice were started on a high fat (Paigen) diet at 8 weeks of life, and continued on this diet for 16 weeks to permit the development of atheromatous lesions and vascular calcification.
  • Figure 12B shows Von Kossa stained sections of the aortic root, valve, and aortic arch showing intense calcification of the media of the minor curvature of the aortic arch in the mutant (LDLr-/-) mice used in Fig. 12A.
  • Figure 13 shows quantitatively that a BMP inhibitor positive control compound can reduce vascular calcification and vascular inflammation in atherogenic mice.
  • Figure 14 shows that macrophage-mediated inflammation is quantitatively decreased in the central arterial vascular bed of atherogenic animals by recombinant or small- molecule BMP inhibitors.
  • Figure 15A shows an Alizarin stained section of the aorta of a 28 day old wild- type mouse.
  • Figure 15B shows an Alizarin stained section of the aorta of a 28 day old MGP-/- mouse.
  • Figure 15C shows an Alizarin stained section of the aorta of a 28 day old MGP-/- mouse treated with a BMP inhibitor positive control compound (LDN); the aorta has less calcification compared to the aorta of the MGP-/- mouse shown in Fig. 15B.
  • LDN BMP inhibitor positive control compound
  • Figure 15D shows an Alizarin stained section of the aorta of a 28 day old MGP-/- mouse treated with an ALK3-Fc polypeptide; the aorta has less calcification compared to the aorta of the MGP-/- mouse shown in Fig. 15B.
  • Figure 16 shows the reduction of arterial calcification, as determined by osteosense fluorescence, in the aorta of MGP-/- mice treated with a BMP inhibitor positive control compound (LDN) or with an ALK3-Fc polypeptide.
  • LDN BMP inhibitor positive control compound
  • Embodiments of the present invention are based on the discovery that ALK3-Fc inhibited activation of the BMP responsive SMAD pathway, which is required for the development of early atheromatous lesions in a hypercholesterolemic small animal model of atherosclerosis.
  • administration of ALK3-Fc inhibited the development of vascular calcification which complicates atherosclerosis as atherosclerosis progresses.
  • ALK3 antagonist can be used to inhibit, prevent, and/or treat the process of atherosclerosis and its associated vascular calcification.
  • ALK3 antagonists can be BMP-ALK3 and/or other functional variants.
  • BMP-ALK3 includes but is not limited to an ALK3-Fc and/or other functional variants thereof.
  • the ALK3-Fc and/or other functional variants thereof function as decoy receptors in binding BMP ligands, thus preventing the BMP ligand from binding the real receptor and activiate the BMP responsive SMAD pathway.
  • ALK3-Fc and/or other functional variants thereof can be administered to individuals with severe atherosclerosis that is progressive despite conventional medical therapies.
  • ALK3-Fc and/or other functional variants thereof can be administered to individuals receiving intravascular stents or angioplasty who have had a history of aggressive restenosis with previous stent or angioplasty procedures.
  • a method of preventing or treating atherosclerosis in a mammalian subject in need thereof comprises administering to the subject an ALK3-Fc or functional variant thereof in a therapeutically effective amount.
  • the therapeutically effective amount of ALK3-Fc and/or functional variant thereof reduced, inhibited, slow down the development of atherosclerotic lesions in the subject.
  • a method of preventing or treating thrombosis of an atherosclerosic plaque in a mammalian subject in need thereof comprises administering to the subject an ALK3-Fc and/or functional variant thereof in a therapeutically effective amount.
  • a method of preventing or treating vascular calcification in a mammalian subject in need thereof comprises administering to the subject an ALK3-Fc and/or functional variant thereof in a therapeutically effective amount.
  • ALK3-Fc and/or other functional variants thereof for prevention or treatment of atherosclerosis.
  • ALK3-Fc and/or other functional variants thereof for prevention or treatment of thrombosis of an atherosclerosic plaque.
  • ALK3-Fc and/or other functional variants thereof for the prevention or treatment of vascular calcification.
  • ALK3-Fc and/or other functional variants thereof for the manufacture of a medicament for the prevention or treatment of atherosclerosis.
  • ALK3-Fc and/or other functional variants thereof for the manufacture of a medicament for the prevention or treatment of thrombosis of an atherosclerosic plaque.
  • ALK3-Fc and/or other functional variants thereof for the manufacture of a medicament for the prevention or treatment of vascular calcification.
  • composition comprising an
  • ALK3-Fc and/or functional variant thereof and at least one agent that is used in the treatment of cardiovascular disease in combination in a pharmaceutically acceptable carrier.
  • Common agent that is used in the treatment of cardiovascular disease include but is not limited to anti- atherosclerosis drugs, anti-thrombosis drugs, anti-inflammatory drugs, angiotensin-converting enzyme (ACE) inhibitors and cholesterol lowering drugs.
  • ACE angiotensin-converting enzyme
  • ALK3-Fc and/or functional variant thereof can be concurrently administering the subject with at least one agent that is used in the treatment of cardiovascular disease, such as anti-atherosclerosis drugs, anti-thrombosis drugs, anti-inflammatory drugs, ACE inhibitors and cholesterol lowering drugs.
  • at least one agent that is used in the treatment of cardiovascular disease such as anti-atherosclerosis drugs, anti-thrombosis drugs, anti-inflammatory drugs, ACE inhibitors and cholesterol lowering drugs.
  • the medicament comprises at least one agent that is used in the treatment of cardiovascular disease, such as anti-atherosclerosis drugs, anti-thrombosis drugs, beta-blockers, anti-inflammatory drugs, ACE inhibitors and cholesterol lowering drugs.
  • the subject is a human subject.
  • ALK3-Fc and/or functional variant thereof or composition can be administered orally, by bolus injection or by parenteral injection.
  • the composition or medicament is formulated for oral, bolus or parenteral delivery.
  • the atherosclerosis is associated with a disease is selected from the group consisting of thrombosis, coronary heart disease, high blood pressure, metabolic syndrome, dyslipidemia, myocardial infartion, stroke, critical limb ischemia, and angina.
  • the method further comprises selecting a subject who has atherosclerosis, cardiovascular disease, vascular calcification and/or thrombosis of an atherosclerosic plaque.
  • the method further comprises selecting a subject who has thrombosis, coronary heart disease, high blood pressure, metabolic syndrome, dyslipidemia, myocardial infartion, stroke, critical limb ischemia, and/or angina.
  • the method further comprises selecting a subject who has hypertension, high cholesterol and/or is obese.
  • Atherosclerosis is the most common form of vascular disease and is a disorder of large arteries that underlies most coronary artery disease, aortic aneurysm, cerebrovascular disease and arterial disease of lower extremities (Libby, in "The Principles of Internal
  • the pathogenesis of atherosclerosis occurs as a reaction to injury (Libby, in "The Principles of Internal Medicine”, 15th ed., Braunward et al. (editors), Saunders, Philadelphia, Pa., 2001, pp. 1377-1382.).
  • the injury to the endothelium may be subtle, resulting in a loss of the ability of the cells to function normally. Examples of types of injury to the endothelium include hypercholesterolemia and mechanical stress (Ross, 1999, N. Engl. J. Med., 340:115).
  • Atherosclerosis can be considered as a form of chronic inflammation occurring within the artery wall.
  • a number of data support the pathogenic or pathogenetic role of inflammation in the etiology of atherosclerosis.
  • fatty streaks the earliest detectable lesions in atherosclerosis, contain macrophage-derived foamy cells which are derived from circulating monocytes.
  • T lymphocytes are also present in the atherosclerotic plaques and secrete IFN gamma, IL-2, TNF alpha and beta which cause vascular macrophage activation, vascular activation and inflammation.
  • IFN gamma is thought to be responsible for plaque destabilization by reducing the fibrous cap.
  • an atheromatous plaque includes nodular accumulation of a soft, flaky, yellowish material at the center of large plaques, composed of macrophages nearest the lumen of the artery, sometimes with underlying areas of cholesterol crystals, and may also include calcification at the outer base of older/more advanced lesions.
  • the atheromatous plaques though compensated for by artery enlargement, can eventually lead to plaque ruptures and stenosis (i.e., narrowing) of the artery and, therefore, an insufficient blood supply to the organ it feeds.
  • the compensating artery enlargement process is excessive, then a net aneurysm results.
  • the complications associated with atherosclerosis are chronic, slowly progressing and cumulative.
  • a common endpoint of coronary thrombosis of a coronary artery is a myocardial infarction (i.e., a heart attack).
  • Atherosclerosis begins when monocytes are activated and move out of the bloodstream into the wall of an artery. There, they are transformed into foam cells, which collect cholesterol and other fatty materials. In time, these fat-laden foam cells accumulate and form atheromas in the lining of the artery's wall, causing a thickening and hardening of the wall. Atheromas may be scattered throughout medium-sized and large arteries, but usually form where the arteries branch presumably because the constant turbulent blood flow at these areas injures the artery's wall, making these areas more susceptible to atheroma formation.
  • white blood cells e.g., lymphocytes, monocytes, and macrophages
  • Atherosclerotic vascular lesions are marked early in their development by the presence of fatty, cholesterol streaks (atheroma) which typically affect high-flow areas of the vascular tree. Over time, these fatty streaks progress or remodel to include inflammatory cells, scar tissue, and eventually calcified tissue.
  • Vascular calcification is thought to be a marker of significant atherosclerosis, and has been associated in multiple large cohorts with an increased risk for myocardial infarction, stroke, and mortality. Vascular calcification is thought to impair vesse function due to the loss of loss of distensibility and lead to compensatory enlargement as a result of damaged vessel integrity.
  • Calcific vascular lesions consist predominantly of hydroxyapatite mineral and have an architecture and cellular make-up which is essentially identical to marrow-filled bone tissue, including the presence of osteoblast- and chondroblast- like cells.
  • the influence of calcification on biomechanics of the vascular wall may promote endothelial injury and plaque rupture through reducing elasticity and increasing shear stresses.
  • Atherosclerotic vascular lesions are also associated with the accumulation of fatty deposits consisting mainly of cholesterol esters in the arteries. Cholesterol accumulates in the foam cells of the arterial wall, thereby narrowing the lumen and decreasing the flow of blood.
  • Hypercholesterolemia can therefore result in very serious cardiovascular pathologies such as infarction, peripheral vascular disease, stroke, sudden death, cardiac decompensation, cerebral vascular accidents and the like.
  • the cholesterol is carried in the blood by various lipoproteins including the low- density lipoproteins (LDL) and the high-density lipoproteins (HDL).
  • LDL low- density lipoproteins
  • HDL high-density lipoproteins
  • the LDL is synthesized in the liver and makes it possible to supply the peripheral tissues with cholesterol.
  • the HDL captures cholesterol molecules from the peripheral tissues and transports them to the liver where they are converted to bile acids and excreted.
  • the development of atherosclerosis and the risk of coronary heart disease (CHD) inversely correlate to the levels of HDL in the serum.
  • CHDD coronary heart disease
  • statins inhibitors of the cholesterols biosynthesis enzyme 3-hydroxyl-3-methylglutary Coenzyme A reductase and thereby this treatment has been used as a successful approach for reducing the risk for atherosclerosis where the primary indication is high LDL level.
  • statins are beneficial for patients whose primary lipid abnormality is low HDL cholesterol.
  • Acute coronary syndromes can be caused by acute destabilization of
  • Atherosclerotic plaques e.g., plaque rupture
  • Acute myocardial ischemia is chest pain due to insufficient blood supply to the heart muscle that results from coronary artery disease (also called coronary heart disease).
  • Patients who have symptoms of acute coronary syndrome may or may not exhibit an ST elevation (also referred to as an ST displacement) by electrocardiogram (ECG or EKG), which is diagnostic of damage to the cardiac muscle or strain on the ventricles.
  • ECG electrocardiogram
  • Most patients who exhibit ST-segment elevation in an ECG ultimately develop a Q-wave acute myocardial infarction (i.e., heart attack).
  • Patients who have ischemic discomfort without ST-segment elevation are generally diagnosed as having unstable angina or a non-ST-segment elevation myocardial infarction (the latter of which can lead to a non-Q-wave myocardial infarction).
  • Acute coronary syndrome thus encompasses the spectrum of clinical conditions ranging from unstable angina to non-Q-wave myocardial infarction and Q-wave myocardial infarction.
  • Atheromatous plaque refers to a condition characterized by the hardening and/or narrowing of the arteries caused by the buildup of athermatous plaque inside the arterial walls.
  • the atheromatous plaque is divided in three components, (1) the atheroma, a nodular accumulation of a soft flaky material at the center of large plaques, composed of macrophages nearest the lumen of the artery; (2) underlying areas of cholesterol crystals; (3) calcification at the outer base of more advanced lesions.
  • Indicators of atherosclerosis include, for example, the development of plaques in the arteries, their calcification, the extent of which can be determined by Sudan IV staining, or the development of foam cells in arteries.
  • the narrowing of the arteries can be determined by coronary angioplasty, ultrafast CT, or ultrasound.
  • Thrombosis and "thrombosis-related disorder” refer to abnormal thrombus formation that causes obstruction of blood vessels and conditions associated with such obstruction. Blood vessels operate under significant shear stresses that are a function of blood flow shear rate. Frequently, there is damage to small blood vessels and capillaries. When these vessels are damaged, hemostasis is triggered to stop the bleeding. Under typical circumstances, such an injury is dealt with through a sequence of events commonly referred to as the "thrombus formation". Thrombus formation is dependent upon platelet adhesion, activation and
  • HDL refers to the high-density lipoproteins.
  • LDL low-density lipoproteins
  • VLDL refers to the very low density lipoproteins.
  • treatment or “treating” includes the administration, to a subject in need, of an amount of an ALK3 antagonist, ALK3-Fc and/or functional variant thereof which will inhibit, decrease or reverse development of, for example, a pathological atherosclerosis, vascular calcification, inflammatory, or thrombosis-related condition as disclosed herein without limitation.
  • Inhibiting in connection with inhibiting atherosclerosis, is intended to mean retarding, stabilizing, or reversing formation or growth of atheromatous plaques, vascular calcification, inflammatory condition, or thrombosis-related indication.
  • Treatment of diseases and disorders herein is intended to also include therapeutic administration of an ALK3 antagonist, ALK3-Fc and/or functional variant thereof (or a pharmaceutical salt, derivative or prodrug thereof) or a pharmaceutical composition containing the ALK3-Fc or functional variant thereof to a subject (i.e., an animal, for example a mammal, such as a human) believed to be in need of treatment for diseases and disorders, such as, for example, inflammation, thrombosis, coronary heart disease, high blood pressure, myocardial infarction, stroke, critical limb ischemia, angina and the like.
  • a subject i.e., an animal, for example a mammal, such as a human
  • diseases and disorders such as, for example, inflammation, thrombosis, coronary heart disease, high blood pressure, myocardial infarction, stroke, critical limb ischemia, angina and the like.
  • the term “prevent” or “prevention” refers to stopping, hindering, and/or slowing down the onset of developing adverse effects and symptoms associated with medical conditions that are associated with atherosclerosis and vascular calcification.
  • the subject is initially diagnosed as at risk by a licensed physician and/or authorized medical practitioner, and a regimen for prophylactic and/or therapeutic treatment via administration of an ALK3 antagonist, ALK3-Fc and/or functional variant thereof is suggested, recommended or prescribed.
  • the phrase "therapeutically effective amount” is the amount of an ALK3 antagonist, ALK3-Fc and/or functional variant thereof that will achieve the goal of improvement in disorder severity and the frequency of incidence.
  • the improvement in disorder severity includes, for example, prevention or inhibition of vascular calcification, and/or accumulation of cholesterol in vessel walls, the reversal of atherosclerosis, as well as slowing down the progression of atherosclerosis, prevention or treatment of inflammatory disorders, and prevention or treatment of thrombosis-relating conditions.
  • the term "subject” is intended to mean a human or other mammal, exhibiting, or at risk of developing, atherosclerosis, an inflammatory condition or thrombosis. Such an individual can have, or be at risk of developing, for example,
  • Atherosclerosis associated with conditions such as thrombosis, coronary heart disease, high blood pressure, myocardial infarction, stroke, critical limb ischemia, angina, peripheral artery disease and the like.
  • conditions such as thrombosis, coronary heart disease, high blood pressure, myocardial infarction, stroke, critical limb ischemia, angina, peripheral artery disease and the like.
  • the prognostic and clinical indications of these conditions are known in the art.
  • Subjects suitable for treatment according to the methods described herein include those who a medical practitioner has diagnosed as having one or more symptoms of
  • Atherosclerosis and particularly those patients who have had or are at risk of an atherosclerotic disease event. Diagnosis may be done by any suitable means known in the art. Methods for diagnosing atherosclerosis are well known in the art, e. g., by measuring systemic atherosclerotic or inflammatory markers such as C-reactive protein, homocysteine, fibrinogen, lipoprotein (a), IL-6, IL-8, IL-17, or those described, for example, in U.S. Pat. No. 6,040,147, which is herein incorporated by reference.
  • systemic atherosclerotic or inflammatory markers such as C-reactive protein, homocysteine, fibrinogen, lipoprotein (a), IL-6, IL-8, IL-17, or those described, for example, in U.S. Pat. No. 6,040,147, which is herein incorporated by reference.
  • Diagnosis and monitoring can also employ an electrocardiogram, chest X-ray, cardiac catheterization, ultrasound (for the measurement of vessel wall thickness), or measurement of blood levels of CPK, CPK-MB, myoglobin, troponin, homocysteine, or C- reactive protein.
  • a subject is diagnosed using computed tomography to detect calcium in the coronary arteries, which is an indicator of plaque progression.
  • a patient at risk of development of an atherosclerotic disease event may have been subjected to the same tests (electrocardiogram, chest X-ray, etc.) or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g., family history, hypertension, diabetes mellitus, high cholesterol levels, smoking, obesity, etc.).
  • risk factors e.g., family history, hypertension, diabetes mellitus, high cholesterol levels, smoking, obesity, etc.
  • Individuals in risk populations can be monitored more rigorously and treatment of atherosclerosis can be started as soon as alterations in the markers or other physiological symptoms can be detected.
  • Atherosclerosis does not produce symptoms until it narrows the interior of an artery by more than 70%. Symptoms depend on location of the narrowing or blockage, which can occur almost anywhere in the body. Symptoms occur because as an artery is narrowed, the tissues supplied by the artery may not receive enough blood and oxygen. The first symptom of a narrowing artery may be pain or cramps at times when blood flow cannot keep up with the tissues' need for oxygen. Typically, symptoms develop gradually as the atheroma slowly narrows an artery. However, sometimes the first symptoms occur suddenly because the blockage occurs suddenly— for example, when a blood clot lodges in an artery narrowed by an atheroma, causing a heart attack or stroke.
  • An atherosclerotic complication or disorder as described herein may include atherosclerosis, ischemic (coronary) heart disease: myocardial ischemia (angina), myocardial infarction; aneurismal disease; atheromatous peripheral vascular disease: aortoiliac disease, chronic and critical lower limb ischemia, visceral ischemia, renal artery disease, cerebrovascular disease, stroke, atherosclerotic retinopathy, thrombosis and aberrant blood clotting and hypertension.
  • Such conditions may be medical or veterinary conditions, and can be treated using the methods and compositions described herein if associated with atherosclerosis.
  • the BMP signaling pathway is known to regulate the function of many cell populations in the arterial vasculature. Endothelial cells transduce signals from a wide variety of BMP ligands. Certain of the BMP ligands, such as BMP9, appear to regulate homeostasis and survival in endothelial cells. Other BMP ligands, such as BMP6, appear to induce the activation and migration of endothelial cells. Vascular smooth muscle cells and vascular pericytes can be induced to transdifferentiate into osteoblast-like, and chondroblast-like cells by BMP2, BMP4, or BMP6 signals in vitro, suggesting that BMP ligands regulate the differentiation of these multipotent cells to for vascular calcific lesions. BMP ligands such as BMP2 are found to be overexpressed in the calcified atherosclerotic lesions from diseased human carotd arteries.
  • BMP ligands regulate the function of monocyte and macrophage populations, perhaps regulating their inflammatory potential or activation.
  • ALK3 is a receptor for members of the transforming growth factor-beta (TGF- beta)/bone morphogenetic protein (BMP) superfamily.
  • TGF-beta/BMP superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates.
  • Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation.
  • TGF- ⁇ signals are mediated by heteromeric complexes of type I and type II serine/ threonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon ligand stimulation (Massague, 2000, Nat. Rev. Mol. Cell Biol. 1:169-178).
  • type I and type II receptors are transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specificity.
  • Type I receptors are essential for signaling; and type II receptors are required for binding ligands and for expression of type I receptors.
  • Type I and II activin receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type II receptors.
  • Activin receptor-like kinase-3 is a type I receptor mediating effects of multiple ligands in the BMP family and is also known as bone morphogenetic protein receptor, type IA (BMPRIA), or activin A receptor, type Il-like kinase (ACVRLK). Unlike several type I receptors with ubiquitous tissue expression, ALK3 displays a restricted pattern of expression consistent with more specialized functionality (ten Dijke, 1993, Oncogene 8:2879-2887). ALK3 is generally recognized as a high affinity receptor for BMP2, BMP4, BMP7 and other members of the BMP family.
  • BMP2 and BMP7 are potent stimulators of osteoblastic differentiation, and are now used clinically to induce bone formation in spine fusions and certain non-union fractures.
  • ALK3 is regarded as a key receptor in mediating BMP2 and BMP4 signaling in osteoblasts (Lavery et al., 2008, J. Biol. Chem. 283:20948-20958).
  • a homozygous ALK3 knockout mouse dies early in embryogenesis (day 9.5).
  • ALK3-Fc is a soluble extracelluar domain of the bone morphogenetic protein
  • BMP immunoglobulin protein type I receptor ALK3, fused to the constant region of the immunoglobulin protein (Fc).
  • ALK3-Fc can to block a variety of ligands which interact with BMP type I receptors, including ALK1, ALK2, ALK3, and ALK6. While not wishing to be bound to any particular mechanism, it is expected that the effect of ALK3 is caused primarily by a BMP antagonist effect, given the very strong BMP2 and BMP4 binding (picomolar dissociation constant) exhibited by the particular soluble ALK3 construct used in these studies.
  • BMP-ALK3 antagonists include, for example, BMP-binding soluble ALK3 polypeptides, antibodies that bind to BMP and disrupt ALK3 binding, antibodies that bind to ALK3 and disrupt BMP binding, non-antibody proteins selected for BMP or ALK3 binding (see e.g., WO/2002/088171, WO/2006/055689,
  • WO/2002/032925 WO/2005/037989, US 2003/0133939, and US 2005/0238646 for examples of such proteins and methods for design and selection of same, the contents of these patent publications are incorporated herein by reference in their entirety
  • randomized peptides selected for BMP or ALK3 binding often affixed to an Fc domain.
  • Two different proteins (or other moieties) with BMP or ALK3 binding activity, especially BMP binders that block the type I (e.g., a soluble type I activin receptor) and type II (e.g., a soluble type II activin receptor) binding sites, respectively, may be linked together to create a bifunctional binding molecule.
  • Nucleic acid aptamers, small molecules and other agents that inhibit the BMP-ALK3 signaling axis are also contemplated. Additionally, nucleic acids, such as antisense molecules, siRNAs or ribozymes that inhibit BMPs, or, particularly, ALK3 expression, can be used as BMP-ALK3 antagonists.
  • Absolute and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values.
  • “approximately” may mean values that are within an order of magnitude, preferably within 5- fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
  • Embodiments of the methods described herein can include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants).
  • Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
  • sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
  • sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
  • homologous in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evolutionary origin,” including proteins from superfamilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.
  • sequence similarity in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.
  • homologous when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
  • the present invention relates to the use of ALK3 polypeptides, preferebaly soluble ALK3 polypeptides that can antagonize the BMP signaling pathway.
  • ALK3 refers to a family of activin receptor-like kinase-3 (ALK3) [also referred to as bone morphogenetic protein receptor, type IA (BMPR1A), or activin A receptor, type Il-like kinase (ACVRLK)] proteins from any species and variants derived from such ALK3 proteins by mutagenesis or other modification.
  • ALK3 herein is understood to be a reference to any one of the currently identified forms.
  • ALK3 family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
  • ALK3 polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ALK3 family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
  • ALK3 polypeptides include polypeptides derived from the sequence of any known ALK3 having a sequence at least about 80% identical to the sequence of an ALK3 polypeptide, and preferably at least 85%, 90%, 95%, 97%, 99% or greater identity.
  • an ALK3 polypeptide, ALK3-Fc and/or functional variant thereof can bind to and inhibit the function of an ALK3 protein and/or BMPs.
  • an ALK3 polypeptide antagonizes the BMP signaling pathway inhibits the formation of atheromatous lesions.
  • ALK3 polypeptides include human ALK3 precursor polypeptide (SEQ. ID. NO: 1) in Fig. 1 and soluble human ALK3 polypeptide (SEQ. ID. NO: 3) in Fig. 3 and in Example 2.
  • Figure 1 and the nucleic acid sequence encoding human ALK3 precursor protein are nucleotides 549-2144 of GENBANKTM entry NM_004329 (SEQ. ID. NO: 2) is shown in Figure 2.
  • the human ALK3 soluble (extracellular), processed polypeptide sequence (SEQ. ID. NO: 3) is shown in Figure 3, and the nucleic acid sequence encoding the human ALK3 extracellular domain are nucleotides 618-1004 of GENBANKTM entry NM_004329) (SEQ. ID. NO: 4) is shown in Figure 4.
  • the invention relates to the use of soluble ALK3 polypeptides.
  • soluble ALK3 polypeptide generally refers to polypeptides comprising an extracellular domain of an ALK3 protein.
  • soluble ALK3 polypeptide includes any naturally occurring extracellular domain of an ALK3 protein as well as any variants thereof (including mutants, fragments and peptidomimetic forms).
  • a BMP-binding ALK3 polypeptide is one that retains the ability to bind to BMPs, particularly BMP2 and BMP4.
  • a functional variant form of an ALK3 polypeptide is one that retains the ability to bind to BMPs, particularly BMP2 and BMP4.
  • a functional variant form of an ALK3 polypeptide can be a soluble protein, e. g., the ectodomain of ALK3 and is therefore not membrane bound.
  • a BMP-binding ALK3 polypeptide will bind to BMP with a dissociation constant of 1 nM or less.
  • the amino acid sequence of human ALK3 precursor protein is provided in Figure 1.
  • the extracellular domain of an ALK3 protein binds to BMP and is generally soluble, and thus can be termed a soluble, BMP-binding ALK3 polypeptide.
  • Methods for determining binding between a BMP and a functional variant form of an ALK3 polypeptide can be performed by any receptor-ligand binding assay known in the art.
  • Examples of soluble, BMP-binding ALK3 polypeptides include the soluble polypeptide illustrated in Figs. 3, 7, 8, 10 and in the Examples.
  • Functionally active fragments of ALK3 polypeptides can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an ALK3 polypeptide.
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function as antagonists (inhibitors) of ALK3 protein or signaling mediated by BMPs.
  • Functionally active variants of ALK3 polypeptides can be obtained by screening libraries of modified polypeptides recombinantly produced from the corresponding mutagenized nucleic acids encoding an ALK3 polypeptide. The variants can be produced and tested to identify those that can function as antagonists (inhibitors) of ALK3 protein or signaling mediated by BMPs.
  • a functional variant of the ALK3 polypeptides comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from Figs. 3, 7, 8, 10 and in the Examples.
  • the functional variant has an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from Figs. 3, 7, 8, 10 and in the Examples.
  • Functional variants can be generated by modifying the structure of an ALK3 polypeptide for such purposes as enhancing therapeutic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo).
  • modified ALK3 polypeptides when selected to retain BMP binding, are considered functional equivalents of the naturally-occurring ALK3 polypeptides.
  • Modified ALK3 polypeptides can also be produced, for instance, by amino acid substitution, deletion, or addition.
  • the present invention contemplates the use of specific mutations of the ALK3 polypeptides so as to alter the glycosylation of the polypeptide.
  • Such mutations can be selected so as to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites.
  • Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine (or asparagines-X-serine) (where "X" is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes.
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the wild-type ALK3 polypeptide (for O-linked glycosylation sites).
  • a variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence.
  • Another means of increasing the number of carbohydrate moieties on an ALK3 polypeptide is by chemical or enzymatic coupling of glycosides to the ALK3 polypeptide.
  • the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of
  • Removal of one or more carbohydrate moieties present on an ALK3 polypeptide may be accomplished chemically and/or enzymatically.
  • Chemical deglycosylation may involve, for example, exposure of the ALK3 polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact.
  • Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal. Biochem. 118:131.
  • Enzymatic cleavage of carbohydrate moieties on ALK3 polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350.
  • the sequence of an ALK3 polypeptide can be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide.
  • ALK3 proteins for use in humans will be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes and insect cells are expected to be useful as well.
  • This disclosure further contemplates a method of generating mutants, particularly sets of combinatorial mutants of an ALK3 polypeptide, as well as truncation mutants; pools of combinatorial mutants are especially useful for identifying functional variant sequences.
  • the purpose of screening such combinatorial libraries may be to generate, for example, ALK3 polypeptide variants which can act as either agonists or antagonist, or alternatively, which possess novel activities altogether.
  • a variety of screening assays are provided below, and such assays may be used to evaluate variants.
  • an ALK3 polypeptide variant may be screened for ability to bind to an ALK3 ligand, to prevent binding of an ALK3 ligand to an ALK3 polypeptide or to interfere with signaling caused by an ALK3 ligand.
  • ALK3 polypeptide or its variants can also be tested in a cell- based or in vivo assay. For example, the effect of an ALK3 polypeptide variant on the phosphorylation of SMAD 1/5/8 as described in the Examples. It is expected that a ALK3 polypeptide or its functional variants thereof will inhibit the activation of SMAD 1/5/8 in the presence of a BMP ligand.
  • Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to a naturally occurring ALK3 polypeptide.
  • mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding a wild-type ALK3 polypeptide.
  • the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction of, or otherwise inactivation of a native ALK3 polypeptide.
  • Such variants, and the genes which encode them can be utilized to alter ALK3 polypeptide levels by modulating the half-life of the ALK3 polypeptides.
  • a short half-life can give rise to more transient biological effects and can allow tighter control of recombinant ALK3 polypeptide levels within the patient.
  • 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 can be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential ALK3 polypeptide sequences.
  • a mixture of synthetic oligonucleotides can be
  • ALK3 polypeptide nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • the library of potential homologs can be generated from a degenerate oligonucleotide sequence.
  • Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate vector for expression.
  • the synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al., (1984) Annu.
  • ALK3 polypeptide variants can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ALK3 polypeptides.
  • the most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • Preferred assays include BMP binding assays and BMP-mediated cell signaling assays.
  • the ALK3 polypeptides for use can further comprise post-translational modifications in addition to any that are naturally present in the ALK3 polypeptides.
  • modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • the modified ALK3 polypeptides may contain non-amino acid elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. Effects of such non-amino acid elements on the functionality of an ALK3 polypeptide may be tested as described herein for other ALK3 polypeptide variants.
  • an ALK3 polypeptide When an ALK3 polypeptide is produced in cells by cleaving a nascent form of the ALK3 polypeptide, post-translational processing may also be important for correct folding and/or function of the protein.
  • Different cells such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293 have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the ALK3 polypeptides.
  • polypeptides for use herein include fusion proteins having at least a portion of the ALK3 polypeptides and one or more fusion domains.
  • fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin.
  • GST glutathione S transferase
  • Fc immunoglobulin heavy chain constant region
  • MBP maltose binding protein
  • human serum albumin human serum albumin.
  • a fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography.
  • matrices for affinity chromatography such as glutathione-, amylase-, and nickel- or cobalt- conjugated resins are used.
  • Many of such matrices are available in "kit” form, such as the Pharmacia GST purification system and the QIAexpressTM system (QIAGEN®) useful with (HIS6) (SEQ ID NO: 2) fusion partners.
  • a fusion domain may be selected so as to facilitate detection of the ALK3 polypeptides. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as "epitope tags," which are usually short peptide sequences for which a specific antibody is available.
  • fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation.
  • an ALK3 polypeptide is fused with a domain that stabilizes the ALK3 polypeptide in vivo (a "stabilizer" domain).
  • stabilizing is meant anything that increases serum half life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains.
  • the present disclosure provides for the use of a fusion protein comprising a soluble extracellular domain of ALK3 fused to an Fc domain (e.g., Figure 5).
  • Fc domains are shown below:
  • the Fc domain has one or more mutations at residues such as Asp-
  • the mutant Fc domain having one or more of these mutations has reduced ability of binding to the Fey receptor relative to a wildtype Fc domain.
  • the numbering of the residues for the Fc domain is based on the amino acid nomenclature according to Kabat et al., "Sequences of Proteins of Immunological Interest", 5th ed., National Institutes of Health, Bethesda, Md. (1991), the contents of which are incorporated herein by reference in its entirety.
  • mutant Fc domain having one or more of these mutations has increased ability of binding to the MHC class I-related Fc-receptor (FcRN) compared to a wildtype Fc domain.
  • FcRN MHC class I-related Fc-receptor
  • an ALK3 polypeptide may be placed C-terminal to a heterologous domain, or, alternatively, a heterologous domain may be placed C-terminal to an ALK3 polypeptide.
  • the ALK3 polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains.
  • the ALK3 polypeptides for use with the methods disclosed herein contain one or more modifications that are capable of stabilizing the ALK3 polypeptides.
  • modifications enhance the in vitro half life of the ALK3 polypeptides, enhance circulatory half life of the ALK3 polypeptides or reduce proteolytic degradation of the ALK3 polypeptides.
  • Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ALK3 polypeptide and a stabilizer domain), modifications of a glycosylation site (including, for example, addition of a glycosylation site to an ALK3 polypeptide), and modifications of carbohydrate moiety (including, for example, removal of carbohydrate moieties from an ALK3 polypeptide).
  • fusion proteins including, for example, fusion proteins comprising an ALK3 polypeptide and a stabilizer domain
  • modifications of a glycosylation site including, for example, addition of a glycosylation site to an ALK3 polypeptide
  • modifications of carbohydrate moiety including, for example, removal of carbohydrate moieties from an ALK3 polypeptide.
  • an ALK3 polypeptide is fused to a stabilizer domain such as an IgG molecule (e.g., an Fc domain).
  • stabilizer domain not only refers to a fusion domain (e.g., Fc) as in the case of fusion proteins, but also includes nonproteinaceous modifications such as a carbohydrate moiety, or nonproteinaceous polymer, such as polyethylene glycol.
  • the present invention provides for the use of isolated and/or purified forms of the ALK3 polypeptides, which are isolated from, or otherwise substantially free of, other proteins.
  • ALK3 polypeptides will generally be produced by expression from recombinant nucleic acids.
  • the invention provides uses for isolated and/or recombinant nucleic acids encoding any of the ALK3 polypeptides (e.g., soluble ALK3 polypeptides), including fragments, functional variants and fusion proteins disclosed herein.
  • ALK3 polypeptides e.g., soluble ALK3 polypeptides
  • Fig. 2 (SEQ. ID. NO: 2) encodes the naturally occurring human ALK3 precursor polypeptide
  • Fig. 4 SEQ. ID. NO: 4
  • the subject nucleic acids can be single-stranded or double stranded.
  • Such nucleic acids can be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for making ALK3 polypeptides or as direct therapeutic agents (e.g., in a gene therapy approach).
  • the subject nucleic acids encoding ALK3 polypeptides are further understood to include nucleic acids that are variants of Fig. 2 or 4.
  • Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants.
  • ALK3 polypeptides may be encoded by isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ. ID. NO: 2 or 4, and the Examples.
  • nucleic acid sequences complementary to SEQ. ID. NOS: 2, 4, 17, 20, 23, 28 and 33 and variants thereof are also within the scope of this invention.
  • the nucleic acid sequences of the ALK3 antagonist, ALK3-Fc and/or functional variant thereof can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library.
  • nucleic acids include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ. ID. NOS: 2, 4, 17, 20, 23, 28 and 33 and complement sequences thereof, or fragments thereof.
  • SEQ. ID. NOS: 2, 4, 17, 20, 23, 28 and 33 and complement sequences thereof, or fragments thereof.
  • One skilled in the art would be able to generate the coding nucleic acid sequences using conventional molecular biology methods.
  • appropriate stringency conditions which promote DNA hybridization can be varied.
  • appropriate stringency conditions which promote DNA hybridization can be varied.
  • 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.
  • the salt 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.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Both temperature and salt can be varied, or temperature or salt concentration may be held constant while the other variable is changed.
  • nucleic acids which hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature.
  • ID. NO: 2, 4, 17, 20, 23, 28 and 33 due to degeneracy in the genetic code are also useful.
  • a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the protein.
  • CAU and CAC are synonyms for histidine
  • nucleotides up to about 3-5% of the nucleotides
  • nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation.
  • the recombinant nucleic acids may be operably linked to one or more regulatory nucleotide sequences in an expression construct.
  • Regulatory nucleotide sequences will generally be appropriate to the host cell used for expression.
  • suitable regulatory sequences are known in the art for a variety of host cells.
  • said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the invention.
  • the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
  • An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.
  • Nucleic acid may be used in an expression vector comprising a nucleotide sequence encoding an ALK3 polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of the ALK3 polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding an ALK3 polypeptide.
  • Such useful expression control sequences include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
  • a recombinant nucleic acid can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both.
  • Expression vehicles for production of a recombinant ALK3 polypeptide include plasmids and other vectors.
  • suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • bacterial plasmids such as pBR322
  • derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein- Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papilloma virus
  • pHEBo Epstein- Barr virus
  • examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems.
  • the various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art.
  • baculovirus expression systems include pVL- derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the ⁇ -gal containing pBlueBac III).
  • pVL- derived vectors such as pVL1392, pVL1393 and pVL941
  • pAcUW-derived vectors such as pAcUWl
  • pBlueBac-derived vectors such as the ⁇ -gal containing pBlueBac III.
  • a vector will be designed for production of the subject ALK3 polypeptides in CHO cells, such as a Pcmv-Script vector (STRATAGENE®, La Jolla, Calif.), pcDNA4 vectors (INVITROGENTM, Carlsbad, Calif.) and pCI-neo vectors (PROMEGA®, Madison, Wise).
  • a Pcmv-Script vector STRATAGENE®, La Jolla, Calif.
  • pcDNA4 vectors INVITROGENTM, Carlsbad, Calif.
  • pCI-neo vectors PROMEGA®, Madison, Wise
  • ALK3 proteins can be expressed using a host cell transfected with a recombinant gene including a coding sequence (e.g., SEQ. ID. NOS: 2 and 4) for one or more of the subject ALK3 polypeptides.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • an ALK3 polypeptide may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.
  • a host cell transfected with an expression vector encoding an ALK3 polypeptide can be cultured under appropriate conditions to allow expression of the ALK3 polypeptide to occur.
  • the ALK3 polypeptide may be secreted and isolated from a mixture of cells and medium containing the ALK3 polypeptide. Alternatively, the ALK3 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 ALK3 polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific for particular epitopes of the ALK3 polypeptides and affinity purification with an agent that binds to a domain fused to the ALK3 polypeptide (e.g., a protein A column may be used to purify an ALK3-Fc fusion).
  • the ALK3 polypeptide may be a fusion protein containing a domain which facilitates its purification.
  • Purification may be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography.
  • the purification could be completed with viral filtration and buffer exchange.
  • ALK3-hFc protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS PAGE. This level of purity was sufficient to achieve desirable effects in mice.
  • 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 ALK3 polypeptide can allow purification of the expressed fusion protein by affinity chromatography using a Ni 2+ metal resin.
  • the purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified ALK3 polypeptide (e.g., see Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972); the contents of these publications are incorporated herein by reference in their entirety.
  • fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
  • BMP antagonists are “ALK3 antagonists”.
  • antagonist refers to any organic or inorganic molecule that opposes the naturally occurring signaling events elicited by BMP2, BMP4, BMP7 and other members of the BMP family by way of the BMP responsive SMAD pathway.
  • antagonist includes an antibody that blocks the interaction of a BMP ligand with the ALK3 receptor, thereby preventing the downstream signaling from ALK3, i.e., BMP-ALK3 signaling.
  • the disclosure provides methods for using BMP-ALK3 antagonists, including, for example, BMP-binding ALK3 polypeptides, anti-BMP antibodies, anti-ALK3 antibodies, BMP- or ALK3-targeted small molecules and aptamers, and nucleic acids that decrease expression of BMP and ALK3, to treat or prevent atherosclerosis and vascular calcification.
  • BMP-ALK3 antagonists including, for example, BMP-binding ALK3 polypeptides, anti-BMP antibodies, anti-ALK3 antibodies, BMP- or ALK3-targeted small molecules and aptamers, and nucleic acids that decrease expression of BMP and ALK3, to treat or prevent atherosclerosis and vascular calcification.
  • BMP-ALK3 signaling can be used for inhibiting the development of early atheromatous lesions formation.
  • soluble ALK3 polypeptides and particularly ALK3-Fc are preferred antagonists, and although such antagonists can affect early atheromatous lesions formation through a mechanism other than BMP antagonism, other types of BMP-ALK3 antagonists are expected to be useful, including anti-BMP (e.g., BMP2 or BMP4) antibodies, anti-ALK3 antibodies, antisense, RNAi or ribozyme nucleic acids that inhibit the production of ALK3, BMP2 or BMP4 and other inhibitors of BMP or ALK3, particularly those that disrupt BMP-ALK3 binding.
  • anti-BMP e.g., BMP2 or BMP4 antibodies
  • anti-ALK3 antibodies e.g., anti-ALK3 antibodies
  • antisense RNAi or ribozyme nucleic acids
  • An antibody that is specifically reactive with an ALK3 polypeptide e.g., a soluble ALK3 polypeptide
  • an antibody that is specifically reactive with a BMP polypeptide and which disrupts ALK3 binding may be used as an antagonist.
  • anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the ALK3 polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • An immunogenic portion of an ALK3 or BMP polypeptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum.
  • antibody-producing cells lymphocytes
  • lymphocytes can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an ALK3 polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with a subject polypeptide. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
  • the antibody of the present invention is further intended to include bispecific, single-chain, chimeric, humanized and fully human molecules having affinity for an ALK3 or BMP polypeptide conferred by at least one CDR region of the antibody.
  • An antibody may further 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).
  • the antibody is a recombinant antibody, which term encompasses any antibody generated in part by techniques of molecular biology, including CDR-grafted or chimeric antibodies, human or other antibodies assembled from library-selected antibody domains, single chain antibodies and single domain antibodies (e.g., human VH proteins or camelid VHH proteins).
  • the antibody is a monoclonal antibody, and in certain embodiments, provided herein are available methods for generating novel antibodies.
  • a method for generating a monoclonal antibody that binds specifically to an ALK3 polypeptide or BMP polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the antigen.
  • antibody-producing cells e.g., cells from the spleen
  • a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to the antigen.
  • the monoclonal antibody may be purified from the cell culture.
  • the adjective "specifically reactive with” as used in reference to an antibody is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (e.g., an ALK3 polypeptide) and other antigens that are not of interest that the antibody is useful for, at minimum, detecting the presence of the antigen of interest in a particular type of biological sample. In certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity in binding may be desirable.
  • Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and cross -reacting polypeptides.
  • One characteristic that influences the specificity of an antibody: antigen interaction is the affinity of the antibody for the antigen.
  • affinity a dissociation constant
  • BMPs and ALK3 it is expected that a neutralizing anti-BMP or anti-ALK3 antibody would generally have a dissociation constant of 10-9 or less.
  • the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding.
  • a variety of different techniques are available for testing interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the BiacoreTM binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN
  • nucleic acid compounds that are BMP or ALK3 antagonists include antisense nucleic acids, RNAi constructs and catalytic nucleic acid constructs.
  • a nucleic acid compound may be single or double stranded.
  • a double stranded compound may also include regions of overhang or non-complementarity, where one or the other of the strands is single stranded.
  • a single stranded compound may include regions of self- complementarity, meaning that the compound forms a so-called "hairpin” or "stem-loop” structure, with a region of double helical structure.
  • a nucleic acid compound may comprise a nucleotide sequence that is complementary to a region consisting of no more than 1000, no more than 500, no more than 250, no more than 100 or no more than 50, 35, 30, 25, 22, 20 or 18 nucleotides of the full-length ALK3 nucleic acid sequence or BMP nucleic acid sequence.
  • the region of complementarity will preferably be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, and optionally between 15 and 25 nucleotides.
  • nucleic acid compound will have a length of about 8 to about 500 nucleotides or base pairs in length, and optionally the length will be about 14 to about 50 nucleotides.
  • a nucleic acid may be a DNA (particularly for use as an antisense), RNA or RNA:DNA hybrid. Any one strand may include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA.
  • a double stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA.
  • a nucleic acid compound may include any of a variety of modifications, including one or modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including internucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid).
  • An antisense nucleic acid compound will preferably have a length of about 15 to about 30 nucleotides and will often contain one or more modifications to improve characteristics such as stability in the serum, in a cell or in a place where the compound is likely to be delivered, such as the stomach in the case of orally delivered compounds and the lung for inhaled compounds.
  • the strand complementary to the target transcript will generally be RNA or modifications thereof.
  • the other strand may be RNA, DNA or any other variation.
  • the duplex portion of double stranded or single stranded "hairpin" RNAi construct will preferably have a length of 18 to 40 nucleotides in length and optionally about 21 to 23 nucleotides in length, so long as it serves as a Dicer substrate.
  • Catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also contain modified forms. Nucleic acid compounds may inhibit expression of the target by about 50%, 75%, 90% or more when contacted with cells under physiological conditions and at a concentration where a nonsense or sense control has little or no effect.
  • Preferred concentrations for testing the effect of nucleic acid compounds are 1, 5 and 10 micromolar.
  • ALK3 antagonist, ALK3-Fc and/or functional variant thereof when used to treat, and prevent atherosclerosis or manage cholesterol, or related disorders such as cardiovascular disease.
  • the ALK3 antagonist, ALK3-Fc and/or functional variant thereof can be used with statins.
  • Statins are drugs that competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase "HMG-CoA reductase,” which is the enzyme that catalyzes an early, rate-limiting step in cholesterol biosynthesis. Hebert et al., JAMA 1997, 278: 313-21. This combination, in addition to raising HDL levels and lowering LDL levels may also lowers triglyceride and reduce inflammation.
  • the combination can have additional therapeutic effects, for example, the combination may lower blood pressure; protect against heart disease, for example, by reducing smooth muscle proliferation, reduce heart attacks, reduce platelet aggregation, and to reduce strokes as well as peripheral arterial disease (clogging of the arteries to the legs).
  • statins that can be used together with the ALK3 antagonist, ALK3-
  • Fc and/or functional variant thereof include, but are not limited to, mevastatin, pitavastatin, rosuvastatin, pentostatin (NIPENT®), nystatin, lovastatin (MEVACOR®), simvastatin
  • statins may exist in an inactive form, such as a lactone (e.g., simvastatin), the invention encompasses using the active form (e.g., b- hydroxy acid form) of them. See Physicians Desk Reference, 54th Ed. (2000) pp. 1917-1920.
  • active form e.g., b- hydroxy acid form
  • Fibrates or fibric acid derivatives are regarded as broad- spectrum lipid- modulating agents in that although their main action is to decrease serum triglycerides they also tend to reduce LDL-cholesterol and to raise HDL-cholesterol. It is believed that the combined use of ALK3 antagonist, ALK3-Fc and/or functional variant thereof and a fibrate can reduce the risk of coronary heart disease events in those with low HDL-cholesterol or with raised triglycerides by speeding up the chemical breakdown (i.e., catabolism) of triglyceride-rich lipoproteins that circulate in the body.
  • ALK3 antagonist, ALK3-Fc and/or functional variant thereof and a fibrate can reduce the risk of coronary heart disease events in those with low HDL-cholesterol or with raised triglycerides by speeding up the chemical breakdown (i.e., catabolism) of triglyceride-rich lipoproteins that circulate in the body.
  • Fibrates include, but are not limited to, bezafibrate, ciprofibrate, fenofibrate, gemfibrozil, clofibrate, or combinations thereof. Fibrates suitable for inclusion in the
  • compositions or administration in the ALK3 antagonist, ALK3-Fc and/or functional variant thereof are disclosed in U.S. Pat. Nos. 4,895,762; 6,074,670; and 6,277,405, and the contents of which are incorporated herein by reference in their entirety.
  • Biguanides for use in the compositions and methods described herein include, but are not limited to, metformin, phenformin, buformin, or combinations thereof. Biguanides suitable for use in the compositions or methods described herein are also disclosed in U.S. Pat. No. 6,303,146, and the contents of which are incorporated herein by reference in its entirety.
  • ALK3 antagonist used in an ALK3 antagonist, ALK3-Fc and/or functional variant thereof and a bigaunide may improve glycemic control by enhancing insulin sensitivity in the liver and in muscle.
  • the combination may reduce or avoid cardiovascular risk factors such as dyslipidemia, elevated plasminogen activator inhibitor 1 levels, other fibrinolytic abnormalities,
  • hyperinsulinemia insulin resistance
  • insulin resistance is an effective and safe therapeutic agent for the treatment of type 2 diabetes.
  • an ALK3 antagonist, ALK3-Fc and/or functional variant thereof can be used in combination with glitazones, which may increase glucose uptake in muscle and reduced endogenous glucose production.
  • Glitazones include 5-((4-(2-(methyl-2-pyri- dinyl amino)ethoxy)-phenyl)methyl)-2,4-thiazolidinedione, troglitazone, pioglitazone, ciglitazone, WAY- 120,744, englitazone, AD 5075, darglitazone, rosiglitazone, combinations thereof, or a pharmaceutically acceptable salt, solvate, clathrate, polymorph, prodrug, or pharmacologically active metabolite thereof.
  • Glitazones suitable for use in the compositions or methods described herein are disclosed in U.S. Pat. Nos. 4,687,777; 5,002,953; 5,741,803;
  • compositions comprising an ALK3 antagonist, ALK3-Fc and/or functional variant thereof and a sulfonylurea or a derivative thereof may increase insulin release from the pancreas and may further insulin levels by reducing hepatic clearance of the hormone.
  • Sulfonylurea-based drugs for use the compositions and methods described herein include, but are not limited to, glisoxepid, glyburide, acetohexamide, chlorpropamide, glibomuride, tolbutamide, tolazamide, glipizide, gliclazide, gliquidone, glyhexamide, phenbutamide, tolcyclamide, combinations thereof, or a pharmaceutically acceptable salt, solvate, or clathrate.
  • Combination compositions can also include agents that inhibit CETP.
  • agents that inhibit CETP are, for example, Torcetrapib, and S-(2-[([l-(2- ethylbutyl)cyclohexyl]carbonyl)amino]phenyl)-2- methylpropanethioate.
  • Additional active agents also include cardiovascular drugs.
  • Cardiovascular drugs for use in combination with the ALK3 antagonist, ALK3-Fc and/or functional variant thereof to prevent or treat cardiovascular diseases include peripheral antiadrenergic drugs, centrally acting antihypertensive drugs (e.g., methyldopa, methyldopa HCl), antihypertensive direct vasodilators (e.g., diazoxide, hydralazine HCl), drugs affecting renin-angiotensin system, peripheral vasodilators, phentolamine, antianginal drugs, cardiac glycosides, inodilators (e.g., aminone, milrinone, enoximone, fenoximone, imazodan, sulmazole), antidysrhythmic drugs, calcium entry blockers, ranitine, bosentan, and rezulin.
  • peripheral antiadrenergic drugs e.g., methyldopa, methyldopa HCl
  • the ALK3 antagonist Depending on the disorder for which treatment is sought, the ALK3 antagonist,
  • ALK3-Fc and/or functional variant thereof and pharmaceutical compositions described herein are used in combination therapy with other therapeutics that achieve a specific biological effect.
  • Various medications can lower blood cholesterol levels. They may be prescribed individually or in combination with other drugs. Some of the common types of cholesterol- lowering drugs include statins, resins and nicotinic acid (niacin), gemfibrozil and clofibrate.
  • combination therapy is contemplated utilizing, for example, clofibrate (ANTARA®, which raises the HDL cholesterol levels and lowers triglyceride levels), gemfibrozil (LOPID®, which raises HDL cholesterol levels), nicotinic acid (which works in the liver by affecting the production of blood fats and is used to lower triglycerides and LDL cholesterol, and raise HDL ("good”) cholesterol), resins (which are also called bile acid-binding drugs and work in the intestines by promoting increased disposal of cholesterol), including cholestyramine
  • statins including atorvastatin (LIPITOR®), fluvastatin (LESCOL®), lovastatin (MEVACOR®), pravastatin (PRAVACHOL®), rosuvastatin calcium (CRESTOR®), and simvastatin (ZOCOR®).
  • the drugs of first choice for elevated LDL cholesterol are the HMG CoA reductase inhibitors, e.g., atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin.
  • Statin drugs are effective for lowering LDL cholesterol levels, have few immediate short-term side effects, are easy to administer, have high patient acceptance and have few drug- drug interactions.
  • Another class of drugs for lowering LDL is the bile acid sequestrants— colesevelam, cholestyramine and colestipol— and nicotinic acid (niacin), which have been shown to reduce the risk for coronary heart disease in controlled clinical trials. Both classes of drugs appear to be free of serious side effects. But both can have troublesome side effects and require considerable patient education to achieve adherence. Nicotinic acid is preferred in patients with triglyceride levels that exceed 250 mg/dL because bile acid sequestrants tend to raise triglyceride levels.
  • Angiotensin II causes blood vessels to contract and thereby narrows the blood vessels. The narrowing of the vessels increases the pressure within the vessels and can cause high blood pressure (hypertension).
  • Angiotensin II is formed from angiotensin I in the blood by the enzyme, angiotensin converting enzyme (ACE). ACE inhibitors decrease the production of angiotensin II. As a result, the blood vessels enlarge or dilate, and the blood pressure is reduced.
  • ACE angiotensin converting enzyme
  • ACE inhibitors that available in the United States include captopril (CAPOTEN®), benazepril (LOTENSIN®), enalapril (VASOTEC®), lisinopril (PRINIVIL®, ZESTRIL®) fosinopril (MONOPRIL®), ramipril (ALTACE®), perindopril (ACEON®), quinapril (ACCUPRIL®), moexipril (UNIVASC®), and trandolapril (MAVIK®).
  • acetylsalicylic acid ASPIRIN®, ECOTRIN®
  • TRILISATE® choline magnesium salicylate
  • VOLTAREN® CATAFLAM®
  • VOLTAREN-XR® diflunisal
  • DOLOBID® diflunisal
  • LODINE® etodolac
  • fenoprofen NALFON®
  • flurbiprofen ANSAID®
  • ibuprofen ADVIL®, MOTRIN®, MEDIPREN®, NUPRIN®
  • indomethacin INDOCIN®, INDOCIN-SR®
  • ketoprofen ORUDIS®, ORUVAIL®
  • meclofenamate MECLOMEN®
  • RELAFEN® naproxen
  • NAPROSYN® NAPRELAN®
  • ANAPROX® ALEVE®
  • oxaprozin DAYPRO®
  • phenylbutazone phenylbutazone
  • NSAIDs selective non-steroidal antiinflammatory drugs
  • BUTAZOLIDINE® piroxicam
  • FELDENE® salsalate
  • SALFLEX® salsalate
  • tolmetin TOLECTIN®
  • valdecoxib valdecoxib
  • COX-2 selective non-steroidal antiinflammatory drugs including Bextra, Celebrex, Naproxen, and Vioxx.
  • Prescription- only NSAIDs include ibuprofen (BRUFEN®), aceclofenac (PRESERVEX®), acemetacin (EMFLEX), azapropazone (RHEUMOX®), celecoxib (CELEBREX), dexketoprofen
  • DOLOBID® DOLOBID®
  • LODIN®E etodolac
  • LEDERFEN® fenbufen
  • FENOPRON® flurbiprofen
  • FROBEN® indometacin
  • ketoprofen ORUDIS, ORUVAIL®
  • mefenamic acid meloxicam
  • MOBIC® nabumetone
  • RELIFEX® naproxen
  • NAPROSYN, SYNFLEX® phenylbutazone
  • piroxicam FELDENE®
  • sulindac CLINORIL®
  • tenoxicam MOBIFLEX®
  • tiaprofenic acid SURGAM®
  • anti-thrombosis drugs such as anticoagulant drugs, which inhibit the ability of blood to clot, or coagulate and include dalteparin (FRAGMIN®), danaparoid (ORGARAN®), enoxaparin (LOVENOX®), heparin (various), tinzaparin
  • antiplatelet drugs such as aspirin, ticlopidine (TICLID®), clopidogrel (PLAVIX®), tirofiban (AGGRASTAT®) and eptifibatide (INTEGRILIN®).
  • Still other methods include the use of bivalirudin (selective and reversible thrombin inhibitor), argatroban (reversible inhibitor of thrombin), and low molecular weight heparins (LMWHs), including enoxaparin (LOVENOX®), dalteparin
  • FRAGMIN® ardeparin
  • NPMIFLO® fondaparinux
  • idraparinux Still other anti- thrombosis drugs contemplated for use in methods described herein include FRAGMIN® (dalteparin sodium injection) LOVENOX® (enoxaparin sodium), NORMIFLO® (ardeparin sodium), ORGARAN® (danaparoid sodium), indirect (Antithrombin-Dependent) FXa inhibitors such as fondaparinux (ARIXTRA®) and idraparinux, direct (Antithrombin-Independent) FXa inhibitors such as BAY 59-7939 [Bayer], DPC-423 [Bristol-Myers Squibb], DX-9065a
  • the disorder that may be treated by the compositions of the present invention are limited only by the fact that the disorder needs a therapeutic intervention which inhibits platelet aggregation.
  • the doses of the agent may be modified for each individual subject.
  • routes of administration and uses those of skill in the art are referred to the Physician's Desk Reference for generalized descriptions of formulations, routes of administration and patient monitoring used for agents such as
  • AGGRASTAT® see e.g., entry at pages 1933-1937, PDR, 57th Edn., 2003), AGGRENOX® (see e.g., entry at pages 1023-1026, PDR, 57th Edn., 2003), AGRYLIN® (see e.g., entry at pages 3142-3143, PDR, 57th Edn., 2003), FLOLAN® (see e.g., entry at pages 1516-1521, PDR, 57th Edn., 2003), INTEGRILIN® (see e.g., entry at pages 2138-2142, PDR, 57th Edn., 2003), PRESANTINE® (see e.g., entry at pages 1052-2053, PDR, 57th Edn., 2003), PLAVIX® (see e.g., entry at pages 1098-1101, PDR, 57th Edn., 2003), PLETAL® (see e.g., entry at pages 2780-2782, PDR, 57
  • BMP-ALK3 antagonists e.g., ALK3 polypeptides
  • ALK3 polypeptides can be formulated with a pharmaceutically acceptable carrier.
  • an ALK3 polypeptide can be administered alone or as a component of a pharmaceutical formulation (therapeutic composition).
  • the ALK3 antagonist, ALK3-Fc and/or functional variant thereof can be formulated for administration in any convenient way for use in human or veterinary medicine.
  • the therapeutic methods described herein include administering the pharmaceutical composition systemically or locally as an implant or device.
  • the therapeutic composition for use in the methods described herein is, of course, in a pyrogen-free, physiologically acceptable form.
  • Therapeutically useful agents other than the ALK3 antagonists that are optionally included in the composition as described above may be administered simultaneously or sequentially with the subject compounds (e.g., ALK3 polypeptides) in the methods.
  • compositions suitable for parenteral administration can comprise one or more ALK3 polypeptides (e.g., ALK3-Fc and/or functional variant thereof) in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • ALK3 polypeptides e.g., ALK3-Fc and/or functional variant thereof
  • aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions comprising an ALK3 antagonist, ALK3- Fc and/or functional variant thereof include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions of the present invention may include a matrix capable of delivering one or more therapeutic compounds (e.g., ALK3 polypeptides, ALK3-Fc and/or functional variant thereof) to a target tissue site, providing a structure for the developing tissue and optimally capable of being resorbed into the body.
  • the matrix can provide slow release of the ALK3 polypeptides.
  • Such matrices may be formed of materials presently in use for other implanted medical applications.
  • matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.
  • the particular application of the subject compositions will define the appropriate formulation.
  • Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate,
  • tricalciumphosphate, hydroxyapatite, polylactic acid and polyanhydrides are biodegradable and biologically well defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are non-biodegradable and chemically defined, such as sintered
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate.
  • the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
  • the ALK3 antagonist, ALK3-Fc and/or functional variant thereof can be administered for orally, e.g., in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water- in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient.
  • An agent may also be administered as a bolus, electuary or paste.
  • one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting
  • pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as star
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Specifically, it refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co., 1990). The formulation should suit the mode of administration.
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • compositions comprising an ALK3 antagonist, ALK3-Fc and/or functional variant thereof can also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It is also desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It is
  • the term “comprising” or “comprises” refers to respective component(s) thereof that are essential to the methods and compositions described herein, yet open to the inclusion of unspecified elements, whether essential or not.
  • the use of “comprising” indicates inclusion rather than limitation.
  • the dosage regimen will be determined by the attending physician considering various factors which modify the action of the ALK3 antagonist, ALK3- Fc and/or functional variant thereof (e.g., ALK3 polypeptides).
  • ALK3 polypeptides are provided herein.
  • Such therapy would achieve its therapeutic effect by introduction of the ALK3 polynucleotide sequences into cells or tissues having the disorders as listed above.
  • Delivery of ALK3 polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.
  • Preferred for therapeutic delivery of ALK3 polynucleotide sequences is the use of targeted liposomes.
  • RNA virus such as a retrovirus
  • retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • a number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target- specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody.
  • a sugar for example, a sugar, a glycolipid, or a protein.
  • Preferred targeting is accomplished by using an antibody.
  • specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the ALK3 polynucleotide, e.g., an ALK3-Fc and/or functional variant thereof.
  • tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • ALK3 polynucleotides Another targeted delivery system for ALK3 polynucleotides is a colloidal dispersion system.
  • Colloidal dispersion systems include macromolecule complexes,
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (see e.g., Fraley, et al., Trends Biochem. Sci., 6:77, 1981).
  • compositions of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,
  • phosphatidylethanolamine sphingolipids
  • cerebrosides sphingolipids
  • gangliosides phosphatidylethanolamine
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
  • the targeting of liposomes is also possible based on, for example, organ-specificity, cell- specificity, and organelle- specificity and is known in the art.
  • the efficacy of a given treatment for atherosclerosis can be determined by the skilled clinician. However, a treatment is considered "effective treatment," as the term is used herein, if any one or all of the signs or symptoms of atherosclerosis, for example, level of atherosclerosis marker or severity of other symptoms are altered in a beneficial manner, other clinically accepted symptoms or markers of disease are improved, or ameliorated.
  • the improvement is seen as a need for fewer drug treatments, improved function, reduced pain, increased energy, reduced angina pain, reduced numbness in affected limbs, fewer episodes of hospitalization or long-term care facility usage, and/or longer intervals between hospitalizations, than the individual has experienced prior to treatment with the peptide following treatment with a peptide as described herein. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization or need for medical
  • Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing progress of atherosclerosis; or (2) relieving the disease, e.g., causing regression of symptoms.
  • the methods can also be used to prevent or reduce the likelihood of the development of a complication or disability relating to atherosclerosis (e.g., angina, myocardial infarction and stroke).
  • the treatment of a disease includes curing the disease in a subject or initiating remission of the disease in a subject.
  • treatment can encompass curing the disease, it is emphasized that treatment can be "effective" without effecting a complete cure.
  • An effective amount for the treatment of atherosclerosis means that amount which, when administered to a mammal in need thereof, is sufficient to result in effective treatment as that term is defined herein.
  • Efficacy of an ALK3-Fc polypeptide can be determined by assessing physical indicators of atherosclerosis, for example levels of an atherosclerotic marker, reduced angina pain or symptoms, pain, improved function, enhanced exercise capability etc.
  • efficacy may be determined by a reduction in macrophage- mediated inflammation or calcification or stenosis of the vessels by at least 10% compared to the levels prior to treatment onset, e.g., in a reduction in Thl7 cells, or IL-17 producing T- lymphocytes (see e.g., Chen, X. et al., (2009) Pathways Issue 10; Cover story).
  • ALK3-Fc or functional variant thereof for use in the prevention or treatment of atherosclerosis in a mammalian subject in need thereof.
  • ALK3-Fc or functional variant thereof for use in the prevention or treatment of vascular calcification in a mammalian subject in need thereof.
  • ALK3-Fc or functional variant thereof for use in the prevention or treatment of thrombosis of an atherosclerosic plaque in a mammalian subject in need thereof.
  • ALK3-Fc or functional variant thereof for the manufacture of a medicament for use in the prevention or treatment of thrombosis of an atherosclerosic plaque in a mammalian subject in need thereof.
  • ALK3-Fc or functional variant thereof of any one of paragraphs [A]- [F] further comprising concurrently use of at least one other agent that is used in the treatment of cardiovascular disease in combination in a pharmaceutically acceptable carrier.
  • [L] A method of preventing or treating atherosclerosis in a mammalian subject in need thereof, comprising administering to the subject an effective therapeutic amount of an ALK3-Fc or functional variant thereof.
  • [M] A method of preventing or treating vascular calcification in a mammalian subject in need thereof, comprising administering to the subject an effective therapeutic amount of an ALK3-Fc or functional variant thereof.
  • [N] A method of preventing or treating thrombosis of an atherosclerosic plaque in a mammalian subject in need thereof, comprising administering to the subject an effective therapeutic amount of an ALK3-Fc or functional variant thereof.
  • a composition comprising: an ALK3-Fc or functional variant thereof and at least one other agent that is used in the treatment of cardiovascular disease in combination with a pharmaceutically acceptable carrier.
  • composition of paragraph [U] wherein the at least one other agent is selected from the group consisting of an anti-atherosclerosis drug, an anti-thrombosis drug, an anti-inflammatory drug, an ACE inhibitor and a cholesterol lowering drug.
  • the at least one other agent is selected from the group consisting of an anti-atherosclerosis drug, an anti-thrombosis drug, an anti-inflammatory drug, an ACE inhibitor and a cholesterol lowering drug.
  • CPMB Protocols in Molecular Biology
  • CPCB Current Protocols in Cell Biology
  • ALK3-Fc fusion protein was generated as described in PCT publication no.
  • FIG. 1 the amino acid sequence and corresponding nucleotide sequence for native human ALK3 are shown in Figures 1, 2.
  • An ALK3-hFc fusion protein in which the extracellular domain (native residues 24-152) of human ALK3 ( Figures 3, 4) is fused C-terminally with a human Fc domain ( Figures 5, 6) via a minimal linker (comprised of amino acid residues TGGG (SEQ ID NO: 1)) to yield the protein shown in Figure 7.
  • the hALK3(24-152)-hFc employs the TPA leader and has the unprocessed amino-acid sequence shown in Figure 8.
  • a sense nucleotide sequence encoding this fusion protein and the corresponding antisense sequence are indicated in Figure 9.
  • the protein was expressed in CHO cell lines, and N-terminal sequencing revealed a primary species with an N-terminal block, indicating a start at the native glutamine (Q) residue, consistent with the protein of Fig.7, and a single minor sequence of GAQNLDSMLHGTGMK (SEQ. ID. NO: 4).
  • Another ALK3-Fc variant comprising the murine ALK3 extracellular domain (native residues 24-152 in the murine precursor) and murine Fc domain was generated by similar methods.
  • the amino acid sequence of this variant, mALK3(24-152)-mFc is shown below with the ALK3 domain in bold and italicized:
  • hALK3-hFc nucleic acid sequences may be used for the purposes described herein.
  • This example summarizes nucleic acid constructs that can be used to express ALK3 constructs in CHO cells, according to the methods provided herein, and provides the mature proteins isolated from cell culture.
  • nucleic acid of hALK3-hFc can be expressed in CHO cells and the following
  • ALK3-Fc species are isolated:
  • hALK3(GA,24-152)-hFc sequence is shown below and it retains an initial glycine- alanine from the leader sequence.
  • a nucleic acid encoding hALK3(24-146)-hFc, shown below, can be expressed in CHO cells:
  • hALK3(24-146)-hFc polypeptide is shown below and it begins with a glutamine (which tends to be blocked for N-terminal sequencing by Edman degradation).
  • GMKSDSDQKK SENGVTLAPE DTLPFLKCYC SGHCPDDAIN NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT
  • hALK3(GA,24-146)-hFc polypeptide sequence is shown below and it retains an initial glycine- alanine from the leader sequence.
  • a nucleic acid encoding hALK3(24-140)-hFc is shown below and it may be expressed in CHO cells:
  • the hALK3(GA,24-140)-hFc sequence is shown below and it retains an initial glycine- alanine from the leader sequence.
  • a nucleic acid encoding hALK3(30-152)-hFc is shown below and it can be expressed in CHO cells:
  • the hALK3(GA,30-152)-hFc is shown below and it retains an initial glycine- alanine from the leader sequence.
  • hALK3(A,30-152)-hFc is shown below and it retains an initial alanine from the leader sequence.
  • hALK3(31-152)-hFc polypeptide sequence is shown below.
  • the leader and the initial leucine are removed, leaving an initial histidine (effectively ⁇ 7).
  • DQKKSENGVT LAPEDTLPFL KCYCSGHCPD DAINNTCITN GHCFAI IEED DQGETTLASG CMKYEGSDFQ CKDSPKAQLR RTIECCRTNL CNQYLQPTLP PVVIGPFFDG SIRTGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAP IEKT I SKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK (SEQ. ID. NO: 16)
  • DQKKSENGVT LAPEDTLPFL KCYCSGHCPD DAINNTCITN GHCFAI IEED DQGETTLASG CMKYEGSDFQ CKDSPKAQLR RTIECCRTNL CNQYLQPTLP PVVIGPFFDG SIRTGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAP IEKT I SKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK (SEQ. ID. NO: 17)
  • a nucleic acid encoding hALK3(30-146)-hFc is shown below. It expressed in CHO cells:
  • the hALK3(GA,30-146)-hFc is shown below and it retains an initial glycine- alanine from the leader sequence.
  • hALK3(A,30-146)-hFc is shown below and it retains an initial alanine from the leader sequence.
  • GMKSDSDQKK SENGVTLAPE DTLPFLKCYC SGHCPDDAIN NTCITNGHCF AIIEEDDQGE TTLASGCMKY EGSDFQCKDS PKAQLRRTIE CCRTNLCNQY LQPTLPPVVI GPFTGGGTHT
  • hALK3(31-146)-hFc sequence is shown below and the leader and the initial leucine are removed, leaving an initial histidine (effectively NA7CA6).
  • a nucleic acid encoding hALK3(30-140)-hFc is shown below and it may be expressed in CHO cells: ATGGAT GCAATGAAGA GAGGGCTCTG
  • the hALK3(GA,30-140)-hFc is shown below and it retains an initial glycine- alanine from the leader sequence.
  • hALK3(A,30-140)-hFc is shown below and it retains an initial alanine from the leader sequence.
  • hALK3(31-140)-hFc sequence is shown below, in which the leader and the initial leucine are removed, leaving an initial histidine (effectively NA7CA12).
  • Example 3 Establishment of a mouse model of atheromatous and vascular calcific disease.
  • the inventors' objectives are to demonstrate the effect of pharmacologic BMP inhibition upon the development of (i) atheromatous disease burden, and (ii) vascular calcification in an accepted animal model of atherosclerosis, in order to provide potential proof- of-concept that BMP inhibition can be an effective strategy for preventing atherosclerosis or limiting its progression.
  • BMPs are multifunctional protein ligands which form a subset of the
  • TGF- ⁇ transforming growth factor- ⁇ family of signaling proteins
  • BMPs originally identified by their ability to induce ectopic bone formation, serve broad roles in gastrulation, developmental patterning, and organ formation. In the adult organism, BMP signals serve principally to mediate injury repair and inflammation. Aberrant BMP signaling may contribute to a number of acquired diseases, perhaps via inappropriate activation of repair or inflammatory responses.
  • BMP signals contribute to atherosclerosis, since BMPs and many of the BMP-induced gene products which affect matrix remodeling are overexpressed in early atherosclerotic lesions, and may promote plaque formation and progression (Bostrom, K. & Demer, L.L., Crit Rev Eukaryot Gene Expr 10, 151-158 (2000); Bostrom, K., et al., J Clin Invest 91, 1800-1809 (1993); Tintut, Y., et al., Circulation 108, 2505-2510 (2003)).
  • BMP signals may also induce resident or circulating progenitors to form the cells of bone, including osteoblasts and chondroblasts, and cause calcification of vessels (Tintut, Y., et al., 2003, supra).
  • severe calcific vascular disease is particularly problematic in that it can interfere with the body' s ability to restore adequate circulation to the coronary vessels by angioplasty or bypass surgery.
  • the inventors investigated whether atherosclerotic and calcific lesions can be ameliorated or prevented, if signals which contribute to their progression can be intercepted during their formation.
  • the proof-of-principle experiments described in this report tested the effects of a novel pharmacologic inhibitor of BMP signaling in an accepted animal model of atheromatous disease.
  • LDLr-/- mice When fed a high fat diet, low density lipoprotein receptor-deficient (LDLr-/-) mice are genetically predisposed to high cholesterol levels, and consequently the development of atherosclerotic and calcific vascular lesions, occurring in a manner of weeks only after challenge with a high cholesterol and high lipd diet (Aikawa, E., et al., Circulation 116, 2841-2850 (2007); Aikawa, E., et al., Circulation 115, 377-386 (2007); Ohshima, S., et al., J Nucl Med 50, 612-617 (2009); Isobe, S., et al., J Nucl Med 47, 1497-1505 (2006)).
  • the inventors employed several novel molecular imaging probes which have been validated to detect the presence of osteogenic or bone-forming activity (Osteosense, a bisphosphonate probe which binds to vessel-associated osteoblasts), and vascular inflammation associated with atheroma (Prosense, a cathepsin substrate which binds vessel-associated macrophages), the intensity of either of which can be quantitated by near-infrared fluorescence reflectance imaging as previously described (Aikawa, E., et al., (2007) and (2006) supra).
  • Ostosense a bisphosphonate probe which binds to vessel-associated osteoblasts
  • Prosense a cathepsin substrate which binds vessel-associated macrophages
  • mice had gross evidence of intimal lesions in the minor curvature of the aorta ( Figure 12A), and developed in addition calcification of the vessel media as detected by Von Kossa mineral staining ( Figure 12B). These findings were found with 100% penetrance in LDLr-/- mice given a high fat diet, and were not found in control mutant mice given a normal diet, or in wild-type (C57BL/6) control mice given a high fat diet. This protocol yielded a robust model of atherosclerosis and atherosclerosis-associated vascular calcification in the context of hypercholesterolemia and an atherogenic diet.
  • a BMP inhibitor can inhibit the development of vascular calcification and macrophage-mediated inflammation associated with atheromatous disease.
  • LDLr-/- mice were treated with a BMP inhibitor positive control compound (2.5 mg/kg/d intraperitoneally) or vehicle (saline) for 20 weeks following the initiation of a high fat diet. Mice were injected with Osteosense (to label sites of bone-forming activity via osteoblast binding of this probe) and Prosense (to label sites of macrophage-mediated inflammation).
  • Example 5 Verification that the BMP inhibitor positive control compound inhibits BMP signaling activity (activated SMAD1/5/8) associated with atheromatous lesion formation.
  • LDLr-/- mice were started on a hypercholesterolemic diet at 8 weeks, and treated with either vehicle (saline) or a BMP inhibitor positive control compound (2.5 mg/kg/d intraperitoneally) for an additional 8 weeks. Aortae were harvested and fixed, and then stained with antibodies sensitive for the BMP effector molecule, phosphorylated-SMADl/5/8, and counterstained with DAPI nuclear stain. Within 6-8 weeks of being subjected to a high fat diet, LDR-/- mice developed fatty lesions in the intima of the aortic root, based on traditional histochemical staining techniques (data not shown).
  • the BMP inhibitor positive control compound -treated animals had reduced intimal atheroma formation as compared to vehicle- treated animals. Atheroma formation was associated with prominent staining of phosphorylated- SMAD 1/5/8 in vehicle-treated animals, which was greatly diminished in the BMP inhibitor positive control compound-treated animals.
  • the aortae of vehicle-treated mice revealed intense nuclear staining in a manner typical of nuclear-localized activated SMAD 1/5/8 (data not shown) (Feng, X.H.
  • Example 6 Demonstration that a soluble recombinant BMP receptor ectodomain inhibits BMP signaling activity (activated SMAD1/5/8) associated with atheromatous lesion formation and also inhibits macrophage-mediated inflammation.
  • Inflammatory activity a surrogate of atherosclerotic plaque burden, was assessed by near-IR fluorescence of Prosense (fluor-cathepsin substrate) at 700 nM.
  • Prosense uptake was significantly reduced by treatment with ALK3-Fc (2 mg/kg IP QOD) or a BMP inhibitor positive control compound (2.5 mg IP QD) as compared to vehicle for 6 weeks following the initiation of an atherogenic (Paigen) diet in adult (8 wk) LDLR-/- C57BL/6 mice, particularly in the aortic root and aortic arch (data not shown).
  • Prosense integrated intensity was significantly inhibited by treatment with ALK3-Fc (2 mg/kg IP QOD) or BMP inhibitor positive control compound (2.5 mg IP QD) versus vehicle for 6 weeks following the initiation of an atherogenic (Paigen) diet in adult (8 wk) LDLR-/- C57BL/6 mice, particularly in the aortic valve, root, arch, and suprarenal areas of the aorta.
  • Figure 14 shows that macrophage-mediated inflammation is quantitatively decreased in the central arterial vascular bed of atherogenic animals by recombinant or small-molecule BMP inhibitors. Each bar represents the mean + SEM of measurements obtained on tissues obtained from 10 individual mice per group with significant differences versus vehicle-treated animals indicated.
  • LDLR-/- deficient mice were intiated on an atherogenic diet (Paigen) at 8 weeks of age, and administered either vehicle, a BMP inhibitor positive control compound (2.5 mg/kg IP daily) or ALK3-Fc (2 mg/kg IP every other day). Each treatment group consisted of a total of 10 mice. After 4 weeks of atherogenic diet and drug or vehicle treatment, the animals were sacrificed. The frontal plane sections of the aortic arch were dissected out and stained for macrophage marker (MAC2) and counterstained with DAPI. The BMP inhibitor positive control compound-treated mice exhibited decreased lesion formation overall, and decreased staining for MAC2. ALK3-Fc-treated mice also exhibited profoundly decreased lesion formation and MAC2 staining. This result indicates that BMP inhibitors can effectively limit the development of early atheromatous lesions in atherogenic mice.
  • Paigen atherogenic diet
  • ALK3-Fc ALK3-Fc
  • LDLR-/- deficient mice were initiated on an atherogenic diet (Paigen) at 8 weeks of age, and administered either vehicle, a BMP inhibitor positive control compound (2.5 mg/kg IP daily) or ALK3-Fc (0.2 mg/kg IP every other day). After 6 weeks of atherogenic diet and treatment, the animals were sacrificed. The frontal plane sections of the aortic arch were dissected out and stained for phosphorylated SMAD 1/5/8 and counterstained with DAPI.
  • mice exhibited early atheromatous lesion formation associated with the activation of SMAD 1/5/8 in endothelial, smooth muscle, as well as MAC2+ foam cell populations (data not shown).
  • Control sections stained with only secondary Ab exhibited weak background fluorescence in the internal elastic lamina (data not shown).
  • the BMP inhibitor positive control compound-treated mice exhibited decreased lesion formation overall, and decreased staining for phosphorylated SMAD 1/5/8 (data not shown).
  • ALK3-Fc-treated mice also exhibited profoundly decreased lesion formation and phosphorylated SMAD 1/5/8 staining (data not shown). This result indicates that BMP inhibitor treatment effectively inhibits activation of BMP-SMAD signaling in the vasculature of atherogenic mice.
  • Example 7 Bone morphogenic protein signaling is required for vascular calcification in a murine model of matrix GLA protein deficiency.
  • Matrix GLA protein is a mineral-binding extracellular matrix protein that is thought to prevent vessel calcification by sequestration of calcium ions.
  • MGP also inhibits bone morphogenetic protein (BMP) signaling.
  • BMP bone morphogenetic protein
  • MGP-/- mice were treated with either vehicle or the small molecule BMP type I receptor inhibitor LDN-193189 (LDN, 2.5 mg/kg once daily IP) from day 1 to 28.
  • LDN is used as a BMP inhibitor positive control compound in these experiments.
  • Whole aortas were harvested for phospho-Smad 1/5/8 (P-Smad) immunohistochemistry, a marker of BMP signaling, and for Alizarin Red staining of calcium. Osteogenic activity in aortas was visualized ex vivo by the uptake of a fluorescent bisphosphonate imaging probe.
  • MGP-/- mice were also treated with vehicle or LDN to ascertain if inhibition of BMP signaling could impact survival, using Kaplan-Meier and Cox regression analysis.
  • MGP-/- aortas demonstrated increased P-Smad compared to wild-type mice (data not shows).
  • LDN treatment of MGP-/- mice reduced aortic P-Smad levelsand was associated with a reduction in tissue calcium levels (Fig. 15C).
  • ALK3-Fc treatment of MGP-/- mice also exhibited significantly reduced tissue calcification in the aorta (Fig. 15D).

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Abstract

Des modes de réalisation de l'invention concernent des procédés et des compositions comprenant un antagoniste de ALK-3, un ALK3-Fc et/ou d'autres variants fonctionnels de ceux-ci pour la prévention et/ou le traitement de l'athérosclérose, et ses calcifications et thrombose vasculaires associées.
PCT/US2011/060365 2010-11-12 2011-11-11 Traitement de l'athérosclérose par des antagonistes de bmp-alk3 WO2012065059A2 (fr)

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WO2020090705A1 (fr) 2018-10-31 2020-05-07 千寿製薬株式会社 Inhibiteur de la mort des cellules ganglionnaires de la rétine
WO2022240948A1 (fr) * 2021-05-11 2022-11-17 Keros Therapeutics, Inc. Procédés d'utilisation d'anticorps alk2 et al3

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WO2020090705A1 (fr) 2018-10-31 2020-05-07 千寿製薬株式会社 Inhibiteur de la mort des cellules ganglionnaires de la rétine
WO2022240948A1 (fr) * 2021-05-11 2022-11-17 Keros Therapeutics, Inc. Procédés d'utilisation d'anticorps alk2 et al3

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