actribb antagonist and administration and uses of antagonists

专利摘要:
  ACTRIIB ANTAGONIST AND ADMINISTRATION AND USES OF ANTAGONISTS In certain respects, the present invention provides compositions and methods for promoting bone growth and increasing bone density, as well as for the treatment of multiple myeloma. In addition, methods are provided for administering an ActRIIb antagonist to a patient.
公开号:BR112012005225A2
申请号:R112012005225-0
申请日:2010-09-09
公开日:2020-09-01
发明作者:Josbir Seehra；John Knopf；Ken Attie
申请人:Acceleron Pharma Inc.；
IPC主号:

专利说明:

Í 1/65 ACTRIIB ANTAGONISTS AND ADMINISTRATION AND USES OF
ANTAGONISTS Related patent applications This patent application claims its priority benefit to US Provisional Patent Application No. 61/276 287, filed on September 9, 2009, the teachings of which are incorporated herein in their entirety by reference in this patent application.
Background of the invention Bone diseases, ranging from osteoporosis to fractures, represent a set of pathological conditions for which there are few effective pharmaceutical agents. Instead, treatment focuses on physical and behavioral interventions, including immobilization, exercise and changes in diet. It would be beneficial to have therapeutic agents that promote bone growth and increase bone density, in order to treat a variety of bone disorders.
Bone growth and mineralization depend on the activities of two types of cells, osteoclasts and osteoblasts, although chondrocytes and vasculature cells also participate in critical aspects of these processes. In terms of development, bone formation occurs through two mechanisms, endochondral ossification and intramembranous ossification, the former responsible for longitudinal bone formation and the latter responsible for the formation of topologically flat bones, such as skull bones. Endochondral ossification requires the formation and, in sequence, degradation of cartilage structures in the growth plates that serve as a template for the formation of osteoblasts, osteoclasts, vasculature and subsequent mineralization. During intramembranous ossification, bone is formed directly in the connective tissues. Both processes demand infiltration of osteoblasts and subsequent deposition of matrix.
Fractures and other structural ruptures of the bone are consolidated through a process that, at least superficially, resembles the sequence of osteogenesis events that occurred during development, including the formation of cartilaginous tissue and subsequent mineralization. The fracture healing process can occur in two ways. Direct or primary bone healing occurs without callus formation. Indirect or secondary bone consolidation includes a precursor callus formation stage. Primary fracture consolidation involves repairing mechanical continuity in a closely established rupture. Under suitable conditions, cells that reabsorb bone around the rupture respond by creating a tunnel through reabsorption and establish pathways for blood vessel penetration and subsequent consolidation. Secondary consolidation - follows a process of inflammation, formation of spongy callus, callus mineralization and callus remodeling. In the inflammation stage, the rupture of blood vessels in the periosteum and the endosteum lead to the formation of hematoma and hemorrhage at the lesion site.
Inflammatory cells invade the area. In the spongy callus formation stage, the cells produce new vessels, fibroblasts, intracellular materials and support cells, and granulomatous tissue is formed in the space between the fracture fragments. The clinical union through rupture is established by fibrous or cartilaginous tissue (spongy callus). Osteo-blasts are formed and mediate the spongy callus mineralization, which is then replaced by lamellar bone and subjected to the normal remodeling process.
In addition to fractures and other physical disruptions in bone structure, loss of bone mineral content and bone mass can be caused by a wide variety of conditions and result in significant medical problems. Changes in bone mass occur relatively predictably throughout an individual's life. Even around the age of 30, bones, both in males and females, increase mass to the maximum by means of linear growth of endochondral growth plates and radial growth plates. After reaching the age of approximately 30 (for trabecular bone, for example, flat bones such as the vertebrae and the pelvis) and 40 years (for cortical bone, for example, long bones found in the extremities), there is slow bone loss both in both men and women. In women, a final phase of substantial bone loss also occurs, probably due to post-menopausal estrogen deficiencies. During this phase, women may lose an additional 10% of bone mass from the cortical bone and 25% from the trabecular compartment. Whether resulting or not from a pathological condition, such as osteoporosis, progressive bone loss depends largely on the individual's initial bone mass and whether or not there are exacerbating conditions.
Bone loss is sometimes CHARACTERIZED as an imbalance in the normal bone remodeling process. Healthy bone is constantly undergoing remodeling. Remodeling begins with bone resorption by osteoclasts. The reabsorbed bone is then replaced by new bone tissue, which is CHARACTERIZED by collagen formation by osteoblasts and subsequent calcification. In healthy individuals, rates of resorption and training are balanced. Osteoporosis is a progressive chronic condition, marked by displacement for resorption, resulting in a global reduction in bone mass and bone mineralization. Osteoporosis in humans is preceded by clinical osteopenia (bone mineral density greater than one standard deviation, but less than 2.5 standard deviations below the mean value for young adult bone). Worldwide, approximately 75 million people are at risk for osteoporosis.
Therefore, methods to control the balance between osteoblast osteoclast activity can be useful to promote the consolidation of fractures and other bone damage, as well as for the treatment of diseases, such as osteoporosis, associated with loss of mass. bone and bone mineralization.
ó 3/65 Regarding osteoporosis, estrogen, calcitonin, osteocalcin with vitamin K or high doses of calcium in the diet are all used as therapeutic interventions.
Other therapeutic approaches to osteoporosis include bisphosphonates, parathyroid hormone, calcimimetics, statins, anabolic steroids, lanthanum and strontium salts and sodium fluoride.
These therapeutic agents, however, are often associated with undesirable side effects.
Consequently, the present invention aims to provide compositions and methods to promote bone growth and mineralization.
Summary of the invention The present invention, in part, demonstrates that ActRilb antagonists, including ActRilb-based molecules (for example, molecules containing a ligand-binding portion of an extracellular domain of ActRilb, or a variant thereof, such as ActRIlb- Fc) and other ActRilb signaling antagonists, can be used to increase bone density, to promote bone growth and / or to increase bone strength.
Specifically, the present invention demonstrates that a soluble form! ActRIlb promotes an increase in bone density, bone growth and bone resistance in vivo.
Although most pharmaceutical agents that promote bone growth or inhibit bone loss, bone acts as anti-catabolic agents (also commonly referred to as “catholic agents”) (eg, bisphosphonates) or anabolic agents (eg, parathyroid hormone, PTH, when administered with the appropriate dose), the soluble ActRilb protein exhibits dual activity, having anti-catabolic and anabolic effects.
Therefore, ActRIlb antagonists can be used to increase bone density and to promote bone growth.
Anti-ActRilb antibodies and small molecules and aptamers directed against ActRilb, in addition to nucleic acids that decrease ActRIlb expression can also be used to treat disorders associated with low bone density or low bone resistance, such as osteoporosis, or to promote growth bone in patients in need of such treatment, such as those with bone fracture (in this descriptive report, these molecules are also included in the term “ActRilb antagonists”). The present invention further indicates that ActRilb antagonists are effective in preventing and / or repairing bone damage caused by multiple myeloma tumors and bone metastases (for example, in breast, esophageal, colon, prostate or lung cancer) and, in addition, that ActRilb antagonists decrease the tumor burden in multiple myeloma.
ActRllb antagonists can also be used to treat a variety of disorders or conditions, especially muscle and neuromuscular disorders (for example, muscular dystrophy, amyotrophic lateral sclerosis (ALS) and muscle atrophy), adipose tissue disorders ( for example, obesity), metabolic disorders (for example, type 2 diabetes), neurodegenerative diseases and loss of muscle mass
'4/65 associated with age (sarcopenia), therapy for prostate cancer and cachexia caused by cancer. In specific embodiments, ActRIlb antagonists (for example, soluble ActRilb polypeptides) are able to antagonize an ActRIlb receptor in any process associated with ActRilb activity. Optionally, ActRIlb antagonists of the invention can be created to preferentially antagonize one or more AciRllb receptor ligands, such as GDF8 (also called myostatin), GDF11, activin A, activin B, activin AB, Nodal and BMP7 (also called OP-1 ), and can therefore be useful in the treatment of additional disorders. Examples of ActRilb antagonists include the natural ActRIlb polypeptides, as well as their functional variants.
In certain aspects, the invention provides polypeptides comprising a soluble ActRilb polypeptide. ActRIlb polypeptides can be formulated as a pharmaceutical preparation, comprising the ActRilb polypeptide and a pharmaceutically acceptable carrier. Optionally, the ActRilb polypeptide binds to activin with Kp less than 1 micromolar or less than 100, 10 or 1 nanomolar. Preferably, the composition is at least 95% pure, with respect to other polypeptide components, as assessed by size exclusion chromatography, and more preferably, the composition is' at least 98% pure. An ActRilb polypeptide for use in such a preparation can be any of those described in this patent application, such as a polypeptide having an amino acid sequence selected from SEQ ID NOs: 2, 3, 13, 17 or 20, or having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 13, 17 or 20. An ActRilb polypeptide may include a functional fragment of a natural ActRilb polypeptide, such as that comprising at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: 1 - 3 or a sequence of SEQIDNO: 2, missing 10 to 15 amino acids at the C-terminal (the “tail”). An ActRIlb polypeptide can be encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID NO: 3 or 5.
An ActRilb polypeptide can include one or more changes in the amino acid sequence (for example, in the linker binding domain), relative to a natural ActRIlb polypeptide. Examples of altered ActRilb polypeptides are provided in WO 2006/012627, p. 59 - 60, incorporated herein by reference in this patent application. Alteration in the amino acid sequence can, for example, alter the glycosylation of the polypeptide when produced in a mammal, insect or other eukaryotic cell or alter the proteolytic cleavage of the polypeptide, relative to the natural ActRilb polypeptide.
An ActRilb polypeptide can be a fusion protein that has, as a domain, an ActRilb polypeptide (for example, a linker-binding portion of an ActRllb or variant thereof) and one or more additional domains that confer a property
'5/65 desirable, such! such as improved pharmacokinetics, easier purification, targeting specific tissues, etc.
For example, a domain of a fusion protein can enhance one or more of in vivo stability, in vivo half-life, uptake / administration, localization or distribution in tissues, formation of protein complexes, protein multimerization fusion and / or purification.
Dimerization or multimerization can increase the binding affinity for the ligand.
An ActRilb fusion protein can include an immunoglobulin Fc domain (wild type or mutant) or serum albumin or other portion of polypeptides, which provide desired properties such as improved pharmacokinetics, improved solubility or improved stability.
Typically, an ActRIlb-Fc fusion protein will be produced in the form of a homodimeric complex.
Optionally, an ActRIlb-Fc fusion comprises a relatively unstructured linker positioned between the Fc domain and the extracellular domain of ActRilb.
This unstructured linker may correspond to the unstructured region of approximately 15 amino acids at the C-terminal end of the extracellular domain of ActRilb (the "tail"), or it may be an artificial sequence of 1,2,3,40u5 amino acids or length between 5 and 15, 20, 30, 50 or more amino acids that are relatively free of secondary structure, or a mixture of both. í A linker may be rich in glycine and proline residues and may, for example, contain a single sequence of threonine / serine and glycines or repeated sequences of threonine: na / serine and glycines (eg singlets or TG3 repeats) , TG ,, SG; or SG, 4). A fusion protein can include a purification substrate, such as an epitope tag, such as a FLAG tag, polyhistidine sequence, and GST fusion.
Optionally, a soluble ActRilb polypeptide includes one or more modified amino acid residues, selected from: glycosylated amino acid, PEGylated amino acid, farnesylated amino acid, acetylated amino acid, biotinylated amino acid, amino acid conjugated to lipid group and conjugated amino acid the organic derivatization agent.
A pharmaceutical preparation can also include one or more additional compounds, such as a compound that is used to treat a bone disorder.
Preferably, a pharmaceutical preparation is substantially free of pyrogens.
In general, it is preferable for an ActRI-lb protein to be expressed in mammalian cell lines that properly mediate - natural daglycosylation of the ActRIlb protein in order to decrease the likelihood of an unfavorable immune response in a patient.
Human and CHO cell lines have been used successfully, and other common mammalian expression systems are expected to be useful.
In certain aspects, the invention provides nucleic acids encoding an ActRilb poly-peptide.
An isolated polynucleotide may comprise a coding sequence for an ActRilb polypeptide, as described above.
For example, an isolated nucleic acid may include an extracellular domain coding sequence (for example,
6/65 (example, ligand-binding domain) of an ActRIlb and a sequence that encodes part or all of the transmembrane domain and / or the cytoplasmic domain of an ActRilb, but that encodes a stop codon positioned within the transmembrane domain or the cytoplasmic domain, or positioned between the extracellular domain and the transmembrane or cytoplasmic domain.
For example, an isolated polynucleotide may comprise the complete ActRilb polynucleotide sequence, such as SEQ ID NO: 4 or 5, or a truncated version, said isolated polynucleotide further comprising a transcription termination codon, containing at least six hundred nucleotides prior to the 3 'terminal or, otherwise, positioned in such a way that the translation of the polynucleotide gives rise to an extracellular domain optionally fused to a truncated part of a complete ActRilb.
A preferred nucleic acid sequence is SEQ ID NO: 18. The nucleic acids described in this patent application can be operably linked to a promoter for expression, and the present invention provides cells transformed with these recombinant polynucleotides.
Preferably, the cell is a mammalian, such as a CHO cell.
In certain aspects, the present invention provides methods for producing an ActRilb polypeptide.
This method may include expression of any of the nucleic acids] (for example, SEQ ID NO: 4, 5 or 18) described in this patent application in an appropriate cell, such as a Chinese hamster ovary (CHO) cell . This method may comprise: a) culturing a cell under conditions suitable for expression of the AcRRbb polypeptide, wherein said cell is transformed with an ActRilb expression construct; and b) recovering the ActRilb polypeptide so expressed.
ActRIlb polypeptides can be recovered in crude, partially purified or highly purified fractions.
Purification can be accomplished by a series of purification steps, including, for example, one, two or three or more of the following, in any order: afini- chromatography -decoma protein A, anion exchange chromatography (eg , Q sepharose), chromatography by hydrophobic interaction (eg phenyl sepharose), size exclusion chromatography and cation exchange chromatography.
In certain aspects, an ActRilb antagonist described in this application, such as an ActRilb polypeptide, can be used in a method to promote bone growth or to increase bone density in an individual.
In certain modalities, the invention provides methods for treating a disorder associated with low bone density, or for promoting bone growth, in patients in need of such treatment.
One method may comprise administering to an individual in need of such treatment an effective amount of ActRilb antagonist.
