Animal Health – Victoria Sung, Randall Stevens, William Smith, Victor Schorr Sloan, Keith Hruska, Yifu Fang, Celgene Corp, Washington University in St Louis WUSTL
Abstract for “Activin-ActRII antagonists, uses for treating bone and other conditions”
“This document contains methods to treat bone disorders associated with kidney disease. The methods include administration of Activin ActRIIA inhibitors. Methods and compositions are also provided for the treatment of low-turnover bone disorders. These methods include administration of Activin ActRIIA inhibitors. Herein are compositions that can be used to treat bone disorders associated with kidney disease, as well as compositions that can be used to treat low turnover bone disorders.
Background for “Activin-ActRII antagonists, uses for treating bone and other conditions”
Bone growth and mineralization depend on two types of cells, osteoclasts or osteoblasts. However, chondrocytes as well as cells of the vasculature play a critical role in these processes. Two mechanisms are involved in bone formation during development: intramembranous and endochondral. The former is responsible for longitudinal bone formation, while the latter forms topologically flat bones such as the skull bones. Endochondral Ossification involves the sequential formation and decay of cartilaginous structures within the growth plates, which serve as templates for osteoblasts, osteoclasts and the vasculature. Intimambranous osteossification is when bone is formed in connective tissues. Both require the infiltration and subsequent matrix deposition of osteoblasts.
“Chronic kidney disease is associated with a progressive deterioration in mineral homeostasis, with a disruption of normal serum and tissue concentrations of phosphorus and calcium, and changes in circulating hormones, such as parathyroid hormone, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, other vitamin D metabolites, fibroblast growth factor-23, and growth hormone. See Chronic Kidney Disease-Mineral and Bone Disorder, Kidney Disease: Improving Global Outcomes, Kidney Disease: Working Group (KDIGO), Kidney Disease: Improving Global Outcomes, In: Kidney Int. Suppl. (2009) 76 (Suppl 113), pages S1-130. Chronic kidney disease can disrupt mineral and hormone homeostasis, which is crucial for bone growth during childhood (bone modeling), and adult bone structure and function (bone remodeling). Patients with chronic kidney disease are more likely to have bone abnormalities. Patients with chronic kidney disease may also experience extraskeletal calcification due to disruptions in mineral and endocrine function. These conditions are called chronic kidney disease-related bone and mineral disorders (?CDK?MBD ?).”).
“Bone is constantly in constant turnover. Bone turnover refers to the process of bone resorption and replacement. Bone turnover is a process of bone resorption and replacement. Osteoblasts, osteoclasts, and other cells are required for this. Adynamic bone diseases, which have low turnover or no resorption, are characterised by a reduced or absent rate of bone replacement and resorption. Low turnover or dynamic bone can both be part of CKD-MBD. (Chronic Kidney Disease, Mineral and Bone Disease (CKD?MBD), Kidney Disease : Improving Global Outcomes(KDIGO), CKD?MBD Work Group. In: Kidney Int. Suppl. (2009) 76 (Suppl 110), pages S1-130 and S34.
Vascular calcification is a condition that causes vessel stiffening to increase by increasing calcium levels in the blood vessels. Vascular calcification can increase the risk of myocardial injury in patients with vascular calcification. This condition is especially common in patients with kidney disease (CKD-MBD). See, e.g., Shanahan et al., 2011, Circ. Res. 109:697-711.”
ActRIIA (and ActRIIB), are type II receptors that activate activins. ActRIIA, ActRIIB, and Nodal can biochemically interact other TGF-beta proteins such as GDF8, GDF8, and GDF11 (Yamashita and al., 1995). Cell Biol. 130:217-226 Lee and McPherron 2001, Proc. Natl. Acad. Sci. 98:9306-9310; Yeo & Whitman, 2001. Mol. Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is the primary receptor type I for activins, especially activin A. ALK-7 could also serve as a receptor, particularly activin B.
“In some embodiments, there are methods to treat an adynamic disorder of CKD-MBD in a subject. The method involves administering a therapeutically efficient amount of ActRII inhibitors to the subject. Methods for treating an adynamic form of CKD/MBD in a subject are also provided. The method involves administering a therapeutically-effective amount of ActRII inhibitor to a patient in need of treatment.
“In some cases, the adynamic bones disorder can be distinguished by the absence of tetracycline incorporation in mineralized bone.”
“In some embodiments, there are methods to treat a low bone turnover CKD/MBD form in a subject. The method involves administering a therapeutically-effective amount of ActRII inhibitor treatment to a subject with low bone turnover CKD/MBD. Osteomalacia is a low-bone turnover form of CKD/MBD.
“In some embodiments, there are methods to treat a bone disorder that is hyperphosphatemia in a subject. The method involves administering a therapeutically-effective amount of ActRII inhibitor treatment to the subject with the bone disorder.
“In some embodiments, there are methods to treat atherosclerotic calcium in a subject. The method involves administering a therapeutically-effective amount of ActRII inhibitor treatment to a subject with atheroscleroticcalcification.”
“In some embodiments, there are methods for treating renal disease in a subject. The method includes administering a therapeutically-effective amount of ActRII inhibitor to the subject who has the renal disease. A more specific example of a renal disease is renalfibrosis.
“In one embodiment, there is a method of treating extraskeletal calcium in a subject. This method involves administering a therapeutically-effective amount ActRII inhibitor. A method of preventing extraskeletal calciumification in a subject is also provided in this embodiment. This method involves administering a therapeutically-effective amount ActRII inhibitors to the subject. Extraskeletal calcification is treated or prevented by specific embodiments. This refers to the accumulation of calcium salts within the blood vessels of the subject (e.g., calcifications of the arteries).
“ActRII inhibitor can be used in certain embodiments. It is a polypeptide that contains an amino acid sequence chosen from the following group: 95% identical or SEQID NO.2; 98% exactly to the SEQID NO.3; 98% similar to the SEQID NO.3; 98% equal to the SEQID NO.6; 98% equal to the SEQID NO.7; 98% exact to the SEQID NO.7; 98% same to the SEQID NO.20; 95% to the SEQID NO.21; 95% to the SEQID NO.21; 98% of the SEQID NO.21; and 98% to the SEQID NO.21; 95% to the SEQID NO.21; 98% to the SEQID NO.21; and 98% to the SEQID NO.21; 98% to the SEQID NO.21; 98% to the SEQID NO.21; and 98% to the SEQID NO; The ActRII inhibitor, in a more precise embodiment, is a polypeptide that contains the amino acid sequence from SEQ ID No:7. The ActRII inhibitor can be administered parentally in a more specific form.
“A specific embodiment of the ActRIIA inhibit that can be used in conjunction with the methods herein is an ActRIIA inhibition. The ActRIIA inhibit comprises or consists a particular polypeptide from the following group: a. A polypeptide less than 90% identical SEQID NO.2; and b. A polypeptide more than 95% identical SEQID NO.3; and c. A polypeptide no greater than 98% identical SEQID NO.3;. e. A polypeptide of at least k. SEQID NO.6; m. A polypeptide that is at least 90% identical with SEQID NO.7; or o. A polypeptide that is at least 98% the same as SEQID NO.7; or p.SEQID NO.7; or q. A polypeptide of at least 98% identical with SEQID NO.12; r. A polypeptide less than 95% identical in SEQID NO.12; s. A polypeptide which is at least 98%ident to SEQID NO.12; s. The ActRIIA inhibitor, in a particular embodiment, is a polypeptide that contains or consists of the amino acid sequence from SEQ ID No:7.
“An ActRIIB inhibit can also be used in conjunction with the methods described herein. It consists or contains a specific polypeptide. The ActRIIB inhibitor can be described as a polypeptide that contains or consists of SEQ ID No:23. Another specific embodiment of the ActRIIB inhibit is a polypeptide consisting or comprising SEQ ID No:25.
An ActRIIA and ActRIIB inhibit can be combined in a specific embodiment. The ActRIIA inhibitor, which is a combination of SEQID NO:7 and ActRIIB inhibitor, is a particular embodiment. The ActRIIA inhibitor, a polypeptide consisting or consisting SEQ ID No:7, and the ActRIIB inhibit, a polypeptide consisting or consisting SEQ ID no:25 are two examples of a specific embodiment.
“In some embodiments, the subject to the treatment is younger than 18 years. The subject who is to be treated using the methods herein may have end-stage renal disease. The subject may need dialysis to be treated using the methods herein. The subject may be raised in certain embodiments.
“In some embodiments, methods are provided for treating or preventing hyperphosphatemia (due increase in phosphorus), additionalskeletal calcification (e.g. vascular calcification) and adynamic bones disorder in a patient. The method includes administering a therapeutically-effective amount of ActRII inhibitor (e.g., to a subject with hyperphosphatemia, secondary hypoparathyroidism due to an increase in phosphorus), and extraskeletal calciumification (e.g. vascular calcification
“5.1 Overview”
“The method described herein is a treatment for Chronic Kidney Disease-Mineral & Bone Disorders (CKDMBD). It involves administering an inhibitor to ActRII to a patient who is in need of treatment. An inhibitor of ActRIIA or ActRIIB can also be used.
“In some embodiments, there are methods for treating low bone turnover forms of CKD/MBD. The method involves administering an inhibitor to ActRII to a patient who is in need of treatment. Methods for treating CKD-MBD characterized as hyperphosphatemia or hypercalcemia are provided in certain embodiments. Methods for treating CKD-MBD characterized with extraskeletal calcification such as atherosclerotic calcification are provided in certain embodiments.
“In certain embodiments, methods are provided for treating CKD-MBD where the chronic kidney disease has reached stage 3 stage 4 stage 5 or 5 stage 5. A specific embodiment refers to kidney disease that is at the end of its life. Methods for treating CKD-MBD are provided in certain embodiments. These methods can be described as having a glomerular filtration of less than 60 ml/min/1.73 m2 for adults and less than 89 ml/min/1.73 m2 for pediatric patients. See, Moe et al., 2006, Kidney International 69:1945-1953. Certain embodiments of the methods are for adults with CKD-MBD. They have glomerular filtration rates of less than 50 ml/min/1.73 m2, and 40 ml/min/1.73 m2, respectively. Certain embodiments provide methods for treating CKD-MBD in children. These include methods that have a glomerular filtration of less than 80 ml/min/1.73 m2, 70 ml/min/1.73 m2, 60 ml/min/1.73 m2, 10 ml/min/1.73 m2, and less than 10 ml/min/1.73 m2.
“Gglomerular filtration rates below 60 ml/min/1.73m2 for adult patients and 89 ml/min/1.73m2 for pediatric patients can cause detectable abnormalities in calcium, phosphorus, and vitamin D metabolism. These abnormalities may lead to bone disease.
“In some embodiments, methods are provided for treating bone pathology related to chronic kidney disease (CKD-MBD). See Moe et al., 2006, Kidney International 69:1945-1953. The CKD -MBD can be low-turnover CKD -MBD in certain cases. The histological features listed in Table 1 below can help you diagnose low-turnover CKD/MBD. National Kidney Foundation, Kidney Disease Outcomes Quality Initiative Guidelines on the website of National Kidney Foundation.
“TABLE 1\nHistological Features of Low-Turnover CKD-MBD\nFeature Adynamic Osteomalacia\nBone Formation\nTrabecular bone volume Normal, low Variable\nLow, normal or high\nOsteoid volume Normal, low High-very high\nOsteoid seam thickness Normal, low High-very high\nNumber of osteoblasts Low Low\nBone formation rate Low-very low Low-very low\nMineralization lag time Normal Prolonged\nBone Resorption\nEroded bone perimeter Normal, low Variable\nOften low, may be high\nNumber of osteoclasts Low Low, may be normal or high\nMarrow fibrosis Absent Absent”
“In one embodiment, there is a method of treating extraskeletal calcium in a subject. This method involves administering a therapeutically-effective amount ActRII inhibitor. A method of preventing extraskeletal calciumification in a subject is also provided in this embodiment. This method involves administering a therapeutically-effective amount ActRII inhibitors to the subject. Extraskeletal calcification is treated or prevented by specific embodiments. This refers to the accumulation of calcium salts within the blood vessels of the subject (e.g., calcifications of the arteries).
“In some embodiments, the methods for treatment or prevention extraskeletal calciumification, e.g. vascular calcification are applied to a subject at risk of developing extraskeletal calcification. The ActRII inhibitor is administered to the subject in accordance the methods described herein. In a specific embodiment, the subject at risk of suffering from extraskeletal calcification, e.g., vascular calcification, has hypercholesterolemia. Another specific embodiment shows that the subject at high risk for extraskeletal calcification (e.g.,vascular calcification) has hypertension. Another specific embodiment has the subject at high risk for extraskeletal calcification (e.g.,vascular calcification). Another specific embodiment states that the subject at high risk for extraskeletal calcification (e.g. vascular calcification) has end-stage renal disease. Another specific embodiment states that the subject at high risk for extraskeletal calcification (e.g.,vascular calcification) has chronic kidney disease. Another specific embodiment states that the subject at high risk for extraskeletal calcification (e.g. vascular calcification) has elevated oxidative stress. This is e.g. an imbalance between antioxidant activity and oxidant production in the vasculature. Another specific embodiment shows that the subject at high risk for extraskeletal calcification (e.g. vascular calcification) has a calcification inhibitor defect (e.g. a deficiency or combination of matrix gla protein (MGP), fetuin A (OPG ).
“Intima calcification” can be described as the result of vascular calcification in certain embodiments. Intima calcification can be associated with atherosclerosis. It progresses along with the progression of atherosclerotic plaques.
“In some embodiments, a subject who is suffering from CKD or extraskeletal calcification (e.g. vascular calcification) has an increased level of FGF23. This is relative to FGF23 levels in subjects who are not suffering or at risk from CKD or extraskeletal calcification (e.g. vascular calcification). FGF23 levels can be determined using techniques that are well-known in the art. For example, ELISA is used to detect FGF23 using blood and serum samples. A specific embodiment of FGF23 is the amount detectable in serum in a subject who has, or may have, a form or extraskeletal calcification. This can be used to determine if the subject is suffering from, and/or at risk from suffering from, CKD/MBD or vascular calcification. Another specific embodiment shows that the FGF23 level (e.g. the level detectable within the serum) of a subject who is suffering from or at risk from CKD/MBD or extraskeletal calcification (e.g. vascular calcification) in a subject who is not suffering from or at risk from suffering from CKD/MBD or extraskeletal calcification (e.g. vascular calcification) is approximately 5-10% to 10%, 10-15%, 20%, 15%, 20%, a serum) in a serum level of FGF23 level detectable inside the serum level) of FGF23 in a serum level in a serum level) in a patient not having CKD/or CKD- or extraskeletal calcification
“In some embodiments, the levels of FGF23 in a patient suffering from CKD or extraskeletal calcification can be used to assess whether the method is effective. These methods include administering an ActRII inhibitor that is therapeutically effective. A subject who has been treated according to one or more of these methods shows a decrease in FGF23 (e.g. as measured in the serum) relative to the level that was detected in the subject before treatment with the method described herein. A specific embodiment shows that the level FGF23, i.e. the level detectable within the serum, in a subject with CKD-MBD or extraskeletal calcification (e.g. vascular calcification) is decreased by approximately 5%, 10% and 20% relative to the level FGF23 (e.g. the serum level) found in the subject before treatment. Another specific embodiment shows that the level FGF23 (e.g. the serum level) in a subject who is suffering from CKD-MBD or extraskeletal calcification (e.g. vascular calcification) is decreased by approximately 5-10% or 10%, 20%, 30%, 40%, 50%, 40%, 50%, 60%, 50-60% and 50-75% relative to the level FGF23 (e.g. the serum level) which was detected in the subject before treatment.
“In a particular embodiment, there is a method for treating CKD/extraskeletal calcification. This involves: (i.) administering an ActRII inhibition to an individual with CKD/extraskeletal calcification. (ii.) determining the amount FGF23 present in a tissue sample (e.g. serum) from the individual. (iii.) Repeating the ActRII inhibit administration. If the FGF23 level is not reduced after administration of the ActRII inhibit, then the ActRII inhibition can be increased in dose. If FGF23 levels are not decreased after administration of ActRII inhibitors, it is possible to increase the frequency with which the ActRII inhibit is administered.
“In some embodiments, a subject who is suffering from CKD/MBD or extraskeletal calcification (e.g. vascular calcification) has higher levels of sclerostin. This protein increases in those subjects who are not at high risk of developing CKD/MBD or extraskeletal calcification (e.g. Graciolli et.al., 2010 J Am Soc Nephrol 21.774A). The methods for detecting sclerostin levels are well-known in the art. For example, ELISA can be used to detect sclerostin using blood and serum samples. A specific embodiment of sclerostin levels can be measured using methods known in the art, e.g. ELISA. Subjects are tested for sclerostin in blood and serum samples. Another specific embodiment shows that the serum level of sclerostin in a subject who is suffering from CKD/MBD or extraskeletal calcification (e.g. vascular calcification) is approximately 5-10% to 10%, 10-15%, 20%, 30%, 40%, 50%, 40%, 50%, 50-60% and 50-75% respectively. This level is greater than the serum level of sclerostin in a person who is not at risk or suffering from CKD/extraskeletal calcification
“In some embodiments, the levels of sclerostin found in a subject with CKD or extraskeletal calcification can be used to assess whether the method is effective. These methods include administering an ActRII inhibitor that is therapeutically effective. A subject who has been treated according to one or more of these methods will have a lower level of sclerostin than the amount found in their serum prior to treatment. Another specific embodiment shows that the serum level of sclerostin in a subject with CKD-MBD or extraskeletal calcification (e.g. vascular calcification) is lower than the level detected in the serum prior to treatment using the method described herein. Another specific embodiment reduces the amount of sclerostin in a subject with CKD-MBD or extraskeletal calcification. This is relative to the serum level that was detected in the subject before treatment.
“In a particular embodiment, there is a method for treating CKD/extraskeletal calcification. This includes: (i.) administering an ActRII inhibition to an individual with CKD/extraskeletal calcification. (ii.) determining the amount sclerostin present in a tissue sample of the individual. (e.g. a different serum sample from the same person) and then repeating the ActRII inhibit administration. The ActRII inhibitor can be given at a higher dose if the amount is not reduced by administration of ActRII inhibitor. If the ActRII inhibit administration does not cause a decrease in sclerostin, then the ActRII inhibition can be administered more frequently.
“In some embodiments, the person suffering from vascular calciumification treated according to the methods described herein has less than 18 years of age. A specific embodiment of vascular calcification is a subject less than 13 years old. Another specific embodiment of vascular calcification is a subject less than 12 years old, less than 11, less that 10, less than 9, less over 8, less about 7, less 6 or less 5 years. Another specific embodiment of vascular calcification is the subject who has been treated according to the methods herein is between 1-3 and 5-7 years. 7-9 years. 7-9 years. 7-9 years. 7-9 years. 7-9 years. 7-9 years. 7-9 years. 7-9 years, 7-9, 7-9, 7-9, 7-9, 7-9, 7-9, 7-9, 7-9, 7-9, 7-9, 6-7, 7-8, 7-8, 7-8, 6-7, 7, 6-7, 6, less than 30 or more than 30 year old. Another specific embodiment of vascular calcification is the subject who has been treated according to the methods herein is between 30-35 and 40 years old. Another specific embodiment of vascular calcification is the 60-65 year old subject, 65-70 year old, 70-75, 75-80, or more than 80 years.
“In some embodiments, the subject with vascular calcification treated according to the methods described herein has end-stage renal disease. The subject with vascular calcification is treated according to the methods herein and requires dialysis.
“In some embodiments, the effectiveness or prevention of extraskeletal calciumification, e.g. vascular calcification is evaluated using one or more of the assays that are known to those skilled in the art. Section 5.3(a),(iv) describes some examples of assays. According to such embodiments, one skilled in the art will know that an ActRII inhibitor-treated subject may be adjusted according to the results of assays. A subject treated with an ActRII inhibit may receive a higher dose or may be given an ActRII inhibition more often (i.e. the time between doses may be decreased). A subject treated with a method herein may receive a lower dose of ActRII inhibitor or may be administered an ActRII inhibit less often (i.e. the time between dose administrations could be increased).
“In some embodiments, the methods herein improve the symptoms of hyperphosphatemia, secondary hyperparathyroidism due to an increase in phosphorus, and extraskeletal calciumification, e.g. vascular calcification. The methods described herein can be combined with any method that is known to the skilled tradesperson to determine the severity of the symptoms. Specific embodiments of the methods described herein improve one or more symptoms associated with vascular calcification. Examples of symptoms include an increase in vascular calcium (e.g. arterial), increased apoptosis in vascular smooth muscles cells, loss arterial elasticity, development of left-ventricular hypertrophy, decreased coronary artery perfusion and myocardial inchaemia.
“In some embodiments, the methods herein result in an increase in the levels vascular calcium in a subject by at minimum 5%, 10% and 15%. Certain embodiments result in a decrease of vascular calcium in a subject, e.g. arterial calcium.
“In a particular embodiment, there is a method for reducing the levels vascular Calcium in a subject. It involves: (i.) administering an ActRII inhibition to a subject with CKD-MBD or extraskeletal calcification; (ii.) determining the amount vascular Calcium in a tissue sample (e.g. serum) from said subject. (iii.) if this amount is less than the amount determined in a similar tissue sample from the subject prior to administration of ActRIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII) then repeating the ActRIIIIIIIIIIIIIIIIIIIIIIIIIIIIiiiIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiiv The ActRII inhibitor can be given at a higher dose if there is no decrease in vascular calcium after administration. If the amount of vascular Calcium following administration of ActRII inhibitor is not decreased, it can be increased the frequency with which the ActRII inhibit is administered.
“In some embodiments, the methods herein result in a decrease of the Agatston score for a subject at risk or having vascular calcification. The methods described herein can result in a 5%-10%, 15%, 20% and 25% decreases in the Agatston score for a subject with vascular calcification or at risk. Another specific embodiment results in a 5%-10% to 15%, 10%-15% to 25%, 25%-30% to 30%, 40%-40% to 40%, 40%-45% or 45%-50% reduction in the Agatston score for a subject with vascular calcification or at risk.
“In another embodiment, the methods herein result in a reduction in calcium levels in the vasculature. This could be a decrease of calcium levels in one or more of the subjects arteries, or a subject at risk for developing vascular calciumification. Another specific embodiment results in a decrease of phosphorus levels in the blood, e.g. a decrease of phosphorus levels in one or more of the subjects’ arteries, e.g. a subject at risk or having vascular calcification.
“In some embodiments, methods are provided for treating low-turnover bone disorders. The tests described in Section 5.3 (a) below can help you determine if your bone turnover is low. The biochemical markers for bone turnover include serum or urine collagen crosslinks (N?telopeptide and C-telopeptide), bone specific alkalinephosphatase and serum osteocalcin. 25 hydroxyvitamins D and parathyroid hormone?. A low turnover bone disorder can be described as a dynamic bone disorder in a particular embodiment. A patient who is treated using the methods described herein will see a decrease in bone turnover of at most 10%, 20% or 25%. This can be achieved by reducing bone turnover by 50%, 60%, 70% and 75% respectively. A patient treated using the methods herein can have a decrease in bone turnover of up to 10%, 20%, 30%, 40% or 50%. This is in addition to a possible reduction of up to 70%, 75% and 80%. A patient who is treated using the methods herein can expect a decrease in bone turnover of between 10% and 25%, 20%, 35%, 30%, 40%, 55%, 50%, 60%, 75% and 70%, 85%, 95%, 90%, and 100%. The reduction in bone turnover can be compared with historical data for the same patient. Other embodiments compare the decrease in bone turnover to the average bone turnover of a population that does not have bone disorders. The population that does not have bone disorders may be the same age as the patient and/or the same sex.
“In one embodiment, there is a method for treating a low-turnover bone disorder. This includes: (i.) administering an ActRII inhibition to a subject with a low-turnover bone disorder; and (ii.) determining the level or bone-turnover in the subject after administration of ActRII inhibit (e.g. by using one of the tests described in Section 5.3(a), or by measuring one or several biochemical markers of bone growth); and (iii). compared to the ActRIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII I’st the ActRIIIIIIIIIIIIIIIIIIIIIIIIIIIiiiIIIIIIIIIIIIIIIIIIIIIIIIIIIII to the ActRIIIIIIIIIIIIIIIIIIIIIIIIII the ActRIIIIIIIIIIIIIIIIIIIIIIIIIII the ActRII the ActRII the ActRII the ActRII the ActRII the ActRII the ActRII the ActRIII inhibitor If the ActRII inhibit does not reduce bone turnover, then the ActRII inhibition can be increased. If the ActRII inhibit does not cause a decrease in bone turnover, certain embodiments allow for an increase in the frequency of administering the ActRII inhibition.
“5.2 Inhibitors to ActRII”
“(a) ActRIIA Inhibitors”
“ActRIIA” is the term used herein. The term “ActRIIA” refers to any family of activin receptor type IIa proteins (ActRIIA), from any species, and variants derived by such ActRIIA proteins through mutagenesis or another modification. ActRIIA is understood to refer to any of the forms currently known. ActRIIA proteins are transmembrane proteins. They consist of a ligand binding extracellular domain with a cysteine rich region, a transmembranedomain, and a domain in the cytoplasm with predicted serine/threoninekinase activity.
“ActRIIA inhibitors” can be used in compositions and methods as described in this document. They include activin-binding soluble ActRIIA proteins; antibodies that bind activin (particularly activin A and B subunits) and disrupt ActRIIA bindin; and antibodies that bind ActRIIA and activate activin binding.
“In some embodiments, two or three different proteins (or moieties) that have activin-ActRIIA binding activity, particularly activin binders that block type I (e.g. a soluble Type I activin receptor), binding sites respectively may be linked to form a bifunctional, multifunctional binding molecule that inhibits ActRIIA. This can be used in the compositions or methods described herein. In some embodiments, Activin – ActRIIA signaling axis antagonists include nucleic acids aptamers, small molecules, and other agents.
“(i) ActRIIA Inhibitors Comprising ActRIIA Polypeptides”
“ActRIIA polypeptide” is a term. “ActRIIA polypeptide” means any naturally occurring polypeptide from an ActRIIA member, as well as any variants (including fragments, fusions and peptidometic forms) that still have a useful activity. ActRIIA Polypeptides, for example, are polypeptides that have a sequence at most 80% similar to an ActRIIA sequence and optionally 85%, 90% or 97% of the sequence of an ActRIIA protein. An ActRIIA polypeptide could bind to or inhibit the function an ActRIIA protein/activin, for example. A polypeptide called ActRIIB may be chosen for its ability to stimulate bone growth and mineralization. ActRIIA peptides are human ActRIIA precursor polypeptides (SEQ ID No: 1) and human ActRIIA soluble polypeptides (12, 7, and 12). The ActRIIA precursor peptide’s amino acid sequence can be found at SEQID NO:1. The signal peptide is located at amino acids 1 to 20, while the extracellular domain is at amino acids 21 to 135 and N-linked glycosylation locations of the human ActRIIA preprod polypeptide (SEQID NO: 1) at amino acids 43 and 56. The SEQ ID No:4 nucleic acid sequence that encodes the human ActRIIB precursor protein is available (nucleotides 163-1705 in Genbank entry NM001616). SEQ ID No:5 is the nucleic acid sequence that encodes the soluble human ActRIIA protein of SEQID NO:2. For a detailed description of these sequences, see Table 6.
“In certain embodiments, ActRIIA soluble polypeptides are used in the compositions or methods described herein. The ActRIIA protein’s extracellular domain can bind activin and can therefore be called a soluble activin-binding ActRIIA peptide. As such, the term “soluble ActRIIA protein” is used in this document. The term?soluble ActRIIA polypeptide? generally refers to any polypeptide containing an ActRIIA extracellular domain. This includes any naturally occurring extracellular Domain of an ActRIIA proteins as well any variants (including fragments, mutants, and peptidometic forms). However, soluble ActRIIA proteins can bind to activin. The wild type ActRIIA protein has a lower selectivity than GDF8/11. By coupling native or modified ActRIIA proteins with an activin-selective, second binding agent, they may be given additional specificity for activin. The soluble polypeptides shown in SEQ ID NOS: 2, 3, 7, 12, and 13 are examples of activin-binding ActRIIA soluble polypeptides. Another example of soluble, activin binding ActRIIA polypeptides is the signal sequence. This sequence can be found in SEQ ID NOS: 2, 3, 7, 12 and 13. TPA is used to illustrate the ActRIIA-hFc peptide.
“In some embodiments, the ActRIIA inhibitors used in the compositions or methods described herein consist of a conjugate/fusion proteins comprising an activin binding domain of ActRIIA linked with an Fc portion an antibody. In some embodiments, an activin-binding protein is linked to an Fc section of an antibody using a linker (e.g., an peptide linker). Optionally, the Fcdomain may contain one or more mutations at residues like Asp-265 and lysine 322, as well as Asn-434. The Fc domain mutant with one or more of these mutations, such as an Asp-265 mutation, may have a decreased ability to bind to it. Fc domain is more sensitive than a wild-type Fc. Other cases show that a mutant Fc domain with one or more mutations (e.g. an Asn-434 substitution) has a greater ability to bind the MHC class 1-related Fc receptor (FcRN), relative to a wild type Fc domain. SEQ ID NOS 6-7, 12, and 13 show examples of fusion proteins that combine an ActRIIA soluble extracellular domain with an Fc domain.
“ActRIIA inhibits are used in specific embodiments. They comprise the extracellular Domain of ActRIIA or a portion thereof linked to an Fc section of an antibody. The ActRIIA inhibition contains an amino sequence that is at minimum 75% identical to one selected from SEQ ID Nos. 6, 7, 12 and 13. Another embodiment of the ActRIIA inhibits used in the compositions, methods and methods described herein comprises the extracellular domain or a portion thereof linked to an Fc section of an antibody. In this case, ActRIIA contains an amino sequence that is at minimum 80%, 85% and 90% identical to an amino sequence chosen from SEQ ID Nos: 6, 7, 12 and 13.
“In some embodiments, ActRIIA inhibitors used in compositions and methods herein contain a truncated version of an extracellular Domain of ActRIIA. The carboxy terminus or the amino terminus can be affected by the truncation of ActRIIA polypeptide. The truncation may be 1, 2, 3, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17-18 19, 20, 21, 22, 23 24, or 25 amino acid long relative to mature ActRIIB protein extracellular domain. The truncation in certain embodiments can be 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23 24, or 25 Nterminal amino acids of mature ActRIIA Polypeptide Extracellular Domain. The truncation may be 1, 2, 3, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17-18, 19, 20, 22, 23, 24, or 25 N-terminal amino acid of the mature ActRIIA Polypeptide Extracellular Domain. The truncated ActRIIA forms include polypeptides containing amino acids 20-128, 20-129, 20-135, 20-138, 20-135, 20-135, 20-135, 20-131, 20-132, 20-135, 20-135, 20-135, 20-131, 20-132, 20-133, 20-134, 20-135, 20-132, 20-131, 20-132, 20-135, 20-135, 20-131, 20-133, 20-134, 20-134, 20-131, 22-131, 23-131, 24-131; and the positions in SEQ ID No:1
“In some embodiments, the ActRIIA inhibitors used in the compositions or methods described herein consist of an extracellular domain of ActRIIA that contains one or more amino acids substitutions. The ActRIIA inhibitors used in certain embodiments include a reduced form of the ActRIIA extracellulardomain that also contains an amino acid substitution.
“ActRIIA inhibitor is a fusion protein that binds to the extracellular domain of human ActRIIA receptor with the Fc portion IgG1. Another embodiment of the ActRIIA inhibit to be used in these compositions and methods is a fusion proteins between the Fc portion IgG1 and the truncated human ActRIIA receptor extracellular domain. Another embodiment of the ActRIIA inhibit to be used in these compositions and methods is a Fusion Protein between the ActRIIA antagonist and the Fc portion IgG1. The fusion protein contains one or more amino acids substitutions.
“Functionally active fragments ActRIIA Polypeptides can be obtained by screening polypeptides that have been recombinantly made from the fragment of the nucleic acids encoding the ActRIIA protein. Chemically, fragments can also be synthesized using methods such as t-Boc and Merrifield solid phase moc chemistry. You can produce the fragments either recombinantly, or chemically synthesis. Then you can test them to determine which peptidyl fragments are capable of acting as antagonists or inhibitors of ActRIIA protein signaling or activin.
“In addition to functionally active variants ActRIIA Polypeptides, it is possible to obtain them by screening libraries modified polypeptides that have been recombinantly made from the mutagenized nucleic acid encoding ActRIIA. These variants can be made and tested to determine if they can inhibit or act as antagonists of ActRIIA protein signaling. A functional variant of ActRIIA polypeptides may contain an amino sequence that is at most 75% identical to one selected from SEQ ID Nos 2 or 3. The functional variant may contain an amino sequence that is at least 80% or 85% or 90% identical to the sequence from SEQ ID Nos 2 or 3.
“Functional variants can be created, for example by altering the structure of an ActRIIA protein for therapeutic efficacy or stability (e.g. ex vivo shelf-life and resistance to proteolytic degrading in vivo). These modified ActRIIA proteins, when they are selected to retain activin binding can be considered functional counterparts to the naturally occurring ActRIIA proteines. Modified ActRIIA proteins can be made by amino acid substitution, deletion or addition. It is reasonable to assume that an isolated substitution of a leucine, isoleucine, valine, aspartate, glutamate, or threonine with serine or similar replacement of an amino acid with a structurally similar amino acid (e.g. conservative mutations), will not have a significant effect on the biological activity. Conservative replacements are those occurring within a family that is related in their side chain. It is possible to determine if an ActRIIA amino acid sequence change results in a functional homolog by looking at the ability of the variant ActRIIA protein to stimulate cells in a manner similar to wild-type ActRIIA.
“In some embodiments, the ActRIIA inhibit to be used with the compositions and techniques described herein might comprise an ActRIIA protein having one or more specific mutations which can alter the glycosylation. These mutations can introduce or remove one or more glycosylation site(s), such as O-linked and N-linked glycosylations sites. Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine (or asparagines-X-serine) (where ?X? Any amino acid that is recognized by appropriate cell glycosylation enzymes. Alterations can also be made to the sequence of wild-type ActRIIA polypeptides by adding or substituting one or more serine and threonine amino acids (for O-linked Glycosylation Sites). Modifying the tripeptide sequence to not allow for glycosylation can be achieved by a variety of amino acids substitutions and deletions. Chemical or enzymatic coupling glycosides to an ActRIIA protein can also increase the number of carbohydrate moieties. The coupling method used will determine whether the sugar(s), arginine or histidine, are attached to the ActRIIA polypeptide. These methods are described by WO 87/05330, published Sep. 11, 1987; and Aplin and Wriston (1981), CRC Crit. Rev. Biochem., pp. Biochem., pp. 259-306, which is incorporated herein by reference. One or more carbohydrate moieties on ActRIIA polypeptides can be removed chemically and/or enzyme-wise. Chemical deglycosylation may involve, for example, exposure of the ActRIIA 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. Hakimuddin and colleagues further describe chemical deglycosylation. (1987) Arch. Biochem. Biophys. 259:52, and by Edge et. al. (1981) Anal. Biochem. 118:131. Thotakura and colleagues described a range of exo- and endo-glycosidasses that can be used to cleave carbohydrate moieties from ActRIIA polypeptides. (1987) Meth. Enzymol. 138:350. 138:350 ActRIIA proteins can be expressed in mammalian cells that provide proper glycosylation (e.g. HEK293 and CHO cell lines), but other expression systems such as mammalian expression lines, yeast cell strains with engineered glycosylation enzymes, or insect cells are also possible.
“Further, herein are methods for generating mutants. Particularly sets of combinatorial mutations of an ActRIIA protein, as well as truncation variants. Pools of combinatorial mutations are particularly useful in identifying functional variants. Screening such combinatorial libraries might be used to create variants of ActRIIA polypeptides that can either act as agonists or antagonists, or even have novel activities. Below are a variety of screening methods that can be used to screen variants. An ActRIIA variant of a polypeptide may be tested for its ability to bind an ActRIIA protein ligand. This could prevent ActRIIA proteins from binding to ActRIIA aminopeptides.
“Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to a naturally occurring ActRIIA polypeptide. Mutagenesis can also result in variants with intracellular half-lives that are dramatically different from the wild-type ActRIIA protein. The altered protein may be made more or less resistant to proteolytic degradation or other cellular processes that result in the destruction or inactivation of native ActRIIA proteins. These variants and their genes can be used to modify the half-life ActRIIA peptides. A shorter half-life, for example, can lead to more transient biological effects. This can enable tighter control over recombinant ActRIIA protein levels within patients. Mutations can be made to an Fc fusion protein’s linker and/or Fc portion in order to alter its half-life.
A combinatorial library can be made by combining a number of genes to encode a collection of polypeptides that each contain at least one of the potential ActRIIA sequences. A mixture of synthetic oligonucleotides may be enzymatically linked into gene sequences to form a combination of polypeptides. The degenerate set potential ActRIIA nucleotide sequences can then be expressed as individual polypeptides or as a set larger fusion proteins (e.g. for phage display).
There are many ways to generate a library of homologs from a degenerate sequence of oligonucleotides. An automatic DNA synthesizer can chemically synthesize a degenerate sequence of genes. The synthetic genes are then ligated into the appropriate vector for expression. The art of synthesizing degenerate Oligonucleotides has been well documented. See, for example, Narang S. (1983) Tetrahedron39:3; Itakura and al. (1981) Recombinant DNA Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al., (1984) Annu Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477). These techniques were used in directed evolution of other proteins (see Scott et.al., (1990), Science 249, 386-390; Roberts, et.al., (1992), PNAS USA, 89:2429-2433; Devlin, et.al. (1990), Science 249, 404-406; Cwirla, et.al. (1990), PNAS USA USA 87, 6378-6382, as well as U.S. Pat Nos. Nos.
You can also use other methods of mutagenesis to create a combinatorial library. By screening for alanine scanning mutation and similar methods, ActRIIA variants of polypeptides can be created and isolated from a library (Ruf et.al., 1994) Biochemistry 33:1565-1572; Wang and co., 1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-29292; Lowman and colleagues (1991) Biochemistry 30, 10832-10838. Cunningham et.al. (1989) Science 244 :1081-1085. By linker scanning mutation (Gustin and colleagues (1993) Virology 193 :653-660; Brown, et.al. (1992) Mol. Cell Biol. 12:2644-2652 McKnight et.al., (1982), Science 232,316); by saturation mutagenesis [Meyers et.al., (1986), Science 232,613]; by PCR mutagenesis [Leung et.al., (1989), Method Cell Mol Biol 1:11-19]); or by random mutagenesis. (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis is a great way to identify truncated or bioactive ActRIIA polypeptides, especially in combinatorial settings.
There are many techniques that can be used to screen gene products from combinatorial libraries created by point mutations or truncations. These techniques can be used to quickly screen gene libraries created by combinatorial mutation of ActRIIA polypeptides. Cloning large gene libraries into replicable expression vectors is the most common method for screening them. The vectors are then transformed into appropriate cells and the genes of combination are expressed under conditions that allow for the detection of desired activity. This allows for relatively easy isolation of the vector coded by the gene. The most popular assays are activin binding assays or activin-mediated cells signaling assays.
“ActRIIA Polypeptides may be used as inhibitors in certain embodiments. These post-translational modifications can also be present in ActRIIA proteins. These modifications can include, but not be limited to, acetylation and carboxylation, glycosylations, phosphorylation. lipidation, acylation, and phosphorylation. The modified ActRIIA proteins may also contain non-amino acids elements such as lipids or polyethylene glycols. Any method that is available to the skilled artisan can be used to test the effects of these non-amino acids elements on the functionality and function of an ActRIIA protein. Post-translational processing can also be crucial for proper folding and/or functioning of an ActRIIA protein when it is created in cells by cleaving the nascent ActRIIA form. Different cells, such as CHO, HeLa and MDCK, 293, W138 or HEK293, have different cellular machinery and specific mechanisms for such post-translational activity. These may be used to modify and process the ActRIIA proteins.
“In some aspects, functional variants of ActRIIA proteins used in the inhibitions of the methods described herein include fusion protein having at least a part of the ActRIIA peptides and one to more fusion domains. Examples of fusion domains are polyhistidine (Glu-Glu), glutathione S Transferase (GST), thioredoxin and protein A. Also, there is an immunoglobulin heavy chains constant region (Fc), maltose binding proteins (MBP) or human serum albumin. You can select a fusion domain to confer desired properties. Some fusion domains can be used to isolate fusion proteins using affinity chromatography. The appropriate matrices for affinity purification are used, including glutathione, amylase, nickel- and cobalt-conjugated reagents. Many of these matrices can be purchased in the?kit? Many of these matrices are available in?kit? form, such the Pharmacia GST purification systems and the QIAexpress? (Qiagen), which can be used with (HIS6) Fusion partners. Another example is the selection of a fusion domain to aid in detection of ActRIIA polypeptides. These domains can be used to detect various fluorescent proteins, such as GFP, as well as?epitope tag?. These are often short peptide sequences that can be used to identify a specific antibody. There are several well-known epitope tags that can be used to identify monoclonal antibodies. These include FLAG, HA and cmyc tags. Some fusion domains contain a protease cleavage location, such as Factor Xa and Thrombin. This allows the relevant protease partial digestion of the fusion proteins, and thereby liberates the recombinant protein from them. By subsequent chromatographic separation, the proteins can be separated from the fusion domain. An ActRIIA protein is fused to a domain that stabilises it in vivo. This is known as a “stabilizer”. domain). By ?stabilizing? Is meant any action that increases serum half-life, regardless of whether it is due to decreased destruction, decreased kidney clearance, or some other pharmacokinetic effect. It is known that fusions with the Fc part of an immunoglobulin can confer desirable pharmacokinetic characteristics on a wide variety of proteins. Also, fusions with human serum albumin may confer desirable properties. You can also choose fusions to human serum albumin that have multimerizing domains, such as dimerizing, trimerizing domains, and functional domains, which confer additional biological functions, such as stimulation of bone or muscle growth.
It is possible to arrange different elements of fusion proteins in any way that achieves the desired functionality. An ActRIIA peptide can be placed Cterminal to a heterologous or alternatively, a heterologous peptide may be placed in the C-terminal of an ActRIIA peptide. The ActRIIA polypeptide and heterologous domains need not be adjacent in the fusion protein. Additional domains and amino acid sequences can be added C- or Nterminal to either of the domains, or between them.
“In some embodiments, ActRIIA peptides may be modified to stabilize the ActRIIA peptides. These modifications can, for example, increase the in vitro half-life of ActRIIA proteins, prolong the circulatory half-life of ActRIIA proteines, or decrease the proteolytic degradation. These stabilizing modifications can include, but not be limited to, fusion protein (including, for instance, fusion proteins consisting of an ActRIIA Polypeptide and a stabilizerdomain), modifications to a glycosylation website (including, as an example, adding a glycosylation location to an ActRIIA Polypeptide), and modifications to carbohydrate moiety (including the removal of carbohydrate moieties in an ActRIIA Polypeptide). An ActRIIA polypeptide and a stabilizer domain, such as an IgG molecular (e.g. an Fc domain) are fused in the case of fusion protein. The term “stabilizer domain” is used herein. The term “stabilizer domain” does not just refer to a fusion domain, as in the case with fusion proteins. It also includes nonproteinaceous modifications, such as a carbohydrate moiety or nonproteinaceous copolymer like polyethylene glycol.
“In some embodiments, purified and/or isolated ActRIIA proteins, which have been isolated from other proteins or are otherwise substantially free of them, can be used in conjunction with the methods and compositions described. ActRIIA can be expressed from recombinant DNA.
“In certain aspects, ActRIIA peptides used for compositions and methods are created using isolated and/or modified nucleic acid encoding any ActRIIA peptides (e.g. soluble ActRIIA peptides), as well as fragments, functional variants, and fusion proteins. SEQ ID No: 4 encodes the ActRIIA precursor, and SEQ ID 5 encodes ActRIIA’s extracellular domain. These nucleic acid can be either single-stranded, or double-stranded. These nucleic acid can be either DNA or RNA molecules. These nucleic acid can be used in various ways, including to make ActRIIA polypeptides, or as therapeutic agents (e.g. in a gene therapy).
“In some aspects, nucleic acid encoding ActRIIA Polypeptides might include nucleic acid variants of SEQID NO: 4 and 5. Variant nucleotide sequences are sequences that differ by one to three nucleotide additions, substitutions, or deletions.
“In some embodiments, the nucleic acids sequences that encode ActRIIA polypeptides can be isolated or recombinant and less than 80%, 85% or 90%. They may also be 95%, 97% or 98%. They may be 99% identical to SEQID NO: 4 or 5. A person of ordinary skill in art will be able to recognize that nucleic acids sequences complementing SEQ ID No: 4 or 5 and variants SEQ ID no: 4 and 5 can be used to produce ActRIIA proteins suitable for use with the methods and compositions discussed herein. Further embodiments allow for such nucleic acids sequences to be isolated, recombinant and/or fused with a heterologous sequence or from a DNA database.
“In other embodiments nucleic acid used in the production ActRIIA polypeptides may include nucleotide sequencings that hybridize under extremely stringent conditions to the nucleotide-sequences designated in SEQID NO: 4 and 5, complement sequence of SEQID NO: 4 and 5, or fragments thereof. An ordinary person with knowledge of the art will know that stringency conditions that promote DNA hybridization may be adjusted. One can do hybridization at approximately 6.0 times sodium citrate/sodium chloride (SSC), at 45 degrees Celsius. Then, one can wash the DNA with 2.0 times SSC at fifty degrees Celsius. You can choose from a low salt concentration of approximately 2.0 times SSC at fifty degrees Celsius to a high salt concentration of about 0.2x SSC at fifty degrees Celsius. The wash step temperature can also be adjusted from room temperature at 22 degrees Celsius (low stringency) to 65 degree Celsius (high stringency). Temperature and salt can be varied or the temperature or salt concentration may change while one variable is maintained constant. One embodiment allows nucleic acid to be hybridized under low stringency conditions, such as 6x SSC at ambient temperature and 2x SSC at night. This can be combined with the methods described herein.
“Isolated nucleic acid that differs from the nucleic acid as described in SEQ ID Nos: 4 and 5 due to degeneracy of the genetic code can also be used in the manufacture of ActRIIA Polypeptides, suitable for use with the methods and compositions discussed herein. A number of amino acids can be identified by multiple triplets, for example. Codons that identify the same amino acid or synonyms may lead to?silent? Mutations that do not alter the amino acid sequence of the protein are possible. It is possible that DNA sequence polymorphisms which do cause changes in the amino acids sequences of subject proteins may exist in mammalian cells. Natural allelic variation may cause variations in nucleotides that encode a specific protein.
“In some embodiments, the regulatory nucleotide sequences of a expression construct may be operably linked with recombinant DNA. The regulatory nucleotide sequences used to express genes will usually be suitable for the host cell. There are many types of suitable expression vectors and regulatory sequences that can be used for different host cells. These regulatory sequences can include, but not be limited to, promoter, leader, signal, or transcriptional sequences, translational start or termination sequences and enhancer/activator sequences. These are known as constitutive and inducible inducers. These promoters can be naturally occurring promoters or hybrid promoters that combine elements from more than one promoter. An expression construct can be found in a cell via an episome (e.g., plasmid) or may be embedded in a genome. A preferred embodiment of the expression vector includes a selectable marker gene that allows for the selection and transformation of host cells. The art is well-informed about the existence of selectable marker genes. They will differ depending on the host cell.
“In certain instances, the nucleic acids used in the production ActRIIA peptides can be provided in expression vectors that contain a sequence of nucleotides encoding ActRIIA peptides and linked to at least one regulatory sequence. Art-recognized regulatory sequences can be used to control the expression of ActRIIA polypeptides. The term regulatory sequence also includes enhancers, promoters, and other expression control components. Goeddel, Gene Expression Technology: Methods In Enzymology Academic Press, San Diego (Calif.) (1990). These vectors can be used to express ActRIIA-encoding DNA sequences by using any of the many expression control sequences. These useful expression control sequences include the tet promoter and adenovirus immediate early promoter. RSV promoters. T7 promoter whose expression can be directed by T7 RNA Polymerase. The major operator and promoter regions for phage lambda. The design of an expression vector can depend on factors such as the host cell that will be transformed and/or what type of protein is being expressed. It is also important to consider the vector’s copy numbers, control over that number, and any other proteins encoded by it, such as antibiotic marks.
Recombinant nucleic acids are used to produce ActRIIA polypeptides. The cloned gene or a portion of it can be ligated into a vector that is suitable for expression in prokaryotic or eukaryotic (yeast, avian or insect) cells or both. Plastids and other vectors can be used to produce a recombinant ActRIIA protein. Plastids that are suitable for expression in prokaryotic cell types such as E.coli, pEMBL, pBR322-derived and pEMBL-derived vectors, pEX, pBTac, pEMBL, pEX, pEMBL, pEMBL, pUC, and pBTac-derived vectors are all examples of these types of vectors. coli.”
“Some mammalian expression sequences include both prokaryotic sequences that allow for the vector to be propagated in bacteria and one or more eukaryotic transcript units that can be expressed in eukaryotic cell. Examples of mammalian expression vectors that can be transfected into eukaryotic cells include the pcDNAI/amp and pcDNAI/neo. These vectors can be modified with sequences of bacterial plasmids such as pBR322, which facilitates replication and drug resistance selection in prokaryotic and non-eukaryotic cells. Alternately, you can transiently express proteins in eukaryotic cell by using derivatives of viruses like the bovine papillomavirus (BPV-1), and Epstein-Barr virus(pHEBo), pREP-derived, and p205. In the section on gene therapy delivery systems, you will find examples of other viral expression systems (including retroviral). It is well-known that there are many methods for preparing plasmids as well as for transforming host organisms. Other suitable expression systems, both for prokaryotic as well as eukaryotic cells are available in Molecular Cloning A Laboratory Manual 3rd Ed. Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press 2001). Sometimes it is possible to express the recombinant proteins using a Baculovirus Expression System. These baculovirus expression system examples include pVL-derived Vectors (such pVL1392, 1393 and pVL941) and pAcUW vectors.
“Vectors are possible to produce the subject ActRIIA proteins in CHO cells. These vectors include a PcmvScript vector (Stratagene La Jolla (Calif.), pcDNA4 Vectors (Invitrogen Carlsbad (Calif.) and pCIneo vectors [Promega Madison, Wis.]. The subject gene constructs can be used, as will be obvious, to induce expression of the subject ActRIIA proteins in cells grown in culture.
“Host cells can be transfected with a gene recombinant including a sequence coding sequence (e.g. SEQ ID NO 4 or 5 for one or more subject ActRIIA proteins. These genes can then be used to produce ActRIIA protein suitable for use in the compositions and methods described herein. Any prokaryotic and eukaryotic cells can be used as the host cell. An ActRIIA polypeptide described herein can be expressed in E. coli, mammalian cells, yeast cells, and insect cells (e.g. using a baculovirus gene expression system). The art has many other suitable host cells.
“The ActRIIA polypeptides are produced in the following methods. A host cell can be transfected with an expression vector that encodes an ActRIIA protein. This can then be grown under the appropriate conditions for expression. You can extract the ActRIIA protein from a mixture containing the ActRIIA protein and cells. The ActRIIA protein may also be retained in the cytoplasm or in a membrane fraction. Cells are then harvested and the protein isolated. Cell culture can include host cells, media, and other byproducts. The art is well-versed in the selection of suitable media for cell culture. You can isolate the subject ActRIIA proteins from host cells or cell culture media using any of the techniques described in the art. The preferred embodiment of the ActRIIA protein is a fusion protein that contains a domain that facilitates its purification. One embodiment of purification involves a series column chromatography steps. These include, for example, protein A chromatography and Q sepharose Chromatography, phenylsepharose Chromatography, size exclusion Chromatography, and cation exchange Chromatography. You can also purify the buffer by buffer exchange or viral filtration. This demonstrates that ActRIIA hFc protein was obtained to a purity of >98% by size exclusion analysis chromatography, and >95% by SDS-PAGE. This purity level was sufficient to produce desirable bone effects in mice and acceptable safety profiles in rats, mice, and other non-human primates.
“In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of a recombinant ActRIIA polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni2+ metal resin. You can then remove the purification leader sequence by treating it with enterokinase, to obtain the purified ActRIIA protein (e.g. Hochuli and colleagues, 1987, J. Chromatography 411,177; Janknecht and colleagues, PNAS USA, 88:8972).
The techniques for creating fusion genes are well-known. The basic principles of fusion genes are that they combine DNA fragments from different sequences. These include using conventional methods, which use blunt-ended and stagger-ended termini to ligation, restriction enzyme digest to ensure appropriate termini, filling in of cohesive ends, alkalinephosphatase treatment to prevent unwanted joining, and enzymatic binding. Another embodiment of the fusion gene is possible to be synthesized using conventional methods, including automated DNA synthesizers. Anchor primers are another option for PCR amplification. These anchor primers give rise to complementary overlaps between two consecutive fragments of DNA. This can then be annealed to create a chimeric sequence of genes (see Current Protocols in Molecular Biology). Ausubel et al., John Wiley & Sons: 1992).”
“ActRIIA -Fc fusion proteins can be expressed in stably-transfected CHO?DUKX Bl1 cells using a pAID4 vector. (SV40 ori/enhancers, CMV promoter), a tissue plasminogen lead sequence of SEQID NO:9. As shown in SEQ ID No:7, the Fc portion of the IgG1 Fc sequence is human. In some embodiments, the protein contains between 1.5 and 2.5 moles sialic acid per molecule ActRIIA?Fc fusion protein.
“In some embodiments, the ActRIIA -Fc fusion’s serum half-life can exceed 25-32 days in humans. The CHO cell-expressed material may have a greater affinity for activin B than the ActRIIA hFc fusion protein expressed by human 293 cells (del Re and al., J Biol Chem). 2004 Dec. 17; 279(51):53126-35). The TPA leader sequence was more productive than any other sequences. It also, unlike ActRIIA?Fc which is expressed with a native leader and may have a very pure N-terminal sequence. The native leader sequence could result in two distinct species of ActRIIA?Fc. Each has a different N-terminal sequencing.
“(b) Inhibitors ActRIIB”
“ActRIIB” is the term used herein. The term “ActRIIB” refers to any family of activin receptor type IIB proteins (ActRIIB), from any species, and variants derived by such ActRIIB proteins through mutagenesis or another modification. Any of the known forms of ActRIIB are referred to herein. ActRIIB members are transmembrane proteins. They consist of a ligand binding extracellular domain with a cysteine rich region, a transmembranedomain, and a domain in the cytoplasm with predicted serine/threoninekinase activity.
“ActRIIB inhibitors” can be used in the compositions described herein. They include activin-binding soluble ActRIIB peptides; activin subunits (activin A and B) that disrupt ActRIIB bindin binding; activin A and B subunits that disrupt ActRIIB binding; activin binding antibodies; activin binding antibodies; activin binding non-antibody proteins; and random peptides that are selected for activin and ActRIIB a Fc domain.
“In some embodiments, two or more proteins (or other moieties with activin/ActRIIB binding activity), especially activin binding sites that block type I (e.g. a soluble Type I activin receptor), and type II (e.g. a soluble Type II activin receptor), may be linked together to form a bifunctional, multifunctional binding molecule that inhibits ActRIIB. This can then be used in the compositions or methods described herein. The compositions and methods herein may include nucleic acids aptamers, small molecules, and other agents that inhibit ActRIIB.
“(i) ActRIIB Inhibitors Comprising ActRIIB Polypeptides”
“ActRIIB polypeptide” is the term used herein. The term “ActRIIB polypeptide” refers to any naturally occurring polypeptide from an ActRIIB member, as well as any variants (including fragments, fusions and peptidometic forms) that maintain a useful activity. ActRIIB Polypeptides, for example, are polypeptides that have a sequence at least 90% identical to an ActRIIB receptor’s sequence and optionally at least 85%-95%, 96%-97%, 98% or 99% more identity. An ActRIIB polypeptide could bind to or inhibit the function an ActRIIB protein/activin, for example. The human ActRIIB precursor protein polypeptide (SEQID NO:16 and SEQ ID No:28) is an example of an ActRIIB peptide. The ActRIIB precursor protein whose amino acid sequence can be referred to as SEQID NO:16 (or SEQID NO:28) (i.e. the human ActRIIB pre-protease polypeptide), has the signal peptide at amino acids 1-18; the extracellular domain at amino acids 19-134, and potential N-linked glycosylation site at amino acid positions 42 to 65. The sequence of nucleic acids that encodes the human ActRIIB precursor protein is known as SEQID NO:19. (SEQID NO:19 contains an alanine at codon 64. However, one skilled in the art could modify it to add an arginine to the codon 64). For a detailed description of these sequences, see Table 6.
“The numbering system for ActRIIB-related amino acids described herein is based upon the amino acid numbering SEQID NO:16 (which only differ in their amino acid expressed at 64), except where otherwise noted. For example, if an ActRIIB polypeptide is described as having a substitution/mutation at amino acid position 79, then it is to be understood that position 79 refers to the 79th amino acid in SEQ ID NO:16 or SEQ ID NO:28, from which the ActRIIB polypeptide is derived. If an ActRIIB protein is described as having an actinine or arginine at amino acids 64, it should be understood that position 64 refers the 64th amino Acid in SEQID NO:16 and SEQID NO:28 from which the ActRIIB Polypeptide was derived.
“In some embodiments, the ActRIIB inhibitors used in the compositions or methods described herein include polypeptides that contain an activin binding domain of ActRIIB. The activin binding domains of ActRIIB may be the extracellular domain or a portion thereof in some embodiments. Specific embodiments make the extracellular domain of ActRIIB soluble. U.S. Patent Application Publication Nos. 62 and 63 disclose examples of modified ActRIIB ActRIIB polypeptides. 20090005308 & 20100068215 are the disclosed forms of ActRIIB polypeptides, and their disclosures are included herein by reference in all respects.
“In certain embodiments, ActRIIB peptides used herein in compositions and methods are soluble ActRIIB peptides. The term “soluble ActRIIB peptide” is used. The term?soluble ActRIIB polypeptide? generally refers to any polypeptides that contain an extracellular domain from an ActRIIB proteins as well any variants (including fragments, mutants, and peptidometic forms). Soluble ActRIIB peptides can bind activin, but the wild-type ActRIIB protein doesn’t have a significant selectivity for binding to activin over GDF8/11. The methods described herein may allow for the use of altered forms or ActRIIB that have different binding properties. These altered forms are described, e.g. in international patent publication Nos. These disclosures are included in the international patent application publication Nos. WO 2006/012627 WO 2010/019261. The addition of an activin-selective binding compound to activate native or modified ActRIIB proteins can give them additional activin specificity. Exemplary soluble ActRIIB peptides include the extracellular Domain of a human ActRIIB Polypeptide (e.g. SEQ ID NOs 17, 18, 23, 26, 27, 29, 30, 31, 32.33.36.37.42 and 43).
Hilden et. al. disclosed an Fc fusion protein that has the ActRIIB extracellular structure. (Blood 1994, 83(8), 2163-70), which contains an alanine in the position that corresponds to amino acid 64 of ActRIIB precursor amino acids sequence. (Herein referred as?A64?) It has been shown to have a low affinity for GDF-11 and activin. An Fc fusion protein containing an arginine at 64 position of the ActRIIB precursor amino acids sequence (herein referred as?R64?) is in contrast. An Fc fusion protein with an arginine at position 64 of the ActRIIB precursor amino acid sequence (herein referred to as?R64?) has an affinity for activin, GDF-11, and low nanomolar to high picomol ranges (see, e.g. U.S. Patent Application Publication Number. 20100068215 is incorporated herein in its entirety. SEQ ID No: 28 presents an ActRIIB precursor amino acids sequence with an arginine position 64. In SEQ ID No:28, an ActRIIB precursor sequence with an arginine position 64 is presented. Other embodiments of ActRIIB polypeptides may contain an amino acid other than arginine or alanine at the position that corresponds to amino acid 64 in the ActRIIB precursor amino acid sequence.
Summary for “Activin-ActRII antagonists, uses for treating bone and other conditions”
Bone growth and mineralization depend on two types of cells, osteoclasts or osteoblasts. However, chondrocytes as well as cells of the vasculature play a critical role in these processes. Two mechanisms are involved in bone formation during development: intramembranous and endochondral. The former is responsible for longitudinal bone formation, while the latter forms topologically flat bones such as the skull bones. Endochondral Ossification involves the sequential formation and decay of cartilaginous structures within the growth plates, which serve as templates for osteoblasts, osteoclasts and the vasculature. Intimambranous osteossification is when bone is formed in connective tissues. Both require the infiltration and subsequent matrix deposition of osteoblasts.
“Chronic kidney disease is associated with a progressive deterioration in mineral homeostasis, with a disruption of normal serum and tissue concentrations of phosphorus and calcium, and changes in circulating hormones, such as parathyroid hormone, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, other vitamin D metabolites, fibroblast growth factor-23, and growth hormone. See Chronic Kidney Disease-Mineral and Bone Disorder, Kidney Disease: Improving Global Outcomes, Kidney Disease: Working Group (KDIGO), Kidney Disease: Improving Global Outcomes, In: Kidney Int. Suppl. (2009) 76 (Suppl 113), pages S1-130. Chronic kidney disease can disrupt mineral and hormone homeostasis, which is crucial for bone growth during childhood (bone modeling), and adult bone structure and function (bone remodeling). Patients with chronic kidney disease are more likely to have bone abnormalities. Patients with chronic kidney disease may also experience extraskeletal calcification due to disruptions in mineral and endocrine function. These conditions are called chronic kidney disease-related bone and mineral disorders (?CDK?MBD ?).”).
“Bone is constantly in constant turnover. Bone turnover refers to the process of bone resorption and replacement. Bone turnover is a process of bone resorption and replacement. Osteoblasts, osteoclasts, and other cells are required for this. Adynamic bone diseases, which have low turnover or no resorption, are characterised by a reduced or absent rate of bone replacement and resorption. Low turnover or dynamic bone can both be part of CKD-MBD. (Chronic Kidney Disease, Mineral and Bone Disease (CKD?MBD), Kidney Disease : Improving Global Outcomes(KDIGO), CKD?MBD Work Group. In: Kidney Int. Suppl. (2009) 76 (Suppl 110), pages S1-130 and S34.
Vascular calcification is a condition that causes vessel stiffening to increase by increasing calcium levels in the blood vessels. Vascular calcification can increase the risk of myocardial injury in patients with vascular calcification. This condition is especially common in patients with kidney disease (CKD-MBD). See, e.g., Shanahan et al., 2011, Circ. Res. 109:697-711.”
ActRIIA (and ActRIIB), are type II receptors that activate activins. ActRIIA, ActRIIB, and Nodal can biochemically interact other TGF-beta proteins such as GDF8, GDF8, and GDF11 (Yamashita and al., 1995). Cell Biol. 130:217-226 Lee and McPherron 2001, Proc. Natl. Acad. Sci. 98:9306-9310; Yeo & Whitman, 2001. Mol. Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is the primary receptor type I for activins, especially activin A. ALK-7 could also serve as a receptor, particularly activin B.
“In some embodiments, there are methods to treat an adynamic disorder of CKD-MBD in a subject. The method involves administering a therapeutically efficient amount of ActRII inhibitors to the subject. Methods for treating an adynamic form of CKD/MBD in a subject are also provided. The method involves administering a therapeutically-effective amount of ActRII inhibitor to a patient in need of treatment.
“In some cases, the adynamic bones disorder can be distinguished by the absence of tetracycline incorporation in mineralized bone.”
“In some embodiments, there are methods to treat a low bone turnover CKD/MBD form in a subject. The method involves administering a therapeutically-effective amount of ActRII inhibitor treatment to a subject with low bone turnover CKD/MBD. Osteomalacia is a low-bone turnover form of CKD/MBD.
“In some embodiments, there are methods to treat a bone disorder that is hyperphosphatemia in a subject. The method involves administering a therapeutically-effective amount of ActRII inhibitor treatment to the subject with the bone disorder.
“In some embodiments, there are methods to treat atherosclerotic calcium in a subject. The method involves administering a therapeutically-effective amount of ActRII inhibitor treatment to a subject with atheroscleroticcalcification.”
“In some embodiments, there are methods for treating renal disease in a subject. The method includes administering a therapeutically-effective amount of ActRII inhibitor to the subject who has the renal disease. A more specific example of a renal disease is renalfibrosis.
“In one embodiment, there is a method of treating extraskeletal calcium in a subject. This method involves administering a therapeutically-effective amount ActRII inhibitor. A method of preventing extraskeletal calciumification in a subject is also provided in this embodiment. This method involves administering a therapeutically-effective amount ActRII inhibitors to the subject. Extraskeletal calcification is treated or prevented by specific embodiments. This refers to the accumulation of calcium salts within the blood vessels of the subject (e.g., calcifications of the arteries).
“ActRII inhibitor can be used in certain embodiments. It is a polypeptide that contains an amino acid sequence chosen from the following group: 95% identical or SEQID NO.2; 98% exactly to the SEQID NO.3; 98% similar to the SEQID NO.3; 98% equal to the SEQID NO.6; 98% equal to the SEQID NO.7; 98% exact to the SEQID NO.7; 98% same to the SEQID NO.20; 95% to the SEQID NO.21; 95% to the SEQID NO.21; 98% of the SEQID NO.21; and 98% to the SEQID NO.21; 95% to the SEQID NO.21; 98% to the SEQID NO.21; and 98% to the SEQID NO.21; 98% to the SEQID NO.21; 98% to the SEQID NO.21; and 98% to the SEQID NO; The ActRII inhibitor, in a more precise embodiment, is a polypeptide that contains the amino acid sequence from SEQ ID No:7. The ActRII inhibitor can be administered parentally in a more specific form.
“A specific embodiment of the ActRIIA inhibit that can be used in conjunction with the methods herein is an ActRIIA inhibition. The ActRIIA inhibit comprises or consists a particular polypeptide from the following group: a. A polypeptide less than 90% identical SEQID NO.2; and b. A polypeptide more than 95% identical SEQID NO.3; and c. A polypeptide no greater than 98% identical SEQID NO.3;. e. A polypeptide of at least k. SEQID NO.6; m. A polypeptide that is at least 90% identical with SEQID NO.7; or o. A polypeptide that is at least 98% the same as SEQID NO.7; or p.SEQID NO.7; or q. A polypeptide of at least 98% identical with SEQID NO.12; r. A polypeptide less than 95% identical in SEQID NO.12; s. A polypeptide which is at least 98%ident to SEQID NO.12; s. The ActRIIA inhibitor, in a particular embodiment, is a polypeptide that contains or consists of the amino acid sequence from SEQ ID No:7.
“An ActRIIB inhibit can also be used in conjunction with the methods described herein. It consists or contains a specific polypeptide. The ActRIIB inhibitor can be described as a polypeptide that contains or consists of SEQ ID No:23. Another specific embodiment of the ActRIIB inhibit is a polypeptide consisting or comprising SEQ ID No:25.
An ActRIIA and ActRIIB inhibit can be combined in a specific embodiment. The ActRIIA inhibitor, which is a combination of SEQID NO:7 and ActRIIB inhibitor, is a particular embodiment. The ActRIIA inhibitor, a polypeptide consisting or consisting SEQ ID No:7, and the ActRIIB inhibit, a polypeptide consisting or consisting SEQ ID no:25 are two examples of a specific embodiment.
“In some embodiments, the subject to the treatment is younger than 18 years. The subject who is to be treated using the methods herein may have end-stage renal disease. The subject may need dialysis to be treated using the methods herein. The subject may be raised in certain embodiments.
“In some embodiments, methods are provided for treating or preventing hyperphosphatemia (due increase in phosphorus), additionalskeletal calcification (e.g. vascular calcification) and adynamic bones disorder in a patient. The method includes administering a therapeutically-effective amount of ActRII inhibitor (e.g., to a subject with hyperphosphatemia, secondary hypoparathyroidism due to an increase in phosphorus), and extraskeletal calciumification (e.g. vascular calcification
“5.1 Overview”
“The method described herein is a treatment for Chronic Kidney Disease-Mineral & Bone Disorders (CKDMBD). It involves administering an inhibitor to ActRII to a patient who is in need of treatment. An inhibitor of ActRIIA or ActRIIB can also be used.
“In some embodiments, there are methods for treating low bone turnover forms of CKD/MBD. The method involves administering an inhibitor to ActRII to a patient who is in need of treatment. Methods for treating CKD-MBD characterized as hyperphosphatemia or hypercalcemia are provided in certain embodiments. Methods for treating CKD-MBD characterized with extraskeletal calcification such as atherosclerotic calcification are provided in certain embodiments.
“In certain embodiments, methods are provided for treating CKD-MBD where the chronic kidney disease has reached stage 3 stage 4 stage 5 or 5 stage 5. A specific embodiment refers to kidney disease that is at the end of its life. Methods for treating CKD-MBD are provided in certain embodiments. These methods can be described as having a glomerular filtration of less than 60 ml/min/1.73 m2 for adults and less than 89 ml/min/1.73 m2 for pediatric patients. See, Moe et al., 2006, Kidney International 69:1945-1953. Certain embodiments of the methods are for adults with CKD-MBD. They have glomerular filtration rates of less than 50 ml/min/1.73 m2, and 40 ml/min/1.73 m2, respectively. Certain embodiments provide methods for treating CKD-MBD in children. These include methods that have a glomerular filtration of less than 80 ml/min/1.73 m2, 70 ml/min/1.73 m2, 60 ml/min/1.73 m2, 10 ml/min/1.73 m2, and less than 10 ml/min/1.73 m2.
“Gglomerular filtration rates below 60 ml/min/1.73m2 for adult patients and 89 ml/min/1.73m2 for pediatric patients can cause detectable abnormalities in calcium, phosphorus, and vitamin D metabolism. These abnormalities may lead to bone disease.
“In some embodiments, methods are provided for treating bone pathology related to chronic kidney disease (CKD-MBD). See Moe et al., 2006, Kidney International 69:1945-1953. The CKD -MBD can be low-turnover CKD -MBD in certain cases. The histological features listed in Table 1 below can help you diagnose low-turnover CKD/MBD. National Kidney Foundation, Kidney Disease Outcomes Quality Initiative Guidelines on the website of National Kidney Foundation.
“TABLE 1\nHistological Features of Low-Turnover CKD-MBD\nFeature Adynamic Osteomalacia\nBone Formation\nTrabecular bone volume Normal, low Variable\nLow, normal or high\nOsteoid volume Normal, low High-very high\nOsteoid seam thickness Normal, low High-very high\nNumber of osteoblasts Low Low\nBone formation rate Low-very low Low-very low\nMineralization lag time Normal Prolonged\nBone Resorption\nEroded bone perimeter Normal, low Variable\nOften low, may be high\nNumber of osteoclasts Low Low, may be normal or high\nMarrow fibrosis Absent Absent”
“In one embodiment, there is a method of treating extraskeletal calcium in a subject. This method involves administering a therapeutically-effective amount ActRII inhibitor. A method of preventing extraskeletal calciumification in a subject is also provided in this embodiment. This method involves administering a therapeutically-effective amount ActRII inhibitors to the subject. Extraskeletal calcification is treated or prevented by specific embodiments. This refers to the accumulation of calcium salts within the blood vessels of the subject (e.g., calcifications of the arteries).
“In some embodiments, the methods for treatment or prevention extraskeletal calciumification, e.g. vascular calcification are applied to a subject at risk of developing extraskeletal calcification. The ActRII inhibitor is administered to the subject in accordance the methods described herein. In a specific embodiment, the subject at risk of suffering from extraskeletal calcification, e.g., vascular calcification, has hypercholesterolemia. Another specific embodiment shows that the subject at high risk for extraskeletal calcification (e.g.,vascular calcification) has hypertension. Another specific embodiment has the subject at high risk for extraskeletal calcification (e.g.,vascular calcification). Another specific embodiment states that the subject at high risk for extraskeletal calcification (e.g. vascular calcification) has end-stage renal disease. Another specific embodiment states that the subject at high risk for extraskeletal calcification (e.g.,vascular calcification) has chronic kidney disease. Another specific embodiment states that the subject at high risk for extraskeletal calcification (e.g. vascular calcification) has elevated oxidative stress. This is e.g. an imbalance between antioxidant activity and oxidant production in the vasculature. Another specific embodiment shows that the subject at high risk for extraskeletal calcification (e.g. vascular calcification) has a calcification inhibitor defect (e.g. a deficiency or combination of matrix gla protein (MGP), fetuin A (OPG ).
“Intima calcification” can be described as the result of vascular calcification in certain embodiments. Intima calcification can be associated with atherosclerosis. It progresses along with the progression of atherosclerotic plaques.
“In some embodiments, a subject who is suffering from CKD or extraskeletal calcification (e.g. vascular calcification) has an increased level of FGF23. This is relative to FGF23 levels in subjects who are not suffering or at risk from CKD or extraskeletal calcification (e.g. vascular calcification). FGF23 levels can be determined using techniques that are well-known in the art. For example, ELISA is used to detect FGF23 using blood and serum samples. A specific embodiment of FGF23 is the amount detectable in serum in a subject who has, or may have, a form or extraskeletal calcification. This can be used to determine if the subject is suffering from, and/or at risk from suffering from, CKD/MBD or vascular calcification. Another specific embodiment shows that the FGF23 level (e.g. the level detectable within the serum) of a subject who is suffering from or at risk from CKD/MBD or extraskeletal calcification (e.g. vascular calcification) in a subject who is not suffering from or at risk from suffering from CKD/MBD or extraskeletal calcification (e.g. vascular calcification) is approximately 5-10% to 10%, 10-15%, 20%, 15%, 20%, a serum) in a serum level of FGF23 level detectable inside the serum level) of FGF23 in a serum level in a serum level) in a patient not having CKD/or CKD- or extraskeletal calcification
“In some embodiments, the levels of FGF23 in a patient suffering from CKD or extraskeletal calcification can be used to assess whether the method is effective. These methods include administering an ActRII inhibitor that is therapeutically effective. A subject who has been treated according to one or more of these methods shows a decrease in FGF23 (e.g. as measured in the serum) relative to the level that was detected in the subject before treatment with the method described herein. A specific embodiment shows that the level FGF23, i.e. the level detectable within the serum, in a subject with CKD-MBD or extraskeletal calcification (e.g. vascular calcification) is decreased by approximately 5%, 10% and 20% relative to the level FGF23 (e.g. the serum level) found in the subject before treatment. Another specific embodiment shows that the level FGF23 (e.g. the serum level) in a subject who is suffering from CKD-MBD or extraskeletal calcification (e.g. vascular calcification) is decreased by approximately 5-10% or 10%, 20%, 30%, 40%, 50%, 40%, 50%, 60%, 50-60% and 50-75% relative to the level FGF23 (e.g. the serum level) which was detected in the subject before treatment.
“In a particular embodiment, there is a method for treating CKD/extraskeletal calcification. This involves: (i.) administering an ActRII inhibition to an individual with CKD/extraskeletal calcification. (ii.) determining the amount FGF23 present in a tissue sample (e.g. serum) from the individual. (iii.) Repeating the ActRII inhibit administration. If the FGF23 level is not reduced after administration of the ActRII inhibit, then the ActRII inhibition can be increased in dose. If FGF23 levels are not decreased after administration of ActRII inhibitors, it is possible to increase the frequency with which the ActRII inhibit is administered.
“In some embodiments, a subject who is suffering from CKD/MBD or extraskeletal calcification (e.g. vascular calcification) has higher levels of sclerostin. This protein increases in those subjects who are not at high risk of developing CKD/MBD or extraskeletal calcification (e.g. Graciolli et.al., 2010 J Am Soc Nephrol 21.774A). The methods for detecting sclerostin levels are well-known in the art. For example, ELISA can be used to detect sclerostin using blood and serum samples. A specific embodiment of sclerostin levels can be measured using methods known in the art, e.g. ELISA. Subjects are tested for sclerostin in blood and serum samples. Another specific embodiment shows that the serum level of sclerostin in a subject who is suffering from CKD/MBD or extraskeletal calcification (e.g. vascular calcification) is approximately 5-10% to 10%, 10-15%, 20%, 30%, 40%, 50%, 40%, 50%, 50-60% and 50-75% respectively. This level is greater than the serum level of sclerostin in a person who is not at risk or suffering from CKD/extraskeletal calcification
“In some embodiments, the levels of sclerostin found in a subject with CKD or extraskeletal calcification can be used to assess whether the method is effective. These methods include administering an ActRII inhibitor that is therapeutically effective. A subject who has been treated according to one or more of these methods will have a lower level of sclerostin than the amount found in their serum prior to treatment. Another specific embodiment shows that the serum level of sclerostin in a subject with CKD-MBD or extraskeletal calcification (e.g. vascular calcification) is lower than the level detected in the serum prior to treatment using the method described herein. Another specific embodiment reduces the amount of sclerostin in a subject with CKD-MBD or extraskeletal calcification. This is relative to the serum level that was detected in the subject before treatment.
“In a particular embodiment, there is a method for treating CKD/extraskeletal calcification. This includes: (i.) administering an ActRII inhibition to an individual with CKD/extraskeletal calcification. (ii.) determining the amount sclerostin present in a tissue sample of the individual. (e.g. a different serum sample from the same person) and then repeating the ActRII inhibit administration. The ActRII inhibitor can be given at a higher dose if the amount is not reduced by administration of ActRII inhibitor. If the ActRII inhibit administration does not cause a decrease in sclerostin, then the ActRII inhibition can be administered more frequently.
“In some embodiments, the person suffering from vascular calciumification treated according to the methods described herein has less than 18 years of age. A specific embodiment of vascular calcification is a subject less than 13 years old. Another specific embodiment of vascular calcification is a subject less than 12 years old, less than 11, less that 10, less than 9, less over 8, less about 7, less 6 or less 5 years. Another specific embodiment of vascular calcification is the subject who has been treated according to the methods herein is between 1-3 and 5-7 years. 7-9 years. 7-9 years. 7-9 years. 7-9 years. 7-9 years. 7-9 years. 7-9 years. 7-9 years, 7-9, 7-9, 7-9, 7-9, 7-9, 7-9, 7-9, 7-9, 7-9, 7-9, 6-7, 7-8, 7-8, 7-8, 6-7, 7, 6-7, 6, less than 30 or more than 30 year old. Another specific embodiment of vascular calcification is the subject who has been treated according to the methods herein is between 30-35 and 40 years old. Another specific embodiment of vascular calcification is the 60-65 year old subject, 65-70 year old, 70-75, 75-80, or more than 80 years.
“In some embodiments, the subject with vascular calcification treated according to the methods described herein has end-stage renal disease. The subject with vascular calcification is treated according to the methods herein and requires dialysis.
“In some embodiments, the effectiveness or prevention of extraskeletal calciumification, e.g. vascular calcification is evaluated using one or more of the assays that are known to those skilled in the art. Section 5.3(a),(iv) describes some examples of assays. According to such embodiments, one skilled in the art will know that an ActRII inhibitor-treated subject may be adjusted according to the results of assays. A subject treated with an ActRII inhibit may receive a higher dose or may be given an ActRII inhibition more often (i.e. the time between doses may be decreased). A subject treated with a method herein may receive a lower dose of ActRII inhibitor or may be administered an ActRII inhibit less often (i.e. the time between dose administrations could be increased).
“In some embodiments, the methods herein improve the symptoms of hyperphosphatemia, secondary hyperparathyroidism due to an increase in phosphorus, and extraskeletal calciumification, e.g. vascular calcification. The methods described herein can be combined with any method that is known to the skilled tradesperson to determine the severity of the symptoms. Specific embodiments of the methods described herein improve one or more symptoms associated with vascular calcification. Examples of symptoms include an increase in vascular calcium (e.g. arterial), increased apoptosis in vascular smooth muscles cells, loss arterial elasticity, development of left-ventricular hypertrophy, decreased coronary artery perfusion and myocardial inchaemia.
“In some embodiments, the methods herein result in an increase in the levels vascular calcium in a subject by at minimum 5%, 10% and 15%. Certain embodiments result in a decrease of vascular calcium in a subject, e.g. arterial calcium.
“In a particular embodiment, there is a method for reducing the levels vascular Calcium in a subject. It involves: (i.) administering an ActRII inhibition to a subject with CKD-MBD or extraskeletal calcification; (ii.) determining the amount vascular Calcium in a tissue sample (e.g. serum) from said subject. (iii.) if this amount is less than the amount determined in a similar tissue sample from the subject prior to administration of ActRIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII) then repeating the ActRIIIIIIIIIIIIIIIIIIIIIIIIIIIIiiiIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiiv The ActRII inhibitor can be given at a higher dose if there is no decrease in vascular calcium after administration. If the amount of vascular Calcium following administration of ActRII inhibitor is not decreased, it can be increased the frequency with which the ActRII inhibit is administered.
“In some embodiments, the methods herein result in a decrease of the Agatston score for a subject at risk or having vascular calcification. The methods described herein can result in a 5%-10%, 15%, 20% and 25% decreases in the Agatston score for a subject with vascular calcification or at risk. Another specific embodiment results in a 5%-10% to 15%, 10%-15% to 25%, 25%-30% to 30%, 40%-40% to 40%, 40%-45% or 45%-50% reduction in the Agatston score for a subject with vascular calcification or at risk.
“In another embodiment, the methods herein result in a reduction in calcium levels in the vasculature. This could be a decrease of calcium levels in one or more of the subjects arteries, or a subject at risk for developing vascular calciumification. Another specific embodiment results in a decrease of phosphorus levels in the blood, e.g. a decrease of phosphorus levels in one or more of the subjects’ arteries, e.g. a subject at risk or having vascular calcification.
“In some embodiments, methods are provided for treating low-turnover bone disorders. The tests described in Section 5.3 (a) below can help you determine if your bone turnover is low. The biochemical markers for bone turnover include serum or urine collagen crosslinks (N?telopeptide and C-telopeptide), bone specific alkalinephosphatase and serum osteocalcin. 25 hydroxyvitamins D and parathyroid hormone?. A low turnover bone disorder can be described as a dynamic bone disorder in a particular embodiment. A patient who is treated using the methods described herein will see a decrease in bone turnover of at most 10%, 20% or 25%. This can be achieved by reducing bone turnover by 50%, 60%, 70% and 75% respectively. A patient treated using the methods herein can have a decrease in bone turnover of up to 10%, 20%, 30%, 40% or 50%. This is in addition to a possible reduction of up to 70%, 75% and 80%. A patient who is treated using the methods herein can expect a decrease in bone turnover of between 10% and 25%, 20%, 35%, 30%, 40%, 55%, 50%, 60%, 75% and 70%, 85%, 95%, 90%, and 100%. The reduction in bone turnover can be compared with historical data for the same patient. Other embodiments compare the decrease in bone turnover to the average bone turnover of a population that does not have bone disorders. The population that does not have bone disorders may be the same age as the patient and/or the same sex.
“In one embodiment, there is a method for treating a low-turnover bone disorder. This includes: (i.) administering an ActRII inhibition to a subject with a low-turnover bone disorder; and (ii.) determining the level or bone-turnover in the subject after administration of ActRII inhibit (e.g. by using one of the tests described in Section 5.3(a), or by measuring one or several biochemical markers of bone growth); and (iii). compared to the ActRIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII I’st the ActRIIIIIIIIIIIIIIIIIIIIIIIIIIIiiiIIIIIIIIIIIIIIIIIIIIIIIIIIIII to the ActRIIIIIIIIIIIIIIIIIIIIIIIIII the ActRIIIIIIIIIIIIIIIIIIIIIIIIIII the ActRII the ActRII the ActRII the ActRII the ActRII the ActRII the ActRII the ActRIII inhibitor If the ActRII inhibit does not reduce bone turnover, then the ActRII inhibition can be increased. If the ActRII inhibit does not cause a decrease in bone turnover, certain embodiments allow for an increase in the frequency of administering the ActRII inhibition.
“5.2 Inhibitors to ActRII”
“(a) ActRIIA Inhibitors”
“ActRIIA” is the term used herein. The term “ActRIIA” refers to any family of activin receptor type IIa proteins (ActRIIA), from any species, and variants derived by such ActRIIA proteins through mutagenesis or another modification. ActRIIA is understood to refer to any of the forms currently known. ActRIIA proteins are transmembrane proteins. They consist of a ligand binding extracellular domain with a cysteine rich region, a transmembranedomain, and a domain in the cytoplasm with predicted serine/threoninekinase activity.
“ActRIIA inhibitors” can be used in compositions and methods as described in this document. They include activin-binding soluble ActRIIA proteins; antibodies that bind activin (particularly activin A and B subunits) and disrupt ActRIIA bindin; and antibodies that bind ActRIIA and activate activin binding.
“In some embodiments, two or three different proteins (or moieties) that have activin-ActRIIA binding activity, particularly activin binders that block type I (e.g. a soluble Type I activin receptor), binding sites respectively may be linked to form a bifunctional, multifunctional binding molecule that inhibits ActRIIA. This can be used in the compositions or methods described herein. In some embodiments, Activin – ActRIIA signaling axis antagonists include nucleic acids aptamers, small molecules, and other agents.
“(i) ActRIIA Inhibitors Comprising ActRIIA Polypeptides”
“ActRIIA polypeptide” is a term. “ActRIIA polypeptide” means any naturally occurring polypeptide from an ActRIIA member, as well as any variants (including fragments, fusions and peptidometic forms) that still have a useful activity. ActRIIA Polypeptides, for example, are polypeptides that have a sequence at most 80% similar to an ActRIIA sequence and optionally 85%, 90% or 97% of the sequence of an ActRIIA protein. An ActRIIA polypeptide could bind to or inhibit the function an ActRIIA protein/activin, for example. A polypeptide called ActRIIB may be chosen for its ability to stimulate bone growth and mineralization. ActRIIA peptides are human ActRIIA precursor polypeptides (SEQ ID No: 1) and human ActRIIA soluble polypeptides (12, 7, and 12). The ActRIIA precursor peptide’s amino acid sequence can be found at SEQID NO:1. The signal peptide is located at amino acids 1 to 20, while the extracellular domain is at amino acids 21 to 135 and N-linked glycosylation locations of the human ActRIIA preprod polypeptide (SEQID NO: 1) at amino acids 43 and 56. The SEQ ID No:4 nucleic acid sequence that encodes the human ActRIIB precursor protein is available (nucleotides 163-1705 in Genbank entry NM001616). SEQ ID No:5 is the nucleic acid sequence that encodes the soluble human ActRIIA protein of SEQID NO:2. For a detailed description of these sequences, see Table 6.
“In certain embodiments, ActRIIA soluble polypeptides are used in the compositions or methods described herein. The ActRIIA protein’s extracellular domain can bind activin and can therefore be called a soluble activin-binding ActRIIA peptide. As such, the term “soluble ActRIIA protein” is used in this document. The term?soluble ActRIIA polypeptide? generally refers to any polypeptide containing an ActRIIA extracellular domain. This includes any naturally occurring extracellular Domain of an ActRIIA proteins as well any variants (including fragments, mutants, and peptidometic forms). However, soluble ActRIIA proteins can bind to activin. The wild type ActRIIA protein has a lower selectivity than GDF8/11. By coupling native or modified ActRIIA proteins with an activin-selective, second binding agent, they may be given additional specificity for activin. The soluble polypeptides shown in SEQ ID NOS: 2, 3, 7, 12, and 13 are examples of activin-binding ActRIIA soluble polypeptides. Another example of soluble, activin binding ActRIIA polypeptides is the signal sequence. This sequence can be found in SEQ ID NOS: 2, 3, 7, 12 and 13. TPA is used to illustrate the ActRIIA-hFc peptide.
“In some embodiments, the ActRIIA inhibitors used in the compositions or methods described herein consist of a conjugate/fusion proteins comprising an activin binding domain of ActRIIA linked with an Fc portion an antibody. In some embodiments, an activin-binding protein is linked to an Fc section of an antibody using a linker (e.g., an peptide linker). Optionally, the Fcdomain may contain one or more mutations at residues like Asp-265 and lysine 322, as well as Asn-434. The Fc domain mutant with one or more of these mutations, such as an Asp-265 mutation, may have a decreased ability to bind to it. Fc domain is more sensitive than a wild-type Fc. Other cases show that a mutant Fc domain with one or more mutations (e.g. an Asn-434 substitution) has a greater ability to bind the MHC class 1-related Fc receptor (FcRN), relative to a wild type Fc domain. SEQ ID NOS 6-7, 12, and 13 show examples of fusion proteins that combine an ActRIIA soluble extracellular domain with an Fc domain.
“ActRIIA inhibits are used in specific embodiments. They comprise the extracellular Domain of ActRIIA or a portion thereof linked to an Fc section of an antibody. The ActRIIA inhibition contains an amino sequence that is at minimum 75% identical to one selected from SEQ ID Nos. 6, 7, 12 and 13. Another embodiment of the ActRIIA inhibits used in the compositions, methods and methods described herein comprises the extracellular domain or a portion thereof linked to an Fc section of an antibody. In this case, ActRIIA contains an amino sequence that is at minimum 80%, 85% and 90% identical to an amino sequence chosen from SEQ ID Nos: 6, 7, 12 and 13.
“In some embodiments, ActRIIA inhibitors used in compositions and methods herein contain a truncated version of an extracellular Domain of ActRIIA. The carboxy terminus or the amino terminus can be affected by the truncation of ActRIIA polypeptide. The truncation may be 1, 2, 3, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17-18 19, 20, 21, 22, 23 24, or 25 amino acid long relative to mature ActRIIB protein extracellular domain. The truncation in certain embodiments can be 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23 24, or 25 Nterminal amino acids of mature ActRIIA Polypeptide Extracellular Domain. The truncation may be 1, 2, 3, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17-18, 19, 20, 22, 23, 24, or 25 N-terminal amino acid of the mature ActRIIA Polypeptide Extracellular Domain. The truncated ActRIIA forms include polypeptides containing amino acids 20-128, 20-129, 20-135, 20-138, 20-135, 20-135, 20-135, 20-131, 20-132, 20-135, 20-135, 20-135, 20-131, 20-132, 20-133, 20-134, 20-135, 20-132, 20-131, 20-132, 20-135, 20-135, 20-131, 20-133, 20-134, 20-134, 20-131, 22-131, 23-131, 24-131; and the positions in SEQ ID No:1
“In some embodiments, the ActRIIA inhibitors used in the compositions or methods described herein consist of an extracellular domain of ActRIIA that contains one or more amino acids substitutions. The ActRIIA inhibitors used in certain embodiments include a reduced form of the ActRIIA extracellulardomain that also contains an amino acid substitution.
“ActRIIA inhibitor is a fusion protein that binds to the extracellular domain of human ActRIIA receptor with the Fc portion IgG1. Another embodiment of the ActRIIA inhibit to be used in these compositions and methods is a fusion proteins between the Fc portion IgG1 and the truncated human ActRIIA receptor extracellular domain. Another embodiment of the ActRIIA inhibit to be used in these compositions and methods is a Fusion Protein between the ActRIIA antagonist and the Fc portion IgG1. The fusion protein contains one or more amino acids substitutions.
“Functionally active fragments ActRIIA Polypeptides can be obtained by screening polypeptides that have been recombinantly made from the fragment of the nucleic acids encoding the ActRIIA protein. Chemically, fragments can also be synthesized using methods such as t-Boc and Merrifield solid phase moc chemistry. You can produce the fragments either recombinantly, or chemically synthesis. Then you can test them to determine which peptidyl fragments are capable of acting as antagonists or inhibitors of ActRIIA protein signaling or activin.
“In addition to functionally active variants ActRIIA Polypeptides, it is possible to obtain them by screening libraries modified polypeptides that have been recombinantly made from the mutagenized nucleic acid encoding ActRIIA. These variants can be made and tested to determine if they can inhibit or act as antagonists of ActRIIA protein signaling. A functional variant of ActRIIA polypeptides may contain an amino sequence that is at most 75% identical to one selected from SEQ ID Nos 2 or 3. The functional variant may contain an amino sequence that is at least 80% or 85% or 90% identical to the sequence from SEQ ID Nos 2 or 3.
“Functional variants can be created, for example by altering the structure of an ActRIIA protein for therapeutic efficacy or stability (e.g. ex vivo shelf-life and resistance to proteolytic degrading in vivo). These modified ActRIIA proteins, when they are selected to retain activin binding can be considered functional counterparts to the naturally occurring ActRIIA proteines. Modified ActRIIA proteins can be made by amino acid substitution, deletion or addition. It is reasonable to assume that an isolated substitution of a leucine, isoleucine, valine, aspartate, glutamate, or threonine with serine or similar replacement of an amino acid with a structurally similar amino acid (e.g. conservative mutations), will not have a significant effect on the biological activity. Conservative replacements are those occurring within a family that is related in their side chain. It is possible to determine if an ActRIIA amino acid sequence change results in a functional homolog by looking at the ability of the variant ActRIIA protein to stimulate cells in a manner similar to wild-type ActRIIA.
“In some embodiments, the ActRIIA inhibit to be used with the compositions and techniques described herein might comprise an ActRIIA protein having one or more specific mutations which can alter the glycosylation. These mutations can introduce or remove one or more glycosylation site(s), such as O-linked and N-linked glycosylations sites. Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine (or asparagines-X-serine) (where ?X? Any amino acid that is recognized by appropriate cell glycosylation enzymes. Alterations can also be made to the sequence of wild-type ActRIIA polypeptides by adding or substituting one or more serine and threonine amino acids (for O-linked Glycosylation Sites). Modifying the tripeptide sequence to not allow for glycosylation can be achieved by a variety of amino acids substitutions and deletions. Chemical or enzymatic coupling glycosides to an ActRIIA protein can also increase the number of carbohydrate moieties. The coupling method used will determine whether the sugar(s), arginine or histidine, are attached to the ActRIIA polypeptide. These methods are described by WO 87/05330, published Sep. 11, 1987; and Aplin and Wriston (1981), CRC Crit. Rev. Biochem., pp. Biochem., pp. 259-306, which is incorporated herein by reference. One or more carbohydrate moieties on ActRIIA polypeptides can be removed chemically and/or enzyme-wise. Chemical deglycosylation may involve, for example, exposure of the ActRIIA 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. Hakimuddin and colleagues further describe chemical deglycosylation. (1987) Arch. Biochem. Biophys. 259:52, and by Edge et. al. (1981) Anal. Biochem. 118:131. Thotakura and colleagues described a range of exo- and endo-glycosidasses that can be used to cleave carbohydrate moieties from ActRIIA polypeptides. (1987) Meth. Enzymol. 138:350. 138:350 ActRIIA proteins can be expressed in mammalian cells that provide proper glycosylation (e.g. HEK293 and CHO cell lines), but other expression systems such as mammalian expression lines, yeast cell strains with engineered glycosylation enzymes, or insect cells are also possible.
“Further, herein are methods for generating mutants. Particularly sets of combinatorial mutations of an ActRIIA protein, as well as truncation variants. Pools of combinatorial mutations are particularly useful in identifying functional variants. Screening such combinatorial libraries might be used to create variants of ActRIIA polypeptides that can either act as agonists or antagonists, or even have novel activities. Below are a variety of screening methods that can be used to screen variants. An ActRIIA variant of a polypeptide may be tested for its ability to bind an ActRIIA protein ligand. This could prevent ActRIIA proteins from binding to ActRIIA aminopeptides.
“Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to a naturally occurring ActRIIA polypeptide. Mutagenesis can also result in variants with intracellular half-lives that are dramatically different from the wild-type ActRIIA protein. The altered protein may be made more or less resistant to proteolytic degradation or other cellular processes that result in the destruction or inactivation of native ActRIIA proteins. These variants and their genes can be used to modify the half-life ActRIIA peptides. A shorter half-life, for example, can lead to more transient biological effects. This can enable tighter control over recombinant ActRIIA protein levels within patients. Mutations can be made to an Fc fusion protein’s linker and/or Fc portion in order to alter its half-life.
A combinatorial library can be made by combining a number of genes to encode a collection of polypeptides that each contain at least one of the potential ActRIIA sequences. A mixture of synthetic oligonucleotides may be enzymatically linked into gene sequences to form a combination of polypeptides. The degenerate set potential ActRIIA nucleotide sequences can then be expressed as individual polypeptides or as a set larger fusion proteins (e.g. for phage display).
There are many ways to generate a library of homologs from a degenerate sequence of oligonucleotides. An automatic DNA synthesizer can chemically synthesize a degenerate sequence of genes. The synthetic genes are then ligated into the appropriate vector for expression. The art of synthesizing degenerate Oligonucleotides has been well documented. See, for example, Narang S. (1983) Tetrahedron39:3; Itakura and al. (1981) Recombinant DNA Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al., (1984) Annu Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477). These techniques were used in directed evolution of other proteins (see Scott et.al., (1990), Science 249, 386-390; Roberts, et.al., (1992), PNAS USA, 89:2429-2433; Devlin, et.al. (1990), Science 249, 404-406; Cwirla, et.al. (1990), PNAS USA USA 87, 6378-6382, as well as U.S. Pat Nos. Nos.
You can also use other methods of mutagenesis to create a combinatorial library. By screening for alanine scanning mutation and similar methods, ActRIIA variants of polypeptides can be created and isolated from a library (Ruf et.al., 1994) Biochemistry 33:1565-1572; Wang and co., 1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-29292; Lowman and colleagues (1991) Biochemistry 30, 10832-10838. Cunningham et.al. (1989) Science 244 :1081-1085. By linker scanning mutation (Gustin and colleagues (1993) Virology 193 :653-660; Brown, et.al. (1992) Mol. Cell Biol. 12:2644-2652 McKnight et.al., (1982), Science 232,316); by saturation mutagenesis [Meyers et.al., (1986), Science 232,613]; by PCR mutagenesis [Leung et.al., (1989), Method Cell Mol Biol 1:11-19]); or by random mutagenesis. (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis is a great way to identify truncated or bioactive ActRIIA polypeptides, especially in combinatorial settings.
There are many techniques that can be used to screen gene products from combinatorial libraries created by point mutations or truncations. These techniques can be used to quickly screen gene libraries created by combinatorial mutation of ActRIIA polypeptides. Cloning large gene libraries into replicable expression vectors is the most common method for screening them. The vectors are then transformed into appropriate cells and the genes of combination are expressed under conditions that allow for the detection of desired activity. This allows for relatively easy isolation of the vector coded by the gene. The most popular assays are activin binding assays or activin-mediated cells signaling assays.
“ActRIIA Polypeptides may be used as inhibitors in certain embodiments. These post-translational modifications can also be present in ActRIIA proteins. These modifications can include, but not be limited to, acetylation and carboxylation, glycosylations, phosphorylation. lipidation, acylation, and phosphorylation. The modified ActRIIA proteins may also contain non-amino acids elements such as lipids or polyethylene glycols. Any method that is available to the skilled artisan can be used to test the effects of these non-amino acids elements on the functionality and function of an ActRIIA protein. Post-translational processing can also be crucial for proper folding and/or functioning of an ActRIIA protein when it is created in cells by cleaving the nascent ActRIIA form. Different cells, such as CHO, HeLa and MDCK, 293, W138 or HEK293, have different cellular machinery and specific mechanisms for such post-translational activity. These may be used to modify and process the ActRIIA proteins.
“In some aspects, functional variants of ActRIIA proteins used in the inhibitions of the methods described herein include fusion protein having at least a part of the ActRIIA peptides and one to more fusion domains. Examples of fusion domains are polyhistidine (Glu-Glu), glutathione S Transferase (GST), thioredoxin and protein A. Also, there is an immunoglobulin heavy chains constant region (Fc), maltose binding proteins (MBP) or human serum albumin. You can select a fusion domain to confer desired properties. Some fusion domains can be used to isolate fusion proteins using affinity chromatography. The appropriate matrices for affinity purification are used, including glutathione, amylase, nickel- and cobalt-conjugated reagents. Many of these matrices can be purchased in the?kit? Many of these matrices are available in?kit? form, such the Pharmacia GST purification systems and the QIAexpress? (Qiagen), which can be used with (HIS6) Fusion partners. Another example is the selection of a fusion domain to aid in detection of ActRIIA polypeptides. These domains can be used to detect various fluorescent proteins, such as GFP, as well as?epitope tag?. These are often short peptide sequences that can be used to identify a specific antibody. There are several well-known epitope tags that can be used to identify monoclonal antibodies. These include FLAG, HA and cmyc tags. Some fusion domains contain a protease cleavage location, such as Factor Xa and Thrombin. This allows the relevant protease partial digestion of the fusion proteins, and thereby liberates the recombinant protein from them. By subsequent chromatographic separation, the proteins can be separated from the fusion domain. An ActRIIA protein is fused to a domain that stabilises it in vivo. This is known as a “stabilizer”. domain). By ?stabilizing? Is meant any action that increases serum half-life, regardless of whether it is due to decreased destruction, decreased kidney clearance, or some other pharmacokinetic effect. It is known that fusions with the Fc part of an immunoglobulin can confer desirable pharmacokinetic characteristics on a wide variety of proteins. Also, fusions with human serum albumin may confer desirable properties. You can also choose fusions to human serum albumin that have multimerizing domains, such as dimerizing, trimerizing domains, and functional domains, which confer additional biological functions, such as stimulation of bone or muscle growth.
It is possible to arrange different elements of fusion proteins in any way that achieves the desired functionality. An ActRIIA peptide can be placed Cterminal to a heterologous or alternatively, a heterologous peptide may be placed in the C-terminal of an ActRIIA peptide. The ActRIIA polypeptide and heterologous domains need not be adjacent in the fusion protein. Additional domains and amino acid sequences can be added C- or Nterminal to either of the domains, or between them.
“In some embodiments, ActRIIA peptides may be modified to stabilize the ActRIIA peptides. These modifications can, for example, increase the in vitro half-life of ActRIIA proteins, prolong the circulatory half-life of ActRIIA proteines, or decrease the proteolytic degradation. These stabilizing modifications can include, but not be limited to, fusion protein (including, for instance, fusion proteins consisting of an ActRIIA Polypeptide and a stabilizerdomain), modifications to a glycosylation website (including, as an example, adding a glycosylation location to an ActRIIA Polypeptide), and modifications to carbohydrate moiety (including the removal of carbohydrate moieties in an ActRIIA Polypeptide). An ActRIIA polypeptide and a stabilizer domain, such as an IgG molecular (e.g. an Fc domain) are fused in the case of fusion protein. The term “stabilizer domain” is used herein. The term “stabilizer domain” does not just refer to a fusion domain, as in the case with fusion proteins. It also includes nonproteinaceous modifications, such as a carbohydrate moiety or nonproteinaceous copolymer like polyethylene glycol.
“In some embodiments, purified and/or isolated ActRIIA proteins, which have been isolated from other proteins or are otherwise substantially free of them, can be used in conjunction with the methods and compositions described. ActRIIA can be expressed from recombinant DNA.
“In certain aspects, ActRIIA peptides used for compositions and methods are created using isolated and/or modified nucleic acid encoding any ActRIIA peptides (e.g. soluble ActRIIA peptides), as well as fragments, functional variants, and fusion proteins. SEQ ID No: 4 encodes the ActRIIA precursor, and SEQ ID 5 encodes ActRIIA’s extracellular domain. These nucleic acid can be either single-stranded, or double-stranded. These nucleic acid can be either DNA or RNA molecules. These nucleic acid can be used in various ways, including to make ActRIIA polypeptides, or as therapeutic agents (e.g. in a gene therapy).
“In some aspects, nucleic acid encoding ActRIIA Polypeptides might include nucleic acid variants of SEQID NO: 4 and 5. Variant nucleotide sequences are sequences that differ by one to three nucleotide additions, substitutions, or deletions.
“In some embodiments, the nucleic acids sequences that encode ActRIIA polypeptides can be isolated or recombinant and less than 80%, 85% or 90%. They may also be 95%, 97% or 98%. They may be 99% identical to SEQID NO: 4 or 5. A person of ordinary skill in art will be able to recognize that nucleic acids sequences complementing SEQ ID No: 4 or 5 and variants SEQ ID no: 4 and 5 can be used to produce ActRIIA proteins suitable for use with the methods and compositions discussed herein. Further embodiments allow for such nucleic acids sequences to be isolated, recombinant and/or fused with a heterologous sequence or from a DNA database.
“In other embodiments nucleic acid used in the production ActRIIA polypeptides may include nucleotide sequencings that hybridize under extremely stringent conditions to the nucleotide-sequences designated in SEQID NO: 4 and 5, complement sequence of SEQID NO: 4 and 5, or fragments thereof. An ordinary person with knowledge of the art will know that stringency conditions that promote DNA hybridization may be adjusted. One can do hybridization at approximately 6.0 times sodium citrate/sodium chloride (SSC), at 45 degrees Celsius. Then, one can wash the DNA with 2.0 times SSC at fifty degrees Celsius. You can choose from a low salt concentration of approximately 2.0 times SSC at fifty degrees Celsius to a high salt concentration of about 0.2x SSC at fifty degrees Celsius. The wash step temperature can also be adjusted from room temperature at 22 degrees Celsius (low stringency) to 65 degree Celsius (high stringency). Temperature and salt can be varied or the temperature or salt concentration may change while one variable is maintained constant. One embodiment allows nucleic acid to be hybridized under low stringency conditions, such as 6x SSC at ambient temperature and 2x SSC at night. This can be combined with the methods described herein.
“Isolated nucleic acid that differs from the nucleic acid as described in SEQ ID Nos: 4 and 5 due to degeneracy of the genetic code can also be used in the manufacture of ActRIIA Polypeptides, suitable for use with the methods and compositions discussed herein. A number of amino acids can be identified by multiple triplets, for example. Codons that identify the same amino acid or synonyms may lead to?silent? Mutations that do not alter the amino acid sequence of the protein are possible. It is possible that DNA sequence polymorphisms which do cause changes in the amino acids sequences of subject proteins may exist in mammalian cells. Natural allelic variation may cause variations in nucleotides that encode a specific protein.
“In some embodiments, the regulatory nucleotide sequences of a expression construct may be operably linked with recombinant DNA. The regulatory nucleotide sequences used to express genes will usually be suitable for the host cell. There are many types of suitable expression vectors and regulatory sequences that can be used for different host cells. These regulatory sequences can include, but not be limited to, promoter, leader, signal, or transcriptional sequences, translational start or termination sequences and enhancer/activator sequences. These are known as constitutive and inducible inducers. These promoters can be naturally occurring promoters or hybrid promoters that combine elements from more than one promoter. An expression construct can be found in a cell via an episome (e.g., plasmid) or may be embedded in a genome. A preferred embodiment of the expression vector includes a selectable marker gene that allows for the selection and transformation of host cells. The art is well-informed about the existence of selectable marker genes. They will differ depending on the host cell.
“In certain instances, the nucleic acids used in the production ActRIIA peptides can be provided in expression vectors that contain a sequence of nucleotides encoding ActRIIA peptides and linked to at least one regulatory sequence. Art-recognized regulatory sequences can be used to control the expression of ActRIIA polypeptides. The term regulatory sequence also includes enhancers, promoters, and other expression control components. Goeddel, Gene Expression Technology: Methods In Enzymology Academic Press, San Diego (Calif.) (1990). These vectors can be used to express ActRIIA-encoding DNA sequences by using any of the many expression control sequences. These useful expression control sequences include the tet promoter and adenovirus immediate early promoter. RSV promoters. T7 promoter whose expression can be directed by T7 RNA Polymerase. The major operator and promoter regions for phage lambda. The design of an expression vector can depend on factors such as the host cell that will be transformed and/or what type of protein is being expressed. It is also important to consider the vector’s copy numbers, control over that number, and any other proteins encoded by it, such as antibiotic marks.
Recombinant nucleic acids are used to produce ActRIIA polypeptides. The cloned gene or a portion of it can be ligated into a vector that is suitable for expression in prokaryotic or eukaryotic (yeast, avian or insect) cells or both. Plastids and other vectors can be used to produce a recombinant ActRIIA protein. Plastids that are suitable for expression in prokaryotic cell types such as E.coli, pEMBL, pBR322-derived and pEMBL-derived vectors, pEX, pBTac, pEMBL, pEX, pEMBL, pEMBL, pUC, and pBTac-derived vectors are all examples of these types of vectors. coli.”
“Some mammalian expression sequences include both prokaryotic sequences that allow for the vector to be propagated in bacteria and one or more eukaryotic transcript units that can be expressed in eukaryotic cell. Examples of mammalian expression vectors that can be transfected into eukaryotic cells include the pcDNAI/amp and pcDNAI/neo. These vectors can be modified with sequences of bacterial plasmids such as pBR322, which facilitates replication and drug resistance selection in prokaryotic and non-eukaryotic cells. Alternately, you can transiently express proteins in eukaryotic cell by using derivatives of viruses like the bovine papillomavirus (BPV-1), and Epstein-Barr virus(pHEBo), pREP-derived, and p205. In the section on gene therapy delivery systems, you will find examples of other viral expression systems (including retroviral). It is well-known that there are many methods for preparing plasmids as well as for transforming host organisms. Other suitable expression systems, both for prokaryotic as well as eukaryotic cells are available in Molecular Cloning A Laboratory Manual 3rd Ed. Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press 2001). Sometimes it is possible to express the recombinant proteins using a Baculovirus Expression System. These baculovirus expression system examples include pVL-derived Vectors (such pVL1392, 1393 and pVL941) and pAcUW vectors.
“Vectors are possible to produce the subject ActRIIA proteins in CHO cells. These vectors include a PcmvScript vector (Stratagene La Jolla (Calif.), pcDNA4 Vectors (Invitrogen Carlsbad (Calif.) and pCIneo vectors [Promega Madison, Wis.]. The subject gene constructs can be used, as will be obvious, to induce expression of the subject ActRIIA proteins in cells grown in culture.
“Host cells can be transfected with a gene recombinant including a sequence coding sequence (e.g. SEQ ID NO 4 or 5 for one or more subject ActRIIA proteins. These genes can then be used to produce ActRIIA protein suitable for use in the compositions and methods described herein. Any prokaryotic and eukaryotic cells can be used as the host cell. An ActRIIA polypeptide described herein can be expressed in E. coli, mammalian cells, yeast cells, and insect cells (e.g. using a baculovirus gene expression system). The art has many other suitable host cells.
“The ActRIIA polypeptides are produced in the following methods. A host cell can be transfected with an expression vector that encodes an ActRIIA protein. This can then be grown under the appropriate conditions for expression. You can extract the ActRIIA protein from a mixture containing the ActRIIA protein and cells. The ActRIIA protein may also be retained in the cytoplasm or in a membrane fraction. Cells are then harvested and the protein isolated. Cell culture can include host cells, media, and other byproducts. The art is well-versed in the selection of suitable media for cell culture. You can isolate the subject ActRIIA proteins from host cells or cell culture media using any of the techniques described in the art. The preferred embodiment of the ActRIIA protein is a fusion protein that contains a domain that facilitates its purification. One embodiment of purification involves a series column chromatography steps. These include, for example, protein A chromatography and Q sepharose Chromatography, phenylsepharose Chromatography, size exclusion Chromatography, and cation exchange Chromatography. You can also purify the buffer by buffer exchange or viral filtration. This demonstrates that ActRIIA hFc protein was obtained to a purity of >98% by size exclusion analysis chromatography, and >95% by SDS-PAGE. This purity level was sufficient to produce desirable bone effects in mice and acceptable safety profiles in rats, mice, and other non-human primates.
“In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of a recombinant ActRIIA polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni2+ metal resin. You can then remove the purification leader sequence by treating it with enterokinase, to obtain the purified ActRIIA protein (e.g. Hochuli and colleagues, 1987, J. Chromatography 411,177; Janknecht and colleagues, PNAS USA, 88:8972).
The techniques for creating fusion genes are well-known. The basic principles of fusion genes are that they combine DNA fragments from different sequences. These include using conventional methods, which use blunt-ended and stagger-ended termini to ligation, restriction enzyme digest to ensure appropriate termini, filling in of cohesive ends, alkalinephosphatase treatment to prevent unwanted joining, and enzymatic binding. Another embodiment of the fusion gene is possible to be synthesized using conventional methods, including automated DNA synthesizers. Anchor primers are another option for PCR amplification. These anchor primers give rise to complementary overlaps between two consecutive fragments of DNA. This can then be annealed to create a chimeric sequence of genes (see Current Protocols in Molecular Biology). Ausubel et al., John Wiley & Sons: 1992).”
“ActRIIA -Fc fusion proteins can be expressed in stably-transfected CHO?DUKX Bl1 cells using a pAID4 vector. (SV40 ori/enhancers, CMV promoter), a tissue plasminogen lead sequence of SEQID NO:9. As shown in SEQ ID No:7, the Fc portion of the IgG1 Fc sequence is human. In some embodiments, the protein contains between 1.5 and 2.5 moles sialic acid per molecule ActRIIA?Fc fusion protein.
“In some embodiments, the ActRIIA -Fc fusion’s serum half-life can exceed 25-32 days in humans. The CHO cell-expressed material may have a greater affinity for activin B than the ActRIIA hFc fusion protein expressed by human 293 cells (del Re and al., J Biol Chem). 2004 Dec. 17; 279(51):53126-35). The TPA leader sequence was more productive than any other sequences. It also, unlike ActRIIA?Fc which is expressed with a native leader and may have a very pure N-terminal sequence. The native leader sequence could result in two distinct species of ActRIIA?Fc. Each has a different N-terminal sequencing.
“(b) Inhibitors ActRIIB”
“ActRIIB” is the term used herein. The term “ActRIIB” refers to any family of activin receptor type IIB proteins (ActRIIB), from any species, and variants derived by such ActRIIB proteins through mutagenesis or another modification. Any of the known forms of ActRIIB are referred to herein. ActRIIB members are transmembrane proteins. They consist of a ligand binding extracellular domain with a cysteine rich region, a transmembranedomain, and a domain in the cytoplasm with predicted serine/threoninekinase activity.
“ActRIIB inhibitors” can be used in the compositions described herein. They include activin-binding soluble ActRIIB peptides; activin subunits (activin A and B) that disrupt ActRIIB bindin binding; activin A and B subunits that disrupt ActRIIB binding; activin binding antibodies; activin binding antibodies; activin binding non-antibody proteins; and random peptides that are selected for activin and ActRIIB a Fc domain.
“In some embodiments, two or more proteins (or other moieties with activin/ActRIIB binding activity), especially activin binding sites that block type I (e.g. a soluble Type I activin receptor), and type II (e.g. a soluble Type II activin receptor), may be linked together to form a bifunctional, multifunctional binding molecule that inhibits ActRIIB. This can then be used in the compositions or methods described herein. The compositions and methods herein may include nucleic acids aptamers, small molecules, and other agents that inhibit ActRIIB.
“(i) ActRIIB Inhibitors Comprising ActRIIB Polypeptides”
“ActRIIB polypeptide” is the term used herein. The term “ActRIIB polypeptide” refers to any naturally occurring polypeptide from an ActRIIB member, as well as any variants (including fragments, fusions and peptidometic forms) that maintain a useful activity. ActRIIB Polypeptides, for example, are polypeptides that have a sequence at least 90% identical to an ActRIIB receptor’s sequence and optionally at least 85%-95%, 96%-97%, 98% or 99% more identity. An ActRIIB polypeptide could bind to or inhibit the function an ActRIIB protein/activin, for example. The human ActRIIB precursor protein polypeptide (SEQID NO:16 and SEQ ID No:28) is an example of an ActRIIB peptide. The ActRIIB precursor protein whose amino acid sequence can be referred to as SEQID NO:16 (or SEQID NO:28) (i.e. the human ActRIIB pre-protease polypeptide), has the signal peptide at amino acids 1-18; the extracellular domain at amino acids 19-134, and potential N-linked glycosylation site at amino acid positions 42 to 65. The sequence of nucleic acids that encodes the human ActRIIB precursor protein is known as SEQID NO:19. (SEQID NO:19 contains an alanine at codon 64. However, one skilled in the art could modify it to add an arginine to the codon 64). For a detailed description of these sequences, see Table 6.
“The numbering system for ActRIIB-related amino acids described herein is based upon the amino acid numbering SEQID NO:16 (which only differ in their amino acid expressed at 64), except where otherwise noted. For example, if an ActRIIB polypeptide is described as having a substitution/mutation at amino acid position 79, then it is to be understood that position 79 refers to the 79th amino acid in SEQ ID NO:16 or SEQ ID NO:28, from which the ActRIIB polypeptide is derived. If an ActRIIB protein is described as having an actinine or arginine at amino acids 64, it should be understood that position 64 refers the 64th amino Acid in SEQID NO:16 and SEQID NO:28 from which the ActRIIB Polypeptide was derived.
“In some embodiments, the ActRIIB inhibitors used in the compositions or methods described herein include polypeptides that contain an activin binding domain of ActRIIB. The activin binding domains of ActRIIB may be the extracellular domain or a portion thereof in some embodiments. Specific embodiments make the extracellular domain of ActRIIB soluble. U.S. Patent Application Publication Nos. 62 and 63 disclose examples of modified ActRIIB ActRIIB polypeptides. 20090005308 & 20100068215 are the disclosed forms of ActRIIB polypeptides, and their disclosures are included herein by reference in all respects.
“In certain embodiments, ActRIIB peptides used herein in compositions and methods are soluble ActRIIB peptides. The term “soluble ActRIIB peptide” is used. The term?soluble ActRIIB polypeptide? generally refers to any polypeptides that contain an extracellular domain from an ActRIIB proteins as well any variants (including fragments, mutants, and peptidometic forms). Soluble ActRIIB peptides can bind activin, but the wild-type ActRIIB protein doesn’t have a significant selectivity for binding to activin over GDF8/11. The methods described herein may allow for the use of altered forms or ActRIIB that have different binding properties. These altered forms are described, e.g. in international patent publication Nos. These disclosures are included in the international patent application publication Nos. WO 2006/012627 WO 2010/019261. The addition of an activin-selective binding compound to activate native or modified ActRIIB proteins can give them additional activin specificity. Exemplary soluble ActRIIB peptides include the extracellular Domain of a human ActRIIB Polypeptide (e.g. SEQ ID NOs 17, 18, 23, 26, 27, 29, 30, 31, 32.33.36.37.42 and 43).
Hilden et. al. disclosed an Fc fusion protein that has the ActRIIB extracellular structure. (Blood 1994, 83(8), 2163-70), which contains an alanine in the position that corresponds to amino acid 64 of ActRIIB precursor amino acids sequence. (Herein referred as?A64?) It has been shown to have a low affinity for GDF-11 and activin. An Fc fusion protein containing an arginine at 64 position of the ActRIIB precursor amino acids sequence (herein referred as?R64?) is in contrast. An Fc fusion protein with an arginine at position 64 of the ActRIIB precursor amino acid sequence (herein referred to as?R64?) has an affinity for activin, GDF-11, and low nanomolar to high picomol ranges (see, e.g. U.S. Patent Application Publication Number. 20100068215 is incorporated herein in its entirety. SEQ ID No: 28 presents an ActRIIB precursor amino acids sequence with an arginine position 64. In SEQ ID No:28, an ActRIIB precursor sequence with an arginine position 64 is presented. Other embodiments of ActRIIB polypeptides may contain an amino acid other than arginine or alanine at the position that corresponds to amino acid 64 in the ActRIIB precursor amino acid sequence.
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