In certain aspects, the invention provides uses of the ActRIlb antagonist to produce a medicament for the treatment of a disorder or condition described in this patent application.
In certain respects, the invention provides a method for identifying an agent that
'7/65 stimulates growth or increases bone mineralization. The method comprises: a) identifying an agent under test that binds to a ligand-binding domain of an ActRilb polypeptide; and b) evaluate the effect of the agent on bone growth or mineralization.
In certain respects, the invention provides methods for administering a patient a dose of ActRilb-Fc fusion protein, comprising administering an AcCtRIlb-Fc fusion protein to the patient in an administration schedule that maintains a serum concentration of the ActRIlIb fusion protein. -Fc of at least 8 µg / mL.
In certain aspects, the invention provides a compound for use in administering an ActRilb-Fc fusion protein to a patient, in which the ActRiIlb-Fc fusion protein is administered in a scheme that maintains a serum concentration of the ActRI fusion protein. - Ib-Fc of at least 8 pg / mL. Other exemplary compounds, uses and administration schedules are described in this specification and in the claims.
Brief description of the drawings The patent or patent application contains at least one drawing executed in color. Copies of this patent or publication of the patent application with drawing (s) in color will be provided by the Office upon request for any payment of the necessary fee.
Figure 1 shows a multiple sequence alignment of various vertebrate AcTRIlb proteins and human AcCtRIIA. Figure 2 shows a pharmacokinetic profile (PK) in human patients of a single dose of ActRilb-Fc administered subcutaneously.
Figure 3 shows the average percentage change in lean body mass after 15, 29 or 57 days of subcutaneous administration to human patients of a single dose of ActRilb-Fc.
Figure 4 shows the percentage of individuals with = 0.5 kg (upper panel) or 2 1.0 kg (lower panel) of increase in lean body mass after 15, 29 or 57 days of subcutaneous administration to human patients of a single dose of ActRilb-Fc.
Figure 5 shows the change in leptin, a serum biomarker of fat metabolism, at various times after subcutaneous administration to human patients of a single dose of ActRllb-Fc.
Figure 6 shows the change in adiponectin, a serum biomarker of fat metabolism, at various times after subcutaneous administration to human patients of a single dose of ActRilb-Fc.
Figure 7 shows the average percentage change in bone-specific alkaline phosphatase (BSAP), a serum bone formation biomarker, at various times after subcutaneous administration to human patients of a single dose of ActRilb-Fc.
Figure 8 shows the average percentage change in collagen type 1 C-terminal telopeptide (CTX), a serum bone reabsorption biomarker, at various times
It is 8/65 after subcutaneous administration to human patients of a single dose of ActRilb-Fc.
Figure 9 shows the average percentage change in serum follicle stimulating hormone (FSH) at various times after subcutaneous administration to human patients of a single dose of ActRilb-Fc.
Figure 10 shows a magnetic resonance imaging (MRI) examination of a cross section of the thigh in a representative human subject, who was administered a single subcutaneous dose of 3 mg / kg of ActRilb-Fc. The upper panel shows an MRI assessment of a thigh, the middle panel shows the baseline on Day 9, the lower panel shows the results on Day 29 after administration. The muscle is marked in gray, subcutaneous fat in pink and intramuscular fat in green.
Figure 11 shows the mean percentage change from baseline in thigh muscle volume on Day 29 for human subjects who were administered a single subcutaneous dose of ActRilb-Fc 1 mg / kg or ActRilb-Fc 3 mg / kg, when compared to placebo.
Figure 12 shows an alignment of the ActRIlb signaling sequence and extracellular domain of SEQ ID NO: 1 (residues 1 - 134 of SEQ ID NO: 1) with the ActRilb extracellular domain, represented by SEQ ID NO : 2. As shown, the signal sequence contains 19 amino acids in length. Therefore, an amino acid at the position X in SEQ ID NO: 1 has the corresponding position in X-19 in SEQ ID NO: 2. As an illustration, position 30 of amino acid in SEQ ID NO: 1 is position 11 amino acid in SEQ ID NO: 2. A similar correlation can be determined for other ActRilb sequences described in this patent application.
Figure 13 shows an alignment of human ActRlla and ActRIlb. Boxed residues represent amino acid residues that are believed to be in direct contact with the ligand (ie, the ligand-binding “pocket”), based on an analysis composed of multiple crystalline structures of ActRilb and AcíRila.
Figure 14 shows the pharmacokinetic profile of ActRIlb-hFc in serum over several days during the multiple ascending dose study described in Example 5.
Figure 15 shows serum concentrations of ActRIlb-hFc on the upper axis and the percentage change in follicle stimulating hormone (FSH) on the lower axis.
Figure 16 shows the percentage change in total lean body mass, caused by varying doses of ActRlib-hFc, as measured by DXA.
Detailed description of the invention
1. Overview The transforming growth factor beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to have biological effects
'9/65 on a wide variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis and epithelial cell differentiation. When manipulating the activity of a member of the TGF-beta family, it is often possible to cause significant physiological changes in an organism. For example, the beef breeds Piedmontese and Belgian Blue carry a loss of function mutation in the GDF8 gene (also called myostatin) that causes a marked increase in muscle mass. Grobet et al., Nat Genet. 1997, 17 (1): 71-4. In addition, in humans, inactive GDFB8 alleles are associated with increased muscle mass and, according to reports, exceptional strength. Schuelke et a /., N Engl J Med 2004, 350: 2682-8.
Activins are growth factors formed by dimeric polypeptides that belong to the TGF-beta superfamily. There are three main forms of activins (A, B and AB) that are homo / heterodimers of two closely related B subunits (BaBa, BsBs and BaBs). The human genome also encodes activin C and activin E, which are primarily expressed in the liver. In the TGF-beta superfamily, activins are unique and multifunctional factors that are capable of stimulating the production of hormones in 'ovarian and placental cells, supporting the survival of neuronal cells, influencing the progress of the cell cycle, positive or negative. negatively depending on the cell type, and induce mesodermal differentiation at least in amphibian embryos (DePaolo et a /., 1991, Proc Soc Ep Biol Med. 198: 500-512; Dyson et al., 1997, Curr Biol. 7: 81-84; Woodruff, 1998, Biochem Pharmacol. 55: 953-963). In addition, the erythroid differentiation factor (EDF), isolated from stimulated human monocytic leukemic cells, has been shown to be identical to activin A (Murata et a /., 1988, PNAS, 85: 2434). It has been suggested that activin A acts as a natural positive regulator of erythropoiesis in the bone marrow. In several tissues, activin signaling is antagonized by its related dimer, inhibin. For example, during the release of follicle stimulating hormone (FSH) from the pituitary gland, activin promotes FSH secretion and synthesis, while inhibin prevents FSH secretion and synthesis. Other proteins that can regulate the bioactivity of activin and / or bind to activin include follistatin (FS), follistatin-related protein (FSRP) and azmacroglobulin.
The TGF-B family signals are mediated by heteromeric complexes of serine / threonine kinase type receptors | and type Il, which phosphorylate and downwardly activate Smad proteins when stimulating the ligand (Massagué, 2000, Nat.
Rev. Mol. Cell Biol. 1: 169-178). These type receivers | and type | they are transmembrane proteins, composed of an extracellular ligand-binding domain with a region rich in cysteine, a transmembrane domain and a cytoplasmic domain with specificity
T 10/65 view for serine / threonine. Type receivers | are essential for signaling, and type receivers | are needed for ligand binding and for expression of | type receptors. Type receivers | and II of activin form a stable complex after binding to the ligand, resulting in phosphorylation of type | by | l type receivers.
Two | type receivers related, ActRila and ActRilb, have been identified as type II receptors for activins (Mathews and Vale, 1991, Cell 65: 973-982; Attisano et al., 1992, Cell 68: 97-108). In addition to activins, ActRlla and ActRIlb can interact biochemically with several other proteins of the TGF-B family, including BMP7, Nodal, GDF8 and GDF11 (Yamashita et al., 1995, J. Cell Biol. 130: 217-226; Lee and McPherron, 2001, Proc. Natl. A-cad. Sci 98: 9306-9311; Yeo and Whitman, 2001, Mol. Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16: 2749- 54). ALK4 is the primary type receptor | for activins, especially for activin A, and ALK-7 can serve as a receptor for activins too, especially for activin B.
As demonstrated in this patent application, an ActRilb polypeptide (ActRI-Ib [20-134] -Fc) is effective in promoting bone growth and increasing bone density in vivo, while also increasing muscle mass. Although not intended to be attached to any particular mechanism, the effect of ActRilb on bone is expected to be caused primarily by an antagonistic effect of activin. Regardless of the mechanism, it will be evident from the data presented here that ActRIlb antagonists increase bone density and affect bone biomarkers in a manner compatible with a combined anabolic / anti-resorptive effect in humans. It should be noted that bone is a dynamic tissue, with growth or retraction and increased or decreased density depending on a balance of factors, which produce bone and stimulate mineralization (osteoblasts primarily) and factors that destroy and demineralize bone ( primarily osteoclasts). Bone growth and mineralization can be increased by decreasing productive factors, increasing destructive factors, or both. The terms "promote bone growth" and "increase bone mineralization" refer to the physical changes that can be seen in the bone and are intended to be neutral as to the mechanism by which bone changes can occur.
In addition to promoting bone growth, the invention contemplates using AciRllb antagonists to treat or prevent diseases or conditions that are associated with abnormal activity of an ActRIlb or ActRilb ligand. For example, AcTIRIlb antagonists can be used to treat disorders or conditions in humans or animals. Examples of these disorders or conditions include, but are not limited to, metabolic disorders such as type 2 diabetes, impaired glucose tolerance, metabolic syndrome (for example, X chromosome syndrome) and trauma-induced insulin resistance (for example, burns or imbalance nitrogen); adipose tissue disorders (for example,
(11/65 (example, obesity); muscular and neuromuscular disorders such as muscular dystrophy (including Duchenne muscular dystrophy); amyotrophic lateral sclerosis (ALS); muscle atrophy; organ atrophy; fragility; carpal tunnel syndrome; congestive obstructive pulmonary disease; and sarcopenia, cachexia and other muscle wasting syndromes. Other examples include osteoporosis, especially in elderly and / or postmenopausal women; glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis; and osteoporosis-related fractures. Still other examples include low bone mass due to chronic glucocorticoid therapy, premature gonadal insufficiency, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, nutritional deficiencies and nervous anorexia.
The present invention provides methods in which ActRilb polypeptides and other ActRilb antagonists are used to promote bone growth and to increase bone density in humans, and also to increase muscle mass. ActRilb antagonists include, for example, ActRilb polypeptides that bind to ligands (for example, ActRilb-Fc), antibodies that bind to ActRilb and alter the binding of one or more ligands, such as activin or myostatin, proteins different antibodies, selected for binding to ActRilb (see, for example, WO / 2002/088171, WO / 2006/055689 and WO / 2002/032925, for examples of these proteins and methods for design and selection of the same), randomized peptides selected for connection to ActRlilb, often attached to an Fc domain. Two different proteins (or other groups) with ActRilb binding activity, especially activin binders that block type-binding sites | (for example, a soluble type 1 activin receptor) and type Il (for example, a type 1 soluble activin receptor), respectively, can bind to create a bifunctional binding molecule. Aptamers of nucleic acids, small molecules and other agents that inhibit ActRIlb signaling are included as ActRilb antagonists.
The terms used in this specification generally have their common meanings in the state of the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the descriptive report, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to use and use them. The scope or meaning of any use of a term will be evident from the specific context in which the term is used.
"Around" and "approximately" will generally mean an acceptable degree of error for the measured quantity, given the nature or accuracy of the measurements. Typically, exemplary degrees of error are inserted in 20 percent (%), preferably inserted in 10% and more preferably inserted in 5% of a given value or range of values.
'12/65 Alternatively and especially in biological systems, the terms "around" and "approximately" may mean values entered in an order of magnitude, preferably within 5 times and more preferably within 2 times a given value . Numerical quantities provided in this specification are approximations, unless stated otherwise, meaning that the term “around” or “approximately” can be inferred when not expressly stated.
The methods of the invention can include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants). These comparisons typically comprise polymeric sequence alignments, for example, using sequence alignment programs and / or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few). The technician versed in the subject can quickly recognize that, in such alignments, in the place where a mutation contains the insertion or deletion of a residue, the alignment of sequences will introduce a gap (typically represented by a dash) “A”) in the polymeric sequence that does not contain the inserted or deleted residue.
“Homologous”, in all its grammatical forms and orthographic variations, refers to the relationship between two proteins with “common evolutionary origin”, including superfamily proteins in the same species of organism, as well as homologous proteins of different species of organism. These proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarities, either in terms of percentage of identity or by the presence of specific residues or motifs and conserved positions.
The term "sequence similarity", in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.
However, in common use and in this patent application, the term "homologous," when modified with an adverb of the type "highly", can refer to the similarity of sequence and may or may not be related to a common evolutionary origin.
2. ActRilb Polypeptides In certain respects, the present invention relates to ActRilb polypeptides. In this specification, the term “ActRilb” refers to a family of activin Ilb receptor type proteins (ActRilb) of any species and variants derived from these ActRllb proteins by mutagenesis or other modification. It is understood that the reference in this specification to ActRilb is to any of the forms currently identified. The members of the ActRIlb family are usually transmembrane proteins, composed of an extracellular ligand-binding domain with a region rich in cysteine, a transmembrane domain and a cytoplasmic domain with predicted chiral serine / threonine activity.
'13/65 nase.
The term “ActRilb polypeptide” includes polypeptides containing any natural polypeptide from a member of the ActRIlb family, as well as any of its variants (including mutants, fragments, fusions and peptidomimetic forms) that retain useful activity. See, for example, WO / 2006/012627, whose ActRilb polypeptides are incorporated herein by reference in this patent application. For example, ActRilb polypeptides include polypeptides derived from the sequence of any known ActRilb having a sequence at least about 80% identical to the sequence of an ActRI-lb polypeptide, and optionally at least 85%, 90%, 95%, 97 %, 99% or greater identity. For example, an ActRilb polypeptide of the invention can bind and inhibit the function of an ActRllb protein. An ActRilb polypeptide can be selected for activity in promoting red cell formation in vivo. Examples of ActRilb polypeptides include human ActRIlb precursor polypeptide (SEQ ID NO: 1) and soluble human AcRIRI polypeptides (for example, SEQ ID NO: 2, 3, 13, 17 or 20).
The protein sequence of the human ActRilb precursor is as follows: MTAPWVALALLWGSLWPGSGRGEAETRECIYYNANWELERTINOSGLERCEGEQ 'DKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNER FTHLPEAGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHED PGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLADKASWQSEREIFSTP GMKHENLLQFIAMEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLS YLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQV GTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQ
HPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSLI RRSVNGTTSDCLVSLVTSVTNVDLPPKESS! (SEQ ID NO: 1) The signal peptide is the only underline; of the extracellular domain is in bold and the potential N-linked glycosylation sites are boxed.
The processed sequence of the soluble (extracellular) polypeptide of human ActRIlb is as follows:
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELV —KKGCWLDDFNCYDRQECVATEENPQVYYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAP T (SEQID NO: 2) Under the same conditions, the protein can be produced in the following sequence. The C-terminal “tail” of the extracellular domain is underlined. The sequence with the deleted “tail” (an A15 sequence) is as follows:
GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELV KKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA (SEQ ID NO: 3) Under the same conditions, the protein can be produced with a sequence
Í 14/65 “SGR ...” in the N-terminal. The nucleic acid sequence encoding a human ActR11b precursor protein is as follows (Nucleotides 5-1543 from Genbank NM 001106): ATGACGGCGCCCTGGETGGCCCTCECCCTCCTCTGEGGATCEGCTETGEGCCCG GCTCTGEGEGCETGGEGAGGCTGAGACACGGGAGTGCATCTACTACAACGCCAACTGGG AGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTECGAAGGCGAGCAGGACAAGC GGCTGCACTGCTACGCCTCCTGGGCCAACAGCTCTGGCACCATCGAGCTCGTGAAGAA GGGCTGCTGGCTAGATGACTTCAACTGCTACGATAGGCAGGAGTGTGTGGCCACTGAG GAGAACCCCCAGGTGTACTTCTGCTGCTGTGAAGGCAACTTCTGCAACGAGCGCTTCA CTCATTTGCCAGAGGCTGGGGEGCCCGGAAGTCACGTACGAGCCACCCCCGACAGCCC
CCACCCTGCTCACGGTGCTGGCCTACTCACTGCTGCCCATOEGGGGGCCTTTCCCTCAT CGTCCTGCTGGCCTTTTGGATGTACCGGCATCGCAAGCCCCCoTACGGTCATGTGGAC ATCCATGAGGACCCTGGGCCTCCACCACCATCCCCTCTGGTGGGCCTGAAGCCACTGC AGCTGCTGGAGATCAAGGCTCGGGGGCGCTTTGGCTEGTEGTCTGGAAGGCCCAGCTCAT GAATGACTTTGTAGCTGTCAAGATCTTCCCACTCCAGGACAAGCAGTCGTGGCAGAGTG AACGGGAGATCTTCAGCACACCTGGCATGAAGCACGAGAACCTGCTACAGTTCATTGCT 'GCCGAGAAGCGAGGCTCCAACCTCGAAGTAGAGCTGTGGCTCATCACGGCCTTCCATG ACAAGGGCTCCCTCACGGATTACCTCAAGGGGAACATCATCACATGGAACGAACTGTGT Ú CATGTAGCAGAGACGATGTCACGAGGCCTCTCATACCTGCATGAGGATGTGCCCTGGT GCCGTGGCGAGGGCCACAAGCCGTCTATTGCCCACAGGGACTTTAAAAGTAAGAATGT ATTGCTGAAGAGCGACCTCACAGCCGTGCTGGCTGACTTTGGCTTGGCTGTTCGATTTG AGCCAGGGAAACCTCCAGGGGACACCCACGGACAGGTAGGCACGAGACGGTACATGG CTCCTGAGEGTGCTCGAGGGAGCCATCAACTTCCAGAGAGATGCCTTCCTGCGCATTGA CATGTATGCCATGGGGTTGGTGCTGTGGGAGCTTGTGTCTCGCTGCAAGGCTGCAGAC GGACCCGTGGATGAGTACATGCTGCCCTTTGAGGAAGAGATTGGCCAGCACCCTTCGT TGGAGGAGCTGCAGGAGGTGGTGGTGCACAAGAAGATGAGGCCCACCATTAAAGATCA CTGGTTGAAACACCCGGGCCTGGCCCAGCTTTGTGTGACCATCGAGGAGTGCTGGGAC CATGATGCAGAGGCTCGCTTGTCCGCGGGCTETETGGAGGAGCGGGTGTCCCTGATTC
GGAGGTCGGTCAACGGCACTACCTCGGACTGTCTCGTTTCCOTGGTGACCTCTGTCAC —CAATGTGGACCTGCCCCCTAAAGAGTCAAGCATCTAA (SEQ ID NO: 4) The nucleic acid sequence encoding a soluble polypeptide is as follows: human ActRilb: TCTGGGCETGGEGAGGCTGAGACACGGGAGTGCATCTACTACAACGCCAACT GGGAGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTGCGAAGGCGAGCAGGAC - AAGCGGCTGCACTGCTACGCCTCCTGGGCCAACAGCTCTGGCACCATCGAGCTCGTGA AGAAGGGCTGCTGGCTAGATGACTTCAACTGCTACGATAGGCAGGAGTGTGTGGCCAC TGAGGAGAACCCCCAGGTGTACTTCTGCTGCTGTGAAGGCAACTTCTGCAACGAGCGC
'15/65
TTCACTCATTTGCCAGAGGCTGGGGGCCCGGAAGTCACGTACGAGCCACCCCCGACAG ACCACC (SEQ ID NO: 5) In a specific embodiment, the invention relates to soluble ActRlilb polypeptides. In this specification, the term "soluble ActRilb polypeptide" generally refers to polypeptides comprising an extracellular domain of an ActRilb protein.
The term "soluble ActRIIb polypeptide" in this specification includes any extracellular domain of an ActRilb protein, as well as any of its variants (including mutants, fragments and peptidomimetic forms). Actin-binding actRilb polypeptide is one that retains the ability to bind to activin, including, for example, activin AA, AB, BB or forms that include a C or E subunit. Optionally, an ActRlilb polypeptide binding to activins will bind to AA activin with a dissociation constant of 1 nM or less. The extracellular domain of an ActRllb protein binds to activin and is generally soluble under physiological conditions and therefore can be called soluble ActRilb polypeptide for binding to activins. Examples of soluble ActRilb activin-binding polypeptides include the soluble polypeptides illustrated in SEQ ID NOs: 2, 3, 13, 17 or 20. SEQ ID NO: 13 is called ActRIlb (20-134) -hFc and is described in more detail - Mind the Examples. Other examples of soluble ActRIlb polypeptides comprise a signal sequence in addition to the extracellular domain of an ActRilb protein,] for example, the leading sequence of bee honey melitin (SEQ ID NO: 14), the leading sequence of the activator of the tissue plasminogen (TPA) (SEQ ID NO: 15) or the leader sequence of native ActRilb (SEQ ID NO: 16). The ActRilb-hFc polypeptide illustrated in SEQ ID NO: 17 uses a TPA leader sequence.
In certain embodiments, the invention relates to variants of ActRI-lb polypeptides (e.g., soluble ActRIlb polypeptides). Optionally, the fragments, functional variants and modified forms have the same biological or similar activities as their corresponding wild-type ActRilb polypeptides. For example, an ActRilb variant of the invention can bind and inhibit the function of an ActRilb ligand (for example, activin A, activin AB, activin B, Nodal, GDF8, GDF11 or BMP7). Optionally, an ActRiIlb polypeptide modulates the growth of tissues, such as bone, cartilage, muscle or fat. Examples of ActRilb polypeptides include human ActRllb precursor polypeptide (SEQ ID NO: 1) and soluble human ActRilb polypeptides (for example, SEQ ID NOs: 2, 3, 13, 17 or 20).
Unless stated otherwise, the amino acid positions in an ActRilb polypeptide discussed in this specification refer to the polypeptide — ActRilb precursor (ie SEQ ID NO: 1). It is understood that the corresponding position in another ActRilb polypeptide can be quickly identified based on the information provided in this specification. For example, the ActRIlb precursor polypeptide contains a leader sequence of 19 amino acids that is not contained in the soluble extracellular portion of ActRilb as shown in SEQ ID NO: 2. Thus, a residue at position X in SEQ ID NO: 1 corresponds to residue X-19 in SEQ ID NO: 2. For example, residue 30 of SEQ ID NO: 1 corresponds to residue 11 in SEQ ID NO: 2 (see Figure 12). A similar correlation can be determined for other ActRilb sequences described in this patent application.
The invention identifies portions of functionally active variants of ActRilb.
Applicants have found that an Fc fusion protein having the sequence described by Hilden et a /. (Blood. 1994 Apr 15; 83 (8): 2163-70), which has an alanine in the position corresponding to amino acid 64 of SEQ ID NO: 1 (A64), exhibits relatively low affinity for activin and GDF-11 . On the other hand, the same Fc fusion protein with arginine at position 64 (R64) exhibits affinity for activin and GDF-11 in the low nanomolar to high picomolar range.
Therefore, a sequence with R64 is used as the wild-type reference sequence for human ActR11b in the present invention.
Attisano et a /. (Cell. 1992 Jan 10; 68 (1): 97-108) showed that a deletion of the proline loop at the C-terminal of the extracellular domain of ActRllb reduced the affinity of the receptor for activin.
An ActRilb-Fc fusion protein containing amino acids 20 - 119 of SEQ ID NO: 1, “ActRIIb (20-119) -Fc” exhibits less binding to GDF-11 and activin than a single ActRIIb (20-134 ) -Fc, which includes the proline loop region and the complete justamembrane domain (see US Publication
No. 2009/0005308). However, an ActRI-Ib (20-129) -Fc protein retains similar but somewhat reduced activity in relation to the wild type, even though the proline loop region is altered.
Therefore, extracellular domains of ActRilb that are disrupted at amino acids 134, 133, 132, 131, 130 and 129 are all expected to be active, but constructs disrupted at 134 or 133 may be more active.
Likewise, mutations in any of the residues are not expected to alter binding affinity to ligands to a large extent.
In support of this prediction, mutations of P129 and P130 do not significantly decrease ligand binding.
Therefore, an ActRilb-Fc fusion protein can end at amino acid 109 (the final cysteine), however, forms ending at or between 109 and 119 are expected to show less binding to ligands.
Amino acid 119 is poorly conserved, so it is quickly changed or truncated.
Forms ending in 28 or later positions retain the activity of binding ligands.
Forms ending in or between 119 and 127 will have intermediate linkage capacity.
ActRIIb (25-118) -hFc has been reported to be effective for inhibiting activin and other ligands and for promoting muscle mass and, consequently, these truncated proteins — each ActRIlb-hFc can be used in the methods described in this application for patent (see Zhou et a /., 2010, Cell 142: 531-543). The use of any of these forms may be convenient, depending on the clinical or experimental environment.
) 17/65 At the ActRilb N-terminus, a protein starting at amino acid 29 or earlier is expected to retain ligand-binding activity. Amino acid 29 represents the initial cysteine. An alanine to asparagine mutation at position 24 introduces a sequence with N-linked glycosylation without substantially affecting binding to ligands. This confirms that mutations in the region between the cleavage signal peptide and the cysteine crosslinked region, corresponding to amino acids 20-29, are well tolerated. In particular, constructs starting at position 20, 21, 22, 23 and 24 will retain activity, and constructs starting at positions 25, 26, 27, 28 and 29 are also expected to retain activity. A construct starting on 22, 23, 24 or 25 will exhibit the most activity (see U.S. Publication No. 2009/0005308).
Taken together, an active portion of ActRIlb comprises amino acids 29 - 109 of SEQ ID NO: 1, and constructs can, for example, start at a residue corresponding to amino acids 20 - 29 and end at a position corresponding to amino acids 109 - 134. Other examples include constructs that start at a position starting from —20-290u21-29 and ending at a position starting at 118 - 134, 118 - 133, 119 - 134, 119-133 or 129 - 134, 129 - 133. Other examples include constructs that start at a position from 20 - 24 (or 21 - 24 or 22 - 25) and end at a position from 109 - 134 (or 109 - 133), 118 - 134 (or 118 - 133), 119 - 134 (or 119-133) or from 129 - 134 (or 129 - 133). Variants within these ranges are also contemplated, especially those having at least 80%, 85%, 90%, 95% or 99% identity to the corresponding portion of SEQ ID NO: 2.
An extensive analysis of the analyzed structural function of ActRilb is provided in U.S. Publication No. 2009/0005308, the content of which is incorporated herein by reference in this patent application. ActRilb residues probably in contact with ligands at the binding site were defined as residues Y31, N33, N35, L38 to T41, E47, E50, Q53 to K55, L57, H58, Y60, S62, K74, W78 to N83, Y85, R87, A92 and E94 to F101 of SEQ ID NO: 1. In these positions, conservative mutations are expected to be tolerated, although a mutation in K74A is well tolerated, as they are in R40A, K55A, F82A and mutations in position L79 . R40 is K in Xenopus, indicating that basic amino acids in this position will be tolerated. Bovine Q53éRem ActRilb and Xenopus Kem ActRllb, therefore, amino acids including R, K, Q, N and H will be tolerated in this position. Apart from these residues, modifications are expected to be relatively well tolerated, as long as such changes do not alter the structure of the protein as a whole. It is quickly evident when a protein structure is altered because the protein will tend to express poorly or to be degraded in the culture medium. Therefore, a general formula for an active protein variant of ActRllb is one that comprises amino acids 29 - 109, but that optionally starts at a position ranging from 20 - 24 or 22 - 25 and ends at a position ranging from
'18/65 129 - 134, and comprising a maximum of 1, 2, 5, 10 or 15 conservative amino acid changes at the ligand binding site, and zero, one or more non-conservative changes at positions 40, 53, 55 , 74, 79 and / or 82 at the linker binding site. Such a protein can retain above 80%, 90%, 95% or 99% sequence identity to the amino acid sequence 29-109 of SEQID NO: 1. Sites outside the binding site, where variability can be especially well tolerated, include the amino and carboxy terminals of the extracellular domain (as noted above), and positions 42 - 46 and 65 - 73 of SEQ ID NO: 1. A change from asparagine to alanine at position 65 (N65A) effectively improves binding to | bindings in the context of A64, and thus are not supposed to have a detrimental effect on binding to ligands in the context of R64. This change probably eliminates glycosylation at N65 in the context of A64, thus demonstrating that a significant change in this region is likely to be tolerated. Although a change in R64A is poorly tolerated, R64K is well tolerated and, therefore, another basic residue, such as H, can be tolerated in position 64.
ActRllb is well maintained in practically all vertebrates, with large stretches of the extracellular domain completely conserved. Many of the binders that bind to ActRilb are also highly conserved. In this way, comparisons of ActRilb sequences from various vertebrate organisms provide indications 1 about residues that can be altered. Therefore, an active variant of human ActRilb can include one or more amino acids at corresponding positions in the ActRlilb sequence of another vertebrate, or it can include a residue that is similar to that in the human or other vertebrate sequence. The following examples illustrate this approach for defining an active variant of ActRilb. L46 of the human ActRIlb is valine in the Xenopus ActRilb, so this position can be changed and, optionally, can be changed to another hydrophobic residue, such as V, | or F, for a non-polar residue such as A. E52 of human ActRIllb is K in the Xenopus ActRilb, indicating that this site can be tolerant of a wide variety of changes, including polar residues, such as E, D, KR, H, S , T, P, G, Y and probably A. T93 of the human ActRilib is K in the Xenopus ActRilb, indicating that a wide structural variation is tolerated in this position, with favored polar residues, such as S, K, R, E, D, H , G, P, GeY.F108 of the human ActRilb is Y in the Xenopus ActRilb and therefore Y or another hydrophobic group, such as |, V or L must be tolerated. Human AcRIRIlb E111 is K in Xenopus ActRllb, indicating that altered residues will be tolerated in this position, including D, R, K and H, as well as Q and N. Human ACTRIlb R112 is K in Xenopus ActRilb, indicating that basic residues are tolerated in this position, including ReH Ana position 119 of the human AciRIlb is relatively poorly conserved and appears as P in the ActRlilb of rodents and as V in the ActRilb of Xenopus, therefore, essentially any amino acid must be tolerated in this position.
. 19/65 The addition of an additional N-linked glycosylation site (NXS / T) increases the serum half-life of an ActRilb-Fc fusion protein compared to the ActRIIb (R64) -Fc form (see Publication US No. 2009/0005308). The introduction of asparagine at position 24 (construct A24N) creates an NXT sequence that gives a longer half-life. Other sequences — NX sequences (T / S) are found in 42 - 44 (NQS) and 65 - 67 (NSS) of SEQ ID NO: 1, although the latter may not be efficiently glycosylated with the R at position 64. Sequence - copies of NXS / T can in general be introduced at positions outside the binding site of ligands defined in Figure 13. Sites especially suitable for the introduction of non-endogenous NXS / T sequences include amino acids 20 - 29, 20 - 24, 22 - 25, 109 - 134,120-134 or 129-134 of SEQ ID NO: 1. NXS / T sequences can also be introduced into the linker between the ActRilb sequence and that of Fc or another fusion component. Such a site can be introduced with minimal effort, by entering N in the correct position with respect to pre-existing S or T, or by introducing N in the correct position with respect to pre-existing S or T, or by introducing S or T in a position corresponding to pre-existing N. Thus, the desired changes that create an N-linked glycosylation site are: A24N, R64N, S67N (possibly combined with N65A change), E106N,, R112N, G120N, E123N, P129N, A132N, R112S and R112T. Any S that is expected to be glycosylated can be changed to T without creating an immunogenic site, thanks to the protection afforded by glycosylation. Likewise, any T that is expected to be glycosylated can be changed to S. Therefore, changes S67T and S44T are considered. Likewise, in an A24N variant, an S26T change can be used. Consequently, an ActR11b variant can include one or more additional, non-endogenous N-linked glycosylation consensus sequences.
The L79 position can be altered to give altered binding properties to —actinomyostatin (GDF-11). L79A or L79P reduces binding to GDF-11 to a greater extent than binding to activin. L79E or L79D retains the connection to GDF-11. Notably, the L79E and L79D variants exhibit greatly reduced binding to activin. In vivo experiments indicate that these non-activin receptors retain significant capacity to increase muscle mass, but exhibit decreased effects on other tissues. These data demonstrate the convenience and viability of obtaining polypeptides with reduced effects on activin. The variations described can be combined in several ways. In addition, the results of the mutagenesis program described in this patent application indicate that there are amino acid positions in ActRilb that are often beneficial to be conserved. These include position 64 (basic amino acid), position 80 (acidic or —hydrophobic amino acid), position 78 (hydrophobic and, especially, tryptophan), position 37 (acid and especially aspartic or glutamic acid), position 56 (basic amino acid ), position 60 (hydrophobic amino acid, especially phenylalanine or tyrosine). Therefore, in each variant
"20/65 described herein, the invention provides a structure of amino acids that can be conserved. Other positions that may be convenient to preserve are the following: position 52 (amino acid acid), position 55 (basic amino acid), position 81 (acid ), 98 (polar or charged, especially E, D, Rou K).
In certain embodiments, isolated fragments of the ActRilb polypeptides can be obtained by tracking polypeptides produced by recombination from the corresponding nucleic acid fragment encoding an ActRilb polypeptide (for example, SEQ ID NOs: 4 and 5). Additionally, the fragments can be chemically synthesized with techniques known in the art, such as conventional chemical reactions def-MocoutBoc in Merrifield solid phase. The fragments can be produced (by recombination or chemical synthesis) and tested to identify those peptide fragments that can act, for example, as antagonists (inhibitors) or agonists (activators) of an ActRilb protein or an ActRilb ligand.
In certain embodiments, a functional variant of the ActRilb polypeptides has an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 13, 17 or 20. In certain cases, the 'functional' variant has an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ 'ID NOs : 2, 3, 13, 17 or 20.
In certain embodiments, the present invention contemplates producing functional variants by modifying the structure of an ActRilb polypeptide for the purpose of enhancing therapeutic efficacy or stability (for example, ex vivo shelf life and resistance to proteolytic degradation in vivo). Modified ActRllb polypeptides can also be produced, for example, by substitution, deletion or addition of amino acids. For example, it is reasonable to predict that an isolated replacement of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine or a similar replacement of an amino acid with a structurally related amino acid (eg, conservative mutations) will not have a important effect on the biological activity of the resulting molecule. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of an ActRilb polypeptide results in a functional homolog or not can be quickly determined by assessing the ability of the ActRilb variant polypeptide to produce a cell response in a similar way to the wild type polypeptide. of ActRilb, or to bind to one or more ligands, such as activin, GDF-11 or myostatin, in - similar to the wild type.
In certain specific embodiments, the present invention contemplates producing mutations in the extracellular domain (also called ligand-binding domain) of a poly
T 21/65 ActRilb peptide, so that the variant (or mutant) polypeptide of ActRilb has altered ligand binding activities (for example, binding affinity or specificity). In cases, such variant ActRilb polypeptides altered (increased or reduced) the binding affinity for a specific ligand. In other cases, the variant ActRllb polypeptides altered the binding specificity by their ligands.
For example, the invention provides variant ActRilb polypeptides that preferentially bind to GDF8 / GDF11 over activins. The invention further establishes the suitability of such polypeptides to reduce off-target effects, although such selective variants may be less convenient for the treatment of serious diseases, in which very large gains in muscle mass may be necessary to the therapeutic effect and in which some level of off-target effects are acceptable. For example, amino acid residues from the ActRilb protein, such as E39, K55, Y60, K74, W78, D80 and F101, are at the ligand-binding site and mediate binding to their ligands, such as activin and GDF8. Therefore, the present invention provides an altered ligand-binding domain (for example,
15. —example, GDF8 binding domain) of an ActRilb receptor, which comprises one or more mutations in these amino acids. Optionally, the altered ligand-binding domain may exhibit increased selectivity for a ligand, such as GDF8, over the wild-type ligand-binding domain of an ActRilb receptor. By way of illustration, these mutations increase the selectivity of the altered ligand-binding domain by GDFB8 over activin. Optionally, the ratio of K, activin-to-K, GDFB8-binding ratio of the altered ligand-binding domain is at least 2, 5, 10 or even 100 times greater than the wild-type ligand-binding domain. Optionally, the reason for ICs, from activin inhibition to ICs9, for inhibiting GDF8 of the altered ligand-binding domain is at least 2, 5, 10, or even 100 times greater than the ligand-binding domain of wild type. Optionally, the altered ligand-binding domain inhibits GDF8 with ICs at least 2, 5, 10 or even 100 times less than ICs, 5 to inhibit activin.
As a specific example, the positively charged amino acid residue Asp (D80) of the ActRilb ligand-binding domain can be mutated to a different amino acid residue so that the variant ActRilb polypeptide binds preferentially to GDF8, but not to activin. Preferably, residue D80 is exchanged for an amino acid residue selected from the group consisting of: uncharged amino acid residue, negative amino acid residue and hydrophobic amino acid residue. As an additional specific example, the hydrophobic residue, L79, can be changed to amino acids — acids, aspartic or glutamic acid, to greatly reduce the binding to the activin, while retaining the binding to GDF11 . As will be recognized by those skilled in the art, most of the mutations, variants or modifications described
22/65 tasks can be performed at the nucleic acid level or, in some cases, by post-translational modification or chemical synthesis.
Such techniques are well known in the art.
In certain embodiments, the present invention contemplates specific mutations of the ActRilb polypeptides in order to alter the glycosylation of the polypeptide.
Exemplary glycosylation sites on ActRIlb polypeptides are shown in SEQ ID NO: 1. These mutations can be selected to introduce or eliminate one or more glycosylation sites, such as O-linked or N- connected.
Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine (where "X" is any amino acid) that is specifically recognized by appropriate cellular glycosylation enzymes.
The change can also be made by adding or replacing one or more serine or threonine residues to the ActRilb wild type polypeptide sequence (for O-linked glycosylation sites). A variety of amino acid substitutions or deletions to one or both positions of the first or third amino acid of a glycosylation recognition site (and / or deletion of amino acid in the second position) results in non-glycosylation in the sequence modified tripeptide.
Another means of increasing the number of carbohydrate groups in an ActRilb polypeptide is by chemical or enzymatic coupling of glycosides to the ActRilb polypeptide.
Depending on the coupling method used, the sugar (s) can be fixed to the arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine.
These methods are described in WO 87/05330, published on September 11, 1987, and in Aplin and Wriston (1981) CRC Crit.
Rev.
Biochem., P. 259-306, incorporated herein by reference in this patent application.
The removal of one or more carbohydrate groups present in an ActRilb polypeptide can be carried out chemically and / or enzymatically.
Chemical deglycosylation may involve, for example, exposure of the ActRIlb polypeptide to the trifluoromethanesulfonic acid compound or an equivalent compound.
This treatment results in the cleavage of most or all sugars except binding sugar (N-acetylglycosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact.
Chemical deglycosylation is described in more detail by Hakimuddin et a /. (1987) Arch.
Biochem.
Biophys. 259: 52 and by Edge et al. (1981) Anal.
Biochem. 118: 131. Enzymatic cleavage of carbohydrate groups into ActRilb polypeptides can be achieved by using a variety of endo and exoglycosidases, as described by Thotakura et al. (1987) Meth.
Enzymol. 138: 350. The sequence of an ActRilb polypeptide can be adjusted, as appropriate, depending on the type of expression system used, since mammalian cells, yeasts, insects and plants can all introduce different patterns of glycosylation that can be
. 23/65 affected by the amino acid sequence of the peptide. In general, AciRIlb proteins for use in humans will be expressed in a mammalian cell that provides adequate glycosylation, such as HEK293 or CHO cell lines, although other mammalian cell lines for expression are supposed to be useful as well.
The present invention further contemplates a method for generating variants, especially sets of combinatorial variants of an ActRilb polypeptide, including, optionally, truncated variants; clusters of combinatorial mutants are especially useful for identifying functional variant sequences. The purpose of screening such combinatorial libraries can be to generate, for example, variants of ActRIlb polypeptide that have altered properties, such as altered pharmacokinetics, or altered binding to ligands. A variety of screening assays are provided below, and these assays can be used to evaluate variants. For example, an ActRllb polypeptide variant can be screened for the ability to bind to an ActRilb polypeptide, to prevent binding of an ActRilb linker to an ActRilb polypeptide.
The activity of an ActRllb polypeptide or its variants can also be tested in a cell or in vivo assay. For example, the effect of an ActRilb polypeptide variant on the expression of genes involved in bone production in osteoblasts or precursors can be assessed. The assay can be performed, as necessary, in the presence of one or more recombinant ActRilb ligand proteins (eg, BMP7), cells can be transfected to produce an ActRilb polypeptide and / or variants thereof, and optionally, an ActRIlb linker. Likewise, an ActRilb polypeptide can be administered to a mouse or other animal, and one or more bone-related properties, such as density or volume, can be evaluated. The rate of bone fracture healing can also be assessed. Likewise, the activity of an ActRIlb polypeptide or its variants can be tested in muscle cells, adipocytes and neuronal cells for any effect on the growth of these cells, for example, by the assays described below. Such assays are well known and routine in the state of the art. A reporter gene responding to SMAD can be used in such cell lines to monitor the effects on downward signaling.
Variants derived in a combinatorial way can be generated those that have selective power in relation to a natural ActRilb polypeptide. Such variant proteins, when expressed from recombinant DNA constructs, can be used in gene therapy protocols. Likewise, mutagenesis can give rise to variants, which have drastically different intracellular half-lives than those corresponding to a wild-type ActRilb polypeptide. For example, the altered protein can be made more or less stable to proteolytic degradation or other processes that
. 24/65 result in destruction or otherwise inactivation of a native polypeptide of Ac-1RIlb. Such variants, and the genes that encode them, can be used to alter levels of ActRilb polypeptides by modulating their half-life. For example, a short half-life can give rise to transient biological effects and, when part of an inducible expression system, can allow for closer control of the levels of ActRilb recombinant polypeptides within the cell.
In certain embodiments, the ActRiIlb polypeptides of the invention may further comprise post-translational modifications in addition to any other naturally present in the ActRilb polypeptides. These modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. As a result, the modified ActRIlb polypeptides may contain elements other than amino acids, such as polyethylene glycols, lipids, poly or monosaccharides and phosphates. The effects of these different amino acid elements on the functionality of an ActRIlb polypeptide can be tested, as described in this patent application, for other variants of ActRI-1b polypeptides. When an ActRilb polypeptide is produced in cells by cleavage in an insipient form of the ActRilb polypeptide, post-translational processing may also be important 7 to correct protein folding and / or function. Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have es-, specific cellular mechanisms and mechanism characteristics for such post-translational activities and can be chosen to ensure the correct modification and processing of the polypeptides of AcTRilb.
In certain aspects, functional variants or modified forms of the ActRilb polypeptides include fusion proteins having at least a portion of the ActRilb polypeptides and one or more fusion domains. Well-known examples of these fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (for example, Fc), maltose-binding protein (MBP) or human serum albumin. A fusion domain can be selected to give a desired property. For example, some fusion domains are especially useful for the isolation of fusion proteins by affinity chromatography. For affinity purification purposes, matrices relevant to affinity chromatography, such as resins conjugated to glutathione, amylase and nickel or cobalt, are used. Many of these matrices are available in kit form, such as the Pharmacia GST purification system and the QlAexpress' "(Qiagen) system, useful with fusion partners (HIS;). As another example, a domain of fusion can be selected - demodoafacilitara detection of ActRilb polypeptides. Examples of such detection domains include the various fluorescent proteins (for example, GFP), as well as "epitope tags", which are normally short peptide sequences for which exist
. 25/65 a specific antibody is available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG tag, virus / nfluenza hemagglutinin (HA) and c-myc tag. In some cases, the fusion domains have a protease cleavage site, such as Factor Xa or Thrombin, which allows partial digestion by the relevant protease of the fusion proteins and, thus, the recombinant proteins are released from them. The released proteins can then be isolated and separated from the fusion domain by subsequent chromatography. In certain preferred embodiments, an ActRilb polypeptide is fused to a domain that stabilizes the ActRilb polypeptide in vivo ("stabilizing" domain). “Stabilize”, in this specification, means something that increases the serum half-life, regardless of whether it was due to less destruction, less clearance (clearance) by the kidney or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types — fusion domains that can be selected include multimerization domains (for example, dimerization, tetramerization) and functional domains (which confer an additional biological function, such as stimulation of muscle growth).
As a specific example, the present invention provides a fusion protein as a GDF8 antagonist, which comprises an extracellular domain (for example, GDF8 deliga) fused to an Fc domain (for example, SEQ ID NO: 6) . THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD (A) VSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK (A) VSNKALPVPIEKT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN (A) HYTQKSLSLSPGK3 is a more or less than -5 domain, preferably in the same domain, or as the preferred domain, it is the most preferred domain. In certain cases, the Fc mutant domain containing one or more of these mutations (eg, Asp-265 mutation) is less able to bind to the Fc receptor] than the wild type Fc domain. In other cases, the Fc mutant domain having one or more of these mutations (for example, Asn-434 mutation) has an increased ability to bind to the Fc receptor related to the MHC class | (FCRN) in relation to the wild type Fc domain.
It is understood that different elements of the fusion proteins can be arranged in any way that is compatible with the desired functionality. For example, an ActR11b polypeptide can be positioned C-terminal with respect to a heterologous domain or, alternatively, a heterologous domain can be positioned with C-terminal with respect to an ActRilb polypeptide. The ActRIlb polypeptide domain and the heterologous domain need not be adjacent on a fusion protein, and domains or sequences
26/65 additional amino acids can be included in C or N-terminal in relation to any domain or between domains.
In certain embodiments, the ActRilb polypeptides of the present invention contain one or more modifications that are capable of stabilizing them. For example, these modifications enhance the in vitro half-life of ActRilb polypeptides, enhance the surrounding half-life of ActRilb polypeptides, or reduce the proteolytic degradation of ActRIlb polypeptides. These stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRilb polypeptide and a stabilizing domain), modifications of a glycosylation site (including, for example, addition of a site glycosylation to an ActRIlb polypeptide) and carbohydrate group modifications (including, for example, removal of carbohydrate groups from an ActRilb polypeptide). In the case of fusion proteins, an ActRilb polypeptide is fused to a stabilizing domain such as an IgG molecule (e.g., Fc domain). In this specification, the term “stabilizing domain” not only refers to a fusion domain (for example, Fc), as in the case of fusion proteins, but also includes non-proteinaceous modifications such as with carbohydrate group, or polymer non-proteinaceous such as with polyethylene glycol 2 col.
In certain embodiments, the present invention provides isolated and / or purified forms of the ActRilb polypeptides, which are isolated or, otherwise, are substantially free of other proteins. ActRilb polypeptides will in general be produced by expression from recombinant nucleic acids.
In certain embodiments, the ActRilb polypeptides (modified or not) of the invention can be produced by a variety of techniques known in the art. For example, these ActRilb polypeptides can be synthesized using techniques — standard protein chemistry, such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant GA (ed.), Synthetic Peptides: A User's Guide, WH Freeman and Company, New York (1992). In addition, automatic peptide synthesizers are commercially available (for example, Advanced ChemTech Model 396; Milligen / Biosearch 9600). Alternatively, —ActRllb polypeptides, their fragments or variants can be produced by recombination, using various expression systems (eg, E. coli, Chinese hamster ovary cells, COS cells, baculovirus), as are well known in the state technique (see also below). In an additional embodiment, modified or unmodified ActRIllb polypeptides can be produced by digesting complete ActRilb polypeptides, natural or produced by recombination, using, for example, a protease, for example, trypsin, thermolysin, chymotrypsin , pepsin or enzyme converting basic amino acid pairs (PACE). Computer analysis (using commercially available software
. 27/65 (for example, MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to identify proteolytic cleavage sites. Alternatively, such ActRilb polypeptides can be produced from complete ActRIIB polypeptides, natural or produced by recombination, using standard techniques known in the art, such as by chemical cleavage (for example, with cyanogen bromide, hydroxylamide). at).
3. Nucleic acids encoding ActRilb polypeptides In certain respects, the invention provides isolated and / or recombinant nucleic acids that encode any of the ActRilb polypeptides (e.g., complete and soluble ActRIlb polypeptides), including fragments, functional variants and fusion proteins described in this patent application. For example, SEQ ID NO: 4 encodes the natural precursor polypeptide of human ActRilb, while SEQ ID NO: 5 encodes the processed extracellular domain of AcítRilb. The nucleic acids in question may be of the single strand or double strand type. Such nucleic acids can be DNA or RNA molecules. These nucleic acids can be used, for example, in methods to produce ActRIlb polypeptides or as direct therapeutic agents (for example, in a gene therapy approach). In certain aspects, it is also understood that nucleic acids in question that co- 'actRilb polypeptides include nucleic acids that are variants of SEQ ID NO: 40or5.Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants. embodiments, the invention provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 4, 5 or 18 Any person skilled in the art will recognize that complementary nucleic acid sequences to SEQ ID NOs: 4, 5 or 18 and variants of SEQ ID NOs: 4, 5 or 18 are also included within the scope of this invention. nuc acid leico of the invention can be isolated, recombinant and / or fused with a heterologous nucleotide sequence or in a DNA library. In other embodiments, the nucleic acids of the invention also include nucleotide sequences, and the ActRilb polypeptides encoded by such nucleic acids, which hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NOs: 4, 5 or 18, the complementary sequence of SEQ ID NOs: 4, 5 or 18, or fragments of any of the foregoing. As discussed above, any technician versed in the subject will quickly understand that the appropriate stringent conditions for - promoting DNA hybridization can be varied. For example, hybridization could be performed at 6.0 x sodium chloride / sodium citrate (SSC) at a temperature around 45 ºC, followed by washing with 2.0 x SSC at 50 ºC. For example, the concentration of salt in the
. 28/65 washing step can be selected from low stringency conditions around 2.0 x SSC at 50 ºC to high stringency conditions around 0.2 x SSC at 50 ºC. In addition, the temperature in the washing step can be increased from low-temperature conditions at room temperature, around 22 ºC, to high-temperature conditions at approximately 65 ºC. Both the temperature and the salt concentration can be varied, or the temperature or salt concentration can be kept constant while the other variable is changed. In one embodiment, the invention provides nucleic acids that hybridize under low stringency conditions of 6 x SSC at room temperature, followed by washing with 2 x SSC at room temperature.
Isolated nucleic acids that differ from the nucleic acids represented in SEQ ID NOs: 4, 5 or 18 due to degeneration in the genetic code are also included in the scope of the invention. For example, some amino acids are referred to as more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) can result in "silent" mutations that do not affect the protein's amino acid sequence. However, it is predicted that DNA sequence polymorphisms that actually lead to changes in the amino acid sequences of the 'proteins in question will exist between mammalian cells. Any person skilled in the art will recognize that these variations in one or more nucleotides (up to around 3 - '5% of the nucleotides) of the nucleic acids that encode a given protein may exist between individuals of a given species due to the natural allelic variation . Any and all of these nucleotide variations and the resulting amino acid polymorphisms are included within the scope of the present invention.
In certain embodiments, the recombinant nucleic acids of the invention may be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, said single or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcription start or stop sequences, translation start or stop sequences and reinforcing or activating sequences. Constitutive or inducible promoters are known in the art and are contemplated by the invention. Promoters can be natural promoters or hybrid promoters that combine elements from more than one promoter. An expression construct can be present in a cell or episome, such as a plasmid, or the expression construct can be inserted into a chromosome. In a preferred embodiment, the expression vector contains a selectable marker gene to allow selection of host cells
-. 29/65 transformed quarries.
Selectable marker genes are well known in the art and will vary with the host cell used.
In certain aspects of the invention, the nucleic acid in question is provided in an expression vector, comprising a nucleotide sequence that encodes an ActRilb polypeptide and that is operably linked to at least one regulatory sequence.
Regulatory sequences are recognized in the prior art and are selected for direct expression of the ActRilb polypeptide.
Therefore, the term regulatory sequence includes promoters, reinforcers and other elements of expression control.
Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of a wide variety of expression control sequences that control the expression of a DNA sequence, when operationally linked to it, can be used in these vectors to express DNA sequences encoding an ActRilb polypeptide.
Such useful expression control sequences include, for example, the SV40 promoters of early and late expression, the tet promoter, the promoter of immediate early expression of adenovirus or cytomegalovirus, RSV promoters, the system ma lac, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by the T7 RNA polymerase, the main operator and promoter regions of lambda phage, the control regions for coating fd protein, the promoter for 3 -phosphoglycerate kinase or other glycolytic enzymes, acid phosphatase promoters, for example, Pho5, promoters of yeast mating factors a, promoter of the baculovirus system polyhedron and other sequences known to control cell expression prokaryotic or eukaryotic or their viruses, and various combinations of these.
It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and / or the type of protein desired to be expressed.
In addition, the vector's copy number, the ability to control this copy number, and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
A recombinant nucleic acid of the invention can be produced by ligating the gene - cloned, or part of it, into a vector suitable for expression in prokaryotic cells, eukaryotic cells (from yeast, birds, insects or mammals), or both.
Expression vehicles for producing a recombinant ActRilb polypeptide include plasmids and other vectors.
For example, suitable vectors include plasmids of the types: plasmids derived from pBR322, plasmids derived from pEMBL, plasmids derived from —pEX, plasmids derived from pBTac and plasmids derived from pUC for expression in prokaryotic cells, such as E. coli.
Some mammalian expression vectors contain both prokaryotic sequences
- 30/65 to facilitate the propagation of vectors in bacteria, such as one or more eukaryotic transcription units that are expressed in eukaryotic cells.
Vectors derived from pcD-NAI / amp, pcDNAI / neo, pRC / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, PSVT7, pko-neo and pHyg are examples of mammalian expression vectors suitable for the transfer of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and selection of drug resistance in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, derived from pREP and p205) can be used for transient expression of proteins in eukaryotic cells.
Examples of other viral systems (including retroviral) of expression can be found below in the description of gene therapy release systems.
The various methods employed in preparing plasmids and transforming host organisms are well known in the art.
For other expression systems suitable for prokaryotic and eukaryotic cells, as well as recombination procedures, see Molecular Cloning A Laboratory Manual, 3rd edition, ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). Í In some situations, it may be convenient to express recombinant polypeptides using a baculovirus expression system.
Examples of these baculovirus expression systems include vectors derived from pVL (such as pVL1392, pVL1393 and pVL941), vectors derived from pACUW (such as pAcUW! 1) and vectors derived from pBlueBac (such as pBlueBac II! Containing (-gal) In a preferred embodiment, a vector will be created to produce the ActRilb polypeptides in question in CHO cells, such as the Pemv-Script vector (Stratagene, La Jolla, California), pcDNA4 vectors (Invitrogen, Carisbad, California) and vectors pCl-neo (Pro-mega, Madison, Wiscosin). As will be evident, the gene constructs in question can be used to cause the expression of the ActRilb polypeptides in question in cells propagated in culture, for example, to produce proteins, including proteins proteins or variant proteins, to be purified.
The present invention also pertains to a host cell transfected with a recombinant gene, including a coding sequence (for example, SEQ ID NO: 4, 5 or 18) of one or more of the ActRilb polypeptides in question.
The host cell can be any prokaryotic or eukaryotic cell.
For example, an ActRI-lb polypeptide of the invention can be expressed in bacterial cells such as E. coli, insect cells (for example, using a baculovirus expression system), yeasts or mammals.
Other suitable host cells are known to those skilled in the art.
Accordingly, the present invention also pertains to methods for producing polymers
. 31/65 ActRilb peptides in question.
For example, a host cell transfected with an expression vector encoding an ActRIlb polypeptide can be cultured under appropriate conditions to allow ActRilb polypeptide expression to occur.
The ActRilb polypeptide can be secreted and isolated from the mixture of cells and the medium containing the ActRilb polypeptide.
Alternatively, the ActR11b polypeptide can be retained in the cytoplasm or a fraction of the membrane and the harvested, lysed cells and the isolated protein.
A cell culture includes host cells, medium and other by-products.
Suitable means for cell culture are well known in the art.
The ActRilb polypeptides in question can be isolated from cell culture medium, host cells or both, using techniques known in the art to purify proteins, including ion exchange chromatography, gel filtration chromatography, ultra filtration, electrophoresis, immunoaffinity purification with specific antibodies against certain epitopes of ActRIlb polypeptides and affinity purification with an agent that binds to a domain fused to the ActRilb polypeptide (for example, a protein A column can be used to purify a fusion of ActRIlb-Fc). In a preferred embodiment, the ActRilb polypeptide is a fusion protein containing a domain that facilitates its purification.
In a preferred embodiment, purification is achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenyl sepharose chromatography , size exclusion chromatography and cation exchange chromatography.
Purification could be completed with viral filtration and buffer exchange.
Optionally, a purification scheme involves the following: MabSelect "Y column of protein A (GE Healthcare) with protein in saline with Tris buffer (pH 8.0) (TBS), washed in TBS (150 mM NaCl, pH 8.0) and TBS (50 mM NaCl, pH 8.0), eluted in 0.1 M glycine at pH 3.0; the eluate can be transferred to a Q sepharose column in TBS pH 8.0 and eluted in TBS containing NaCl at a concentration ranging from 200 - 250, 210 - 250, 220 - 250, 210 - 230, 215 - 225 or 225 - 235 mM; the eluate can be exchanged for NH, SO, 1.1 M, Tris 50 MM pH 8.0, washed in the same buffer and eluted in saline with phosphate buffer at pH 7.2 or 7.4 at a concentration ranging from 200 - 250, 210 - 250, 220 - 250, 210 - 230, 215- 2250u225-235 mM NH, SO ,. The eluted material can be dialyzed, subjected to viral filtration and used for final formulation.
In another embodiment, a fusion gene encoding a purification leader sequence, such as a sequence with cleavage site for poly- (His / N-terminal enterokinase of the desired portion of the recombinant ActRilb polypeptide, may allow purification. - cation of the expressed fusion protein by affinity chromatography using a Ni * metal resin. The leading purification sequence can then be removed by treatment with enterokinase to provide the purified ActRilb polypeptide (for example,
. 32/65 example, see Hochuli et a /., (1987) J. Chromatography 411: 177; and Janknecht et al, PNAS USA 88: 8972).
Techniques for producing fusion genes are well known. Essentially, several segments of DNA, encoding different polypeptide sequences, are joined according to conventional techniques, employing blunt-ended terminations or stagger-ended terminations for ligation, di-management by restriction enzymes to provide the appropriate terminations, padding of cohesive ends as appropriate, treatment with alkaline phosphatase to prevent unwanted bonds and enzymatic binding. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automatic DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed, using anchoring primers that cause complementary overhangs to emerge between two consecutive gene fragments that can be subsequently annealed to generate a chimeric gene (see , for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
4. Alternative activin Acryllb antagonists Although soluble ActRilb polypeptides and especially ActRylil-Fc fusions are preferred antagonists, it is anticipated that other types of ActRilb antagonists will be useful, including anti-ActRllb antibodies, nucleic acids antisense, RNAi or ribozymes that inhibit the production of ActRilb, and other ActRilb inhibitors, especially those that compromise the binding of AciRilb.
An antibody that is specifically reactive with an ActRilb polypeptide (for example, a soluble ActRilb polypeptide) and that competitively binds the ligand with the ActRilb polypeptide or that otherwise inhibits ActRilb-mediated signaling - can be used as an antagonist activities of the ActRilb polypeptide.
The use of immunogens derived from an ActRilb polypeptide makes it possible to produce anti-protein / anti-peptide antisera or monoclonal antibodies by standard protocols (see, for example, Antibodies: A Laboratory Manual, ed. By Harlow and Lane (Cold Harbor) Press: 1988)). A mammal, such as a mouse, hamster or rabbit, can be immunized with an immunogenic form of the ActRilb polypeptide, an antigenic fragment that is capable of eliciting an antibody response or a fusion protein. Techniques for imparting immunogenicity to a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of an ActRilb polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detecting antibody titers in plasma or serum. Standard ELISA assays or other immunoassays can be used as the immunogen as an antigen to assess antibody levels.
. 33/65 After immunizing an animal with an antigenic preparation of an ActRilb polypeptide, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard procedures for fusing somatic cells with immortalization of cells, such as myeloma cells, to produce hybridoma cells. These techniques are well known in the art and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique ( Kozbar et al., (1983) Immunology Today, 4: 72) and the EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened by immunochemistry for the production of antibodies specifically reactive with an ActRIlb polypeptide, and monoclonal antibodies isolated from the culture comprising these hybridoma cells. The term “antibody” in this specification is intended to include whole antibodies, for example, of any isotype (IgG, IgA, I! GM, IgE, etc.), and includes fragments or domains of immunoglobulins that are reactive with a selected antigen. Antibodies can be fragmented using conventional techniques, and fragments screened for usefulness and / or interaction with a specific epitope of interest. Therefore, the term includes portion segments, proteolytically cleaved or prepared by recombination of an antibody molecule, which are able to react selectively with a certain protein. Non-limiting examples of such proteolytic and / or recombinant fragments included in Fab, F (ab ') 2, Fab', Fv and single chain antibodies (scFv) containing a VIL] and / or HIV] domain joined by a peptide linker . ScFv fragments can be linked covalently or not to form antibodies having one or more binding sites. The term antibody also includes polyclonal, monoclonal antibodies or other purified antibody preparations and recombinant antibodies. The term "recombinant antibody" means an antibody or antigen-binding domain of an immunoglobulin, expressed from a nucleic acid that has been constructed using molecular biology techniques, such as humanized antibody or entirely human antibody developed from an anti - single chain body. Single domain and single chain antibodies are also included in the term “recombinant antibody”.
In certain embodiments, an antibody of the invention is monoclonal and, in certain modalities, the invention provides methods for generating innovative antibodies. For example, a method for generating monoclonal antibody that specifically binds to an ActRIlb polypeptide may comprise administering to a mouse an amount of an immunogenic composition, comprising the antigenic polypeptide, effective in stimulating a detectable immune response, obtaining antibody-producing cells (e.g. spleen cells) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify one that produces a monocolonal antibody that specifically binds to the antigen. Once obtained, a hybridoma can be propagated in cell culture, optionally under culture conditions in which cells derived from the hybridoma produce the monoclonal antibody that specifically binds to the antigen. The monoclonal antibody can be purified from cell culture.
The adjective "specifically reactive with", used in reference to an antibody, is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective for the antigen of interest (for example, ActRilb polypeptide) among other antigens that are not of interest so that the antibody is useful to, at a minimum, detect the presence of the antigen of interest in a particular type of biological sample. In certain methods employing the antibody, such as therapeutic applications, high levels of specificity in binding may be convenient. Monoclonal antibodies generally have a greater tendency (when compared to polyclonal antibodies) to efficiently criminalize the desired antigens among cross-reacting polypeptides. A characteristic that influences the specificity of an antibody: antigen interaction is the affinity of the antibody for the antigen. Although the desired specificity can be achieved with a range of different affinities, generally preferred antibodies will have an affinity (constant of dissociation) around 10º, 107, 10º, 10º M or less.
In addition, the techniques used to screen antibodies, in order to identify a desirable antibody, can influence the properties of the obtained antibody. For example, if an antibody is to be bound to an antigen in solution, it may be convenient to test for binding in solution. A variety of different techniques are available to test interactions between antibodies and antigens to identify especially desirable antibodies. These techniques include ELISA assays, surface plasmon resonance binding assays (eg, the "Biacore" "assay, Biacore AB, Uppsala, Sweden), sandwich assays (for example, the IGEN paramagnetic sphere system International, Inc., Gaithersburg, Maryland), Western Blotting assays, immunoprecipitation and immunohistochemistry assays.
Examples of categories of nucleic acid-based compounds that are antagonists to ActRilb include antisense nucleic acids, RNAi constructs and catalytic nucleic acid constructs. A nucleic acid-based compound can be single-stranded or double-stranded. A double-stranded compound may also include overhang or non-complementary regions, where one or other of the strands is single-stranded. A single ribbon compound can include regions of self-complementarity, meaning that the compound forms the
- 35/65 has a “hairpin” structure (hair clip) or “stem-loop” (stem-loop) with a helical double structure region.
A nucleic acid-based compound can comprise a nucleotide sequence that is complementary to a region consisting of not more than 1000, not more than 500, not more than 250, not more than 100 or not more than 50, 35, 25, 22 , 20,18 0 or 15 nucleotides of the complete nucleic acid sequence of ActRilb or the nucleic acid sequence of Ba, Bs, Bc or Be activin.
The complementarity region will preferably contain at least 8 nucleotides and, optionally, around 18 to 35 nucleotides.
A complementarity region can fall within the intron, coding sequence or non-coding sequence of the target transcript, such as the portion of the coding sequence.
In general, a nucleic acid compound will be around 8 to about 500 nucleotides or base pairs in length, and optionally, the length will be around 14 to about 50 nucleotides.
A nucleic acid can be DNA (especially for use as an antisense), RNA or hybrid RNA: DNA.
Any of the strands can include a mixture of DNA and RNA, as well as modified forms that cannot be quickly classified as DNA or RNA.
Likewise, a double-stranded compound can be DNA: DNA, DNA: RNA or RNA: RNA, and any of the strands can also include a mixture of DNA and RNA, as well as modified forms that cannot be quickly classified as DNA or RNA.
A nucleic acid compound can include any one of a variety of modifications, including one or more modifications to the backbone (the sugar-phosphate moiety in a natural nucleic acid, including bonds between the nucleotides) or the moiety. bases (the purine or pyrimidine portion of a natural nucleic acid). An antisense nucleic acid compound will preferably be around 15 to about 30 nucleotides in length and will often contain one or more modifications to improve characteristics such as stability in the serum, in a cell or in a place where the compound is likely to be released, such as the stomach, for compounds released orally, and the lung for inhaled compounds.
In the case of an RNAi construct, the strand complementary to the target transcript will generally be RNA or modifications thereof.
The other strand can be RNA, DNA or any other variation.
The duplex portion of the double-stranded or single-stranded hairpin RNAi construct will generally be 18 to 40 nucleotides in length and, optionally, around 21 to 23 nucleotides in length, provided that it serves as a substrate for Dicer enzyme.
Catalytic or enzymatic nucleic acids can be ribozymes or DNA enzymes and can also contain modified forms.
Nucleic acid-based compounds can inhibit target expression by approximately 50%, 75%, 90% or more when brought into contact with cells under physiological conditions and a concentration at which the nonsense or sense control has little - co or no effect.
Preferred concentrations for testing the effect of nucleic acid compounds are 1, 5 and 10 micromolar.
Nucleic acid compounds can also be tested
- 36/65 as well as effects on, for example, red blood cell levels.
In certain embodiments, an ActRIlb antagonist may be a pholistatin polypeptide that antagonizes activin bioactivity and / or binds to activin. The term "follistatin polypeptide" includes polypeptides containing any natural folistatin polypeptide, as well as any of its variants (including mutants, fragments, fusions and peptide-dometic forms) that retain a useful activity, and further includes any monomer or many of folistatin. Variants of follistatin polypeptides that retain activin-binding properties can be identified based on previous studies involving interactions of follistatin and activin. For example, WO2008 / 030367 describes specific domains of follistatin ("FSDs") that have been shown to be important for activin binding. As shown below in SEQ ID NOs: 9-11, the N-terminal domain of follistatin ("FSND", SEQ ID NO: 9), FSD2 (SEQ ID NO: 10) and, to a lesser extent, FSD1 (SEQ ID NO: 11) represent exemplary domains within follistatin that are important for activin binding. In addition, methods for producing and testing polypeptide libraries are described above in the context of ActRilb polypeptides, and these methods also belong to the production and testing of folistatin variants. Follistatin polypeptides include polypeptides derived from the sequence of any known follistatin having a sequence at least about 80% identical to the sequence of a follistatin polypeptide and, optionally, at least 85%, 90 %, 95%, 97%, 99% or with a larger identity. Examples of follistatin polypeptides include the mature follistatin polypeptide or shorter isoforms or other variants of the human folistatin precursor polypeptide (SEQ ID NO: 7), as described, for example, in WO2005 / 025601.
The FST344 isoform of the human follistatin precursor polypeptide is as follows: MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNOWLRQOAKNGRCQVLYKTELSKEE CCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKP RCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCP GSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAY
EGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASEC AMKEAACSSGVLLEVKHGSCNSISEDTEEEEEDEDQDYSFPISSILEW (SEQ ID NO: 7; —NP 037541.1 FOLLISTATINIS is the unique sign; the last 27 residues in bold represent additional amino acids, when compared to a shorter isoform of pholistatin, FST317 (NP 006341), below.
The FST317 isoform of the human follistatin precursor polypeptide is as follows: MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEE CCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKP RCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVOYQGRCKKTCRDVFCP
. 37/65 GSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAY
EGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASEC AMKEAACSSGVLLEVKHSGSCN (SEQ ID NO: 8) The signaling peptide is the only underline.
The sequence of the N-terminal domain of follistatin (FSND) is as follows:
GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNG GAPNCIPCK (SEQ ID NO: 9; FSND) The strings of FSD1 and FSD2 are as follows: ETCENVDCGPGKKCRMNKKNKPRCV (SEQTD) (NOQT) in this patent application may be a related follistatin-like gene (FLRG) that antagonizes the bioactivity of activin and / or binds to activin and other ligands with a spectrum similar to that of ActRilb-Fc. The term "FLRG polypeptide" includes polypeptides comprising any natural FLRG polypeptide, as well as any variants thereof (including mutants, fragments, fusions and peptidomimetic forms) that retain useful activity. Variants of FLRG polypeptides that retain activin-binding activities can be identified using routine methods to analyze interactions of FLRG and activin. See, for example, US 6. 537 966. In addition, methods for producing and testing polypeptide libraries are described above in the context of ActRilb polypeptides, and such methods also pertain to the production and testing of FLRG variants. FLRG polypeptides include polypeptides derived from the sequence of any known FLRG having a sequence at least about 80% identical to the sequence of an FLRG polypeptide and, optionally, at least 85%, 90%, 95 %, 97%, 99% or greater identity. The human FLRG precursor polypeptide is as follows: MRPGAPGPLWPLPWGALAWAVGFEVSSMGSGNPAPGGVCWLQQGQEATCSLVL QTDVTRAECCASGNIDTAWSNLTHPGNKINLLGFLGLVHCLPCKDSCDGVECGPGKACRML GGRPRCECAPDCSGLPARLQVCGSDGATYRDECELRAARCRGHPDLSVMYRGRCRKSCE
HVVCPRPQSCVYVDOTGSAHCVVCRAAPCVPSSPGQELCGNNNVTYISSCHMRQATCFLG —RSIGVRHAGSCAGTPEEPPGGESAEEEENFV (SEQ ID NO: 12; NP. 005851) The signaling peptide is the only underline.
In certain embodiments, functional variants or modified forms of folistatin polypeptides and FLRG polypeptides include fusion protein having at least a portion of the folistatin polypeptides or FLRG polypeptides and one or —more fusion domains, such as for example, domains that facilitate the isolation, detection, stabilization or multimerization of the polypeptide. Suitable fusion domains are discussed in detail above with reference to the ActRilb polypeptides. In
. 38/65 an embodiment, an antagonist is a fusion protein, comprising an activin-binding portion of a follistatin polypeptide fused to an Fc domain. In another mode, an antagonist is a fusion protein comprising an activin-binding portion of an FLRG polypeptide fused to an Fc domain. Folistatin and FLRG have shown in the literature, and by Applicants with respect to FLRG, to have affinities for Activiv A in the picomolar interval, indicating that these agents will inhibit activin A signaling to a similar degree as AciRilb-Fc.
5. Screening assays In certain respects, the present invention relates to the use of Actrilb polypeptides (for example, soluble ActRilb polypeptides) and activin or other ActrRIlb ligands to identify compounds (agents) that are agonists or antagonists of the ActRIlb signaling pathway. Compounds identified through this screening can be tested to assess their ability to modulate bone growth or mineralization or to stimulate muscle growth in vitro. Optionally, these compounds can be further tested on animal models to assess their ability to modulate tissue growth in vivo. : There are numerous approaches for screening therapeutic agents for modulating tissue growth by targeting ActRlilb polypeptides. In certain modalities, high-throughput screening of compounds can be performed to identify agents that alter ActRIlb-mediated effects on bone or muscle. In certain embodiments, the assay is performed to screen and identify compounds that specifically inhibit or reduce the binding of an ActRilb polypeptide to the activity or another linker. Alternatively, the assay can be used to identify compounds that enhance the binding of an ActR11b polypeptide to activin or another linker. In an additional embodiment, the compounds can be identified by their ability to interact with an ActRilb polypeptide.
A variety of test formats will suffice and, in light of the present invention, those not expressly described in this patent application will nevertheless be understood by any person skilled in the art. In this specification, the test compounds (agents) of the invention can be created by combinatorial chemistry. Alternatively, the compounds in question can be natural biomolecules synthesized in vivo or in vitro. Compounds (agents) to be tested for their ability to act as tissue growth modulators can be produced, for example, by bacteria, yeast, plants or other organisms (for example, natural products), chemically produced (for example, small molecules, including peptidomimetics) or produced by recombination. The test compounds contemplated by the present invention include non-peptide organic molecules, peptides, polypeptides, peptidomimetics,
. 39/65 cos, sugars, hormones and nucleic acid molecules. In a specific embodiment, the agent under test is a small organic molecule having a molecular weight of less than approximately 2,000 daltons.
The test compounds of the invention can be provided as unique, distinct entities, or provided in libraries of greater complexity, such as produced by combinatorial chemistry. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. The presentation of test compounds to the test system can be in isolated form or mixtures of compounds, especially in the initial stages of screening. Optionally, the compounds can be optionally derived with other compounds and have derivation groups that facilitate the isolation of the compounds. Non-limiting examples of derivative groups include biotin, fluorescein, digoxigenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S transferase (GST), photoactable crosslinking agents or any combinations thereof. In many drug screening programs that test libraries of compounds and natural extracts, high-throughput assays are convenient in order to maximize the number of compounds researched in a given period of time. Tests carried out on cell-free systems, so that they can be derived with purified or semi-purified Á proteins, are often referred to as “primary” scans in the sense that they can be generated to allow rapid development and relatively easy detection of a change in a molecular target that is mediated by a test compound. In addition, the effects of cell toxicity or the bioavailability of the test compound can generally be ignored in the in vitro system, the assay, instead, being primarily focused on the drug's effect on the molecular target as it can be ma- manifested in a change in the binding affinity between an AciRilb polypeptide and activin.
By way of illustration only, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified ActRilb Polypeptide polypeptide, which is normally capable of binding to activin. To the mixed compound and the ActRilb polypeptide is then added a composition containing an ActRilb linker. The detection and quantification of ActRilb / activin complexes allows determining the effectiveness of the compound in inhibiting (or potentiating) the formation of the complex between the ActRilb polypeptide and activin. The effectiveness of the compound can be assessed by response curves generated from the data obtained, using various concentrations - of the test compound. In addition, a control trial can be performed to provide a baseline value for comparison. For example, in a control assay, isolated and purified activin is added to a composition containing the ActRilb polypeptide, and the
- 40/65 tion of the ActRilb / activin complex is quantified in the absence of the test compound. It will be understood that, in general, the order in which the reagents can be mixed can be varied, and can be mixed simultaneously. In addition, instead of purified proteins, extracts and cell lysates can be used to make an assay system adequately free of cells.
The complex formation between the ActRilb polypeptide and activin can be detected by a variety of techniques. For example, modulation of complex formation can be quantified using, for example, labeled proteins that can be detected such as ActRIlb polypeptide or radiolabeled activin (eg * P, * ºS, * C or ºH), fluorescence-labeled (eg FITC) or enzyme-labeled, immunoassay or chromatographic detection.
In certain embodiments, the present invention contemplates the use of polarized fluorescence assays and fluorescence resonance energy transfer (FRET) assays that measure, directly or indirectly, the degree of interaction between a —ActRilbe polypeptide its Link. In addition, other detection modes, such as those based on optical waveguides (PCT Publication WO 96/26432 and US Patent No. 5 677 196), surface plasmon resonance (SPR), surface charge sensors and force sensors on the surface, they are compatible with many embodiments of the invention. Furthermore, the present invention contemplates the use of an interaction trap test, also known as the “two hybrid assay”, to identify agents that alter or enhance the interaction between an ActRllb polypeptide and its protein binding. See, for example, U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72: 223-232; Madura et al. (1993) J Biol Chem 268: 12046-12054; Bartel et al. (1993) Biotechniques 14: 920-924; and Iwabuchi et a /. (1993) Oncogene 8: 1693-1696. In a specific embodiment, the present invention contemplates the use of reverse systems of two hybrids to identify compounds (for example, small molecules or peptides) that dissociate interactions between an ActRllb polypeptide and its binding protein. See, for example, Vidal and Legrain, (1999) Nucleic Acids Res 27: 919-29; Vidal and Legrain, (1999) Trends Biotechnol 17: 374-81; and U.S. Patents No. 5,525,490; 5,955,280; and 5 965
368.
In certain embodiments, the compounds in question are identified by their ability to interact with an ActRilb polypeptide or activin of the invention. The interaction between the compound and the ActRilb or activin polypeptide can be covalent or non-covalent. For example, this interaction can be identified at the protein level using methods - biochemical in vitro, including photo-crosslinking, binding to radiolabeled ligand and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46: 1). In certain cases, compounds can be screened in a mechanism-based assay, such as a
: 41/65 assay to detect compounds that bind to an ActRI-lb activin or polypeptide. This can include a solid phase or liquid phase bonding event. Alternatively, the coding gene for activin or an ActRilb polypeptide can be transfected with a reporter system (eg, B-galactosidase, luciferase or fluorescent green protein) in a cell and screened against the library, preferably by high-performance screening or with individual library members. Other mechanism-based bonding tests can be used, for example, bonding tests that detect changes in free energy. The binding assays can be performed with the target fixed to a cavity, sphere or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. Bound compounds can be detected normally using colorimetric techniques or by fluorescence or surface plasmon resonance.
In certain respects, the present invention provides methods and agents for modulating (stimulating or inhibiting) bone formation and increasing bone mass. Therefore, any compound identified can be tested on whole cells or tissues, in vitro or in vivo, to confirm its ability to modulate bone or cartilaginous growth. Various methods known in the art can be used for this purpose.
For example, the effect of ActRllb or activin polypeptides or test compounds on bone or cartilaginous growth can be determined by measuring the induction of Msx2 or the differentiation of osteoprogenitor cells into osteoblasts in cell assays (see, for example , Daluiski et al., Nat Genet. 2001, 27 (1): 84-8; Hino et al, Front Biosci. 2004, 9: 1520-9). Another example of cellular assays includes analyzing the osteogenic activity of the polypeptides in question of ActRilb or activin and of compounds under test in mesenchymal progenitor cells and osteoblastic cells. For purposes of illustration, recombinant adenoviruses expressing an ActRilb activin or polypeptide can be constructed to infect C3H10T1 / 2 mesenchymal pluripotent progenitor cells, C2C12 preosteoblastic cells and TE-85 osteoblastic cells. Osteogenic activity is then determined by measuring the induction of alkaline phosphatase, osteocalcin and matrix mineralization (see, for example, Cheng et al., J bone Joint Surg Am. 2003, 85-A (8): 1544-52).
The present invention also contemplates in vivo assays to measure bone or cartilaginous growth. For example, Namkung-Matthai et al., Bone, 28: 80-86 (2001) describes an osteoporotic rat model in which bone repair during the initial period after fracture is studied. Kubo et al., Steroid Biochemistry & Molecular Biology, 68: 197-202 (1999) also describe an osteoporotic rat model in which bone repair during the late period after fracture is studied. Andersson et al., J. Endocrinol. 170: 529-537 describe a model of osteoporosis in mice in which mice are ovariectomized, which causes mice to lose bone mineral content and density.
. 42/65 considerable bone mineral density, with trabecular bone losing approximately 50% of bone mineral density. Bone density could be increased in ovariectomized mice by administering factors such as parathyroid hormone. In certain aspects, the present invention makes use of fracture healing tests that are known in the art. These tests include fracture technique, histological analysis and biochemical analysis and are described in, for example, US Patent No. 6 521 750, the content of which describes its description of experimental protocols to cause, as well as to measure the extent of fractures and the repair process, is incorporated by reference in this patent application.
In certain aspects, the present invention provides methods and agents for stimulating muscle growth and increasing muscle mass, for example, by antagonizing functions of an ActRilb polypeptide and / or an ActRilb ligand. Therefore, any compound identified can be tested on whole cells or tissues, in vitro or in vivo, to confirm its ability to modulate muscle growth. Various methods known in the art can be used for this purpose. For example, methods of the invention are performed in such a way that the signal transduction through a protein to the ActRIlb activated when binding to an ActRIlb ligand (for example, GDF8) has been reduced or inhibited. It will be recognized that the growth of muscle tissue in the body would result in increased muscle mass in the body, when compared to the muscle mass of a corresponding organism (or population of organisms) in which signal transduction by an ActRllb protein has not thus been affected.
For example, the effect of ActRilb polypeptides or test compounds on muscle cell growth / proliferation can be determined by measuring the expression of Pax-3 and Myf-5 genes, which are associated with cell proliferation —Myogenic, and the expression of the MyoD gene, which is associated with muscle differentiation (eg, Amthor et a /., Dev Biol. 2002, 251: 241-57). It is known that GDF8 negatively regulates the gene expression of Pax-3 and Myf-5, and that it prevents the gene expression of MyoD. ActRilb polypeptides or test compounds are expected to antagonize this GDF8 activity. Another example of cell assays includes measuring the proliferation of —myoblasts, such as C (2) C (12) myoblasts, in the presence of ActRilb polypeptides or test compounds (eg, Thomas et a / l., J Biol Chem 2000, 275: 40235-43).
The present invention also contemplates in vivo tests to measure muscle mass and strength. For example, Whittemore et al. (Biochem Biophys Res Commun. 2003, 300: 965-71) describe a method for measuring increased skeletal muscle mass and increased grip strength in mice. Optionally, this method can be used to determine the therapeutic effects of test compounds (eg ActRilb polypeptides) on muscle diseases or conditions, for example, those diseases for
«43/65 which muscle mass is limiting.
It is understood that the screening assays of the present invention apply not only to the ActRIlb polypeptides in question and to variants of the ActRilb polypeptides, but also to any test compounds, including agonists and antagonists of the ActRIlb polypeptides. In addition, these screening assays are useful for checking target drugs and for quality control purposes.
6. Exemplary therapeutic uses In certain embodiments, ActRilb antagonists (for example, ActRilb polypeptides) of the present invention can be used to stimulate aabolic bone growth, treat an osteolytic bone tumor and / or treat or prevent a disease or condition that would benefit from muscle growth. In certain modalities, AntRIlb antagonists and, especially ActRilb-Fc constructs, can be used to treat or prevent bone loss related to cancer. In certain embodiments, the present invention provides methods for treating or preventing bone damage in an individual in need of such treatment by administering to the individual a therapeutically effective amount of an ActRilb antagonist, especially an ActRilb polypeptide. In certain modalities, the present invention provides methods for promoting bone growth or mineralization in an individual in need of such treatment by administering to the individual a therapeutically effective amount of an ActRilb antagonist, especially of an ActRiIlb polypeptide. These methods are preferentially directed to therapeutic and prophylactic treatments for animals and, more preferably, for humans. In certain embodiments, the invention provides for the use of ActRllb antagonists (especially soluble ActRilb polypeptides and neutralizing antibodies directed against ActRilb) for the treatment of disorders associated with low bone density or reduced bone resistance.
In this descriptive report, a therapeutic agent that “prevents” a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample in relation to an untreated control sample. that slows the appearance or reduces the severity of one or more symptoms of the disorder or condition in relation to the untreated control sample. The term "treat" in this specification includes the prophylaxis of the condition mentioned or the improvement or elimination of the condition once it has been established. In any case, prevention or treatment can be discerned in the medical diagnosis and in the intended result of the administration of the therapeutic agent.
The invention provides methods for inducing bone and / or cartilage formation, preventing bone loss, increasing bone mineralization or preventing bone demineralization. For example, the ActRilb antagonists in question can be applied in the treatment
- 44/65 osteoporosis and in the consolidation of bone fractures and cartilage defects in humans and other animals. ActRllb polypeptides may be useful in patients who are diagnosed with low subclinical bone density as a protective measure against the development of osteoporosis.
In a specific modality, methods and compositions of the present invention can find medical utility in the consolidation of bone fractures and cartilage defects in humans and other animals. The methods and compositions in question can also have prophylactic use in reducing closed as well as open fractures and, additionally, in improving the fixation of artificial joints. Newly induced bone formation — by an osteogenic agent contributes to the repair of congenital craniofacial defects, induced by trauma or induced by tumor resection and, additionally, is useful in cosmetic plastic surgery. In certain cases, the ActRilb antagonists in question can provide an environment that attracts bone-forming cells, stimulate the growth of bone-forming cells or induce differentiation of progenitors from bone-forming cells. The ActRilb antagonists of the invention may also be useful in the treatment of osteoporosis.
'Methods and compositions of the invention can be applied to CHARACTERIZED conditions or causes of bone loss, such as osteoporosis (including osteo-, secondary porosis), hyperparathyroidism, Cushing's disease, Paget's disease, thyrotoxicosis, chronic diarrheal state or desorption, chronic kidney disease, renal tubular acidosis or anorexia nervosa.
Osteoporosis can be caused or be associated with several factors. Belonging to the female population, especially if in the post-menopausal period, having low body weight and leading a sedentary lifestyle are all risk factors for osteoporosis (loss of bone mineral density, leading to the risk of fractures). People with any of the following profiles may be candidates for treatment with an ActRilb antagonist: a postmenopausal woman who is not taking estrogen or other hormone replacement therapy; person with personal or maternal history of hip fracture or smoking; tall (over 5 feet and 7 inches [1.70 n]) or thin (less than 125 pounds [56.7 kg]) women in postmenopausal; man with clinical conditions associated with bone loss; person using drugs that are known to cause bone loss, including corticosteroids like Prednisone "Y, various anticonvulsant medications like Dilantin'M and certain barbiturates, or high-dose drugs for thyroid hormone replacement; people with illness liver or family history of osteoporosis; person with high bone turnover (renewal) (eg, excess collagen in urine samples); person with thyroid condition, such as hyperthyroidism; person who has suffered a fracture after only minor trauma; person with radiological evidence of vertebral fracture or other
"45/65 more osteoporosis.
As noted above, osteoporosis can also result from a condition associated with another disorder or from the use of certain medications. Osteoporosis resulting from drugs or other medical conditions is known as secondary osteoporosis. In a condition known as Cushing's disease, the excessive amount of cortisol produced by the body results in osteoporosis and fractures. The most common medications associated with secondary osteoporosis are corticosteroids, a class of drugs that act similarly to cortisol, a hormone naturally produced by the adrenal glands. Although adequate levels of thyroid hormones (which are produced by the thyroid gland) are necessary for the development of the skeleton, excess thyroid hormone can decrease bone mass over time. Other medications that can cause secondary osteoporosis include phenytoin (Dilantin) and barbiturates that are used to prevent seizures; methotrexate (Rheumatrex, Immunex, Folex PFS), a drug for some forms of arthritis, cancer and immune disorders; cy- closporin (Sandimmune, Neoral), a drug used to treat some autoimmune diseases and to suppress the immune system in organ transplant patients; agonists of luteinizing hormone-releasing hormones (Lupron, Zoladex), used to treat prostate cancer and endometriosis; heparin (Calciparina, Liquaemin), an anti-coagulant medication; and cholestyramine (Questran) and colestipol (Colestid), used to treat high cholesterol.
Bone loss resulting from cancer therapy is widely recognized and called cancer therapy-induced bone loss (CTIBL). Bone metastases can create cavities in the bone that can be corrected by treatment with AcTRilb antagonists.
In a preferred embodiment, ActRilb antagonists, especially a soluble ActRilb, described in this patent application can be used in cancer patients. As demonstrated in this specification, an ActRIlb-Fc fusion protein is capable of promoting anabolic bone growth in human patients. Anabolic bone agents have been shown to be useful for treating multiple myeloma (see, for example, Yacbety et al, Blood, 109 (5): 2106-11 (2007); Oyajobi et al, Br J Haematol. 139 ( 3): 434-8 (2007); Fulciniti et al., Blood, 114 (2): 371-9 (2009); Stewart et al. Journal of Cellular Biochemistry 98: 1-13 (2006)). Therefore, activin-ActRilb antagonists described in this application are believed to be useful for treating multiple myeloma and other bone tumors. Patients with certain tumors (eg, prostate, breast, multiple myeloma, or any tumor causing hyperparathyroidism) are at high risk for bone loss due to tumor-induced bone loss, as well as bone metastases and therapeutic agents. These patients can be treated with ActRIlb antagonists, even in the
- 46/65 evidence of bone loss or bone metastases. Patients can also be monitored for evidence of bone loss or bone metastases, and can be treated with ActRIlb antagonists if indications suggest increased risk. In general, DEXA tests are used to assess changes in bone density, while bone remodeling indicators can be used to assess the likelihood of bone metastases. Serum markers can be monitored. Bone specific alkaline phosphatase (BSAP) is an enzyme that is present in osteoblasts. Blood levels of BSAP are increased in patients with bone metastases and other conditions that result in increased bone remodeling. Osteocalcin and pro-collagen peptides are also associated with bone formation and bone metastases. Increases in BSAP have been detected in patients with bone metastasis caused by prostate cancer and, to a lesser extent, in bone metastases from breast cancer. Levels of bone morphogenetic protein 7 (BMP-7) are high in prostate cancer that has metastasized to bone, but not in bone metastases caused by bladder, skin, liver or lung cancer. Carbo-terminal telopeptide Type 1 (ICTP) is a crosslinker found in collagen formed during bone resorption. Since bone is constantly being decomposed and reformed, ICTP will be found throughout the body. However, at the bone metastasis site, the level will be significantly higher than in a normal bone area. ICTP was found at high levels in bone metastasis due to prostate, lung and breast cancer. Another collagen crosslinker, the Type N-terminal telopeptide | (NTx), is produced together with ICTP during bone turnover. The amount of NTx is increased in bone metastasis caused by many different types of cancer, including lung, prostate and breast cancer. Additionally, NTx levels increase with the progression of bone metastasis. Therefore, this marker can be used both to detect metastasis and to measure the extent of the disease. Other markers of resorption include pyridinoline and deoxypyridinoline. Any increase in resorption markers or bone metastasis markers indicates the need for therapy with ActiRllb antagonists in a patient. In view of ActRIlb-Fc having an evident effect on activity markers — osteoblastic (for example, BSAP levels), ActRilb-Fc molecules may be especially useful in cases of cancer-related bone loss, in which bone disease it is osteolytic in nature, rather than osteoblastic in nature. This is typically true for multiple myeloma and bone metastases caused by a variety of tumors. However, bone metastases from prostate cancer, especially, tend to be more osteo-blast in nature, and ActRilb-Fc fusion proteins may not be beneficial in this setting.
ActRIlb antagonists can be administered in conjunction with other
- 47/65 pharmaceutical agents.
Joint administration can be achieved by administering a single joint formulation, by simultaneous administration or by administration at separate times.
ActRilb antagonists can be especially advantageous if administered with other bone-acting agents.
A patient can benefit from receiving an ActRIlb antagonist and jointly taking calcium, vitamin D supplements, exercising properly and / or, in some cases, using another medication.
Examples of other medications include bisphosphonates (alendronate, ibandronate and risedronate), calcitonin, estrogens, parathyroid hormone and raloxifene.
Bisphosphonates (alendronate, ibandronate and risedronate), calcitonin, estrogens and raloxifene affect the bone remodeling cycle and are classified as anti-resorptive medications.
Bone remodeling consists of two distinct stages: bone resorption and bone formation.
Anti-resorptive medications delay or interrupt the bone resorption part of the bone remodeling cycle, but do not delay the bone formation part of the cycle.
As a result, new formation proceeds at a higher rate than bone resorption, and bone density may increase over time.
Teriparatide, a form of parathyroid hormone, increases the rate of bone formation in the bone remodeling cycle.
Alendronate is approved for prevention (5 mg daily or 35 mg once weekly) and treatment (10 mg daily or 70 mg once weekly) for post-, menopausal osteoporosis.
Alendronate reduces bone loss, increases bone density and reduces the risk of fractures of the spine, wrist and hips.
Alendronate is also approved for the treatment of glucocorticoid-induced osteoporosis in men and women, resulting from the prolonged use of these medications (ie, prednisone and cortisone) and for the treatment of male osteoporosis.
Alendronate plus vitamin D is approved for the treatment of osteoporosis in postmenopausal women (70 mg once a week plus vitamin D) and for treatment to improve bone mass in men with osteoporosis.
Ibandronate is approved for the prevention and treatment of postmenopausal osteoporosis.
Ingested in pill form once a month (150 mg), ibandronate should be taken on the same day each month.
Ibandronate reduces bone loss, increases bone density and reduces the risk of fractures of the spine.
Risedronate is approved for the prevention and treatment of postmenopausal osteoporosis.
Ingested daily (5 mg dose) or once a week (35 mg dose or 35 mg dose with calcium), risedronate slows bone loss, increases bone density and reduces the risk of fractures in the spine or other locations.
Risedronate is also approved for male and female use to prevent and / or treat the glucocorticoid-induced osteoporosis that results from the prolonged use of these medications (ie, prednisone or cortisone). Calcitonin is a natural hormone involved in calcium regulation and bone metabolism.
In women who have been in menopause for more than five years, calcitonin slows bone loss, increases
. 48/65 bone density in the spine and can relieve pain associated with bone fractures.
Calcitonin reduces the risk of spine fractures.
Calcitonin is available as an injection (50-100 IU daily) or as a nasal spray (200 IU daily). Estrogen therapy (ET) / hormone therapy (HT) is approved for the prevention of osteoporosis.
ET has been shown to reduce bone loss, increase bone density in both the spine and hip and reduce the risk of hip and spine fractures in postmenopausal women.
ET is most commonly administered as a pill or skin patch that releases a low dose of approximately 0.3 mg per day or a standard dose of approximately 0.625 mg per day and is effective even when started after the age of 70 .
When taken alone, estrogen can increase the female risk of developing cancer of the uterine lining (endometrial cancer). In order to eliminate this risk, health professionals prescribe the progestin hormone combined with estrogen (hormone replacement therapy or HT) for women with an intact uterus.
ET / HT relieves menopausal symptoms and has been shown to have a beneficial effect on bone health.
Side effects include vaginal bleeding, breast hypersensitivity to touch, mood swings and gallbladder disease.
Raloxifene, 60 mg daily, is approved for the prevention and treatment of postmenopausal osteoporosis.
It belongs to a class of drugs called selective estrogen receptor modulators (SERMs), which was developed to provide the beneficial effects of estrogens without their potential disadvantages.
Raloxifene increases bone mass and reduces the risk of spine fractures.
There is still no data available to demonstrate that raloxifene is able to reduce the risk of hip fractures and elsewhere in the spine.
Theraparatide, a form of parathyroid hormone, is approved for the treatment of osteoporosis in postmenopausal women and in men at high risk for fracture.
This medication stimulates new bone formation and significantly increases bone mineral density.
In postmenopausal women, fracture reduction was noted in the spine, hips, feet, ribs and wrist.
In men, fracture reduction was noted in the spine, but data are insufficient to assess fracture reduction in others.
other places.
Teriparatide is self-administered as a daily injection for up to 24 hours.
As shown in this specification, ActRilb antagonists are also useful in promoting muscle growth and, as such, can be useful in host of muscle-related disorders.
Exemplary muscle-related conditions include neuromuscular disorders (eg, muscular dystrophy and atrophy), congestive obstructive pulmonary disease (COPD) (and muscle wasting associated with —COPD), muscle wasting syndrome, sarcopenia, cachexia, adipose tissue disorders (eg, obesity), type 2 diabetes and degenerative bone disease (eg, osteoporosis). Other exemplary conditions include musculodegenerative disorders
. 49/65 and neuromuscular, tissue repair (for example, wound healing), neurodegenerative diseases (for example, amyotrophic lateral sclerosis), immunological disorders (for example, disorders related to proliferation or abnormal lymphocyte function) and obesity or disorders related to abnormal adipocyte proliferation.
In certain embodiments, ActRilb antagonists are used as part of a treatment for muscular dystrophy. The term “muscular dystrophy” refers to a group of degenerative muscle diseases, CHARACTERIZED by weakening and gradual deterioration of skeletal muscles and, at times, cardiac and respiratory muscles. Muscular dystrophies are genetic disorders CHARACTERIZED by progressive loss and weakness of muscle mass that start with microscopic changes in the muscle. As the muscles degenerate over time, the person's muscle endurance declines. Exemplary muscular dystrophies that can be treated with a regimen including the ActRilb polypeptides in question include: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy (EDMD), waist-type muscular dystrophy (LGMD), facioescapuloumeral muscular dystrophy (FSH or FSHD) (also known as Landouzy-Dejerine), myotonic dystrophy (MMD) (also known as Steinert's disease), oculopharyngeal muscular dystrophy (OPMD), dystrophy muscular distta! (DD), congenital muscular dystrophy (CMD). ã Duchenne muscular dystrophy (DMD) was first described by French neurologist Guillaume Benjamin Amand Duchenne in the 1860s. Becker muscular dystrophy (BMD) is named after the German doctor Peter Emil Becker, the first to describe this variant of DMD in the fifties. DMD is one of the most frequently inherited diseases in the male population, affecting one in 3,500 boys. DMD occurs when the dystrophin gene, located on the short arm of the x chromosome, is broken. In order to carry only one copy of the X chromosome, the male population has only one copy of the dystrophin gene. Without the dystrophin protein, the muscle is easily damaged during cycles of contraction and relaxation. Although early in the disease, the muscle compensates for regeneration, afterwards, the muscle progenitor cells cannot keep pace with continuous damage and the healthy muscle - it is replaced by non-functional fibro-fatty tissue. A double effect on muscle and bone can be useful in patients with muscular dystrophy.
BMD results from different mutations in the dystrophin gene. BMD patients have some dystrophin, but it is insufficient in quantity or of low quality. Having some dystrophin protects the muscles of those with BMD from degenerating as badly - as quickly as those of people with DMD.
For example, recent research shows that blocking or eliminating the function of GDF8 (an ActRilb ligand) in vivo can effectively treat at least certain symptoms.
. 50/65 but in patients with DMD and BMD. Therefore, the ActRIlb antagonists in question can act as inhibitors (antagonists) of GDF8 and constitute an alternative resource to block the functions of GDF8 and / or ActRilb in vivo in patients with DMD and BMD. An ActRIlb-Fc protein has been shown to increase muscle mass in a model of dystrophy - muscle in mice (see U.S. Publication No. 2009/0005308).
Likewise, the ActRilb antagonists in question offer an effective way to increase muscle mass in other disease conditions that need muscle growth. For example, ALS, also called Lou Gehrig's disease (motor neuron disease) is a chronic, incurable and inexorable disorder of the central nervous system that attacks motor neurons, components of the central nervous system that connect the brain to skeletal muscles. In ALS, motor neurons deteriorate and die in the end, and while a person's brain remains functioning and fully alert, the command to move never reaches the muscles. Most people with ALS are between 40 and 70 years old. The first motor neurons that weaken - they warm up those that go to the arms and legs. Those with ALS may have problems with walking, may drop objects, fall, their speech is unclear 'and laugh or cry uncontrollably. In the end, the muscles in the extremities begin to atrophy as a result of lack of use. This muscle weakness will become disabling, and a person will need a wheelchair or become incapable of activities outside of bed.
—Most patients with ALS die of respiratory failure or complications of assisted ventilation such as pneumonia, from 3 to 5 since the onset of the disease. An ActRIlb-Fc protein has been shown to improve the physical appearance, muscle mass and life expectancy of an ALS model in mice (see U.S. Publication No. 2009/0005308).
The increased muscle mass induced by ActRillb antagonists could also benefit those suffering from diseases with loss of muscle mass. It has been observed that GDF8 expression is inversely correlated with free fat mass in humans and that increased GDF8 gene expression is associated with weight loss in male patients with AIDS wasting syndrome. By inhibiting the “GDF8 function in AIDS patients, at least certain symptoms of AIDS can be relieved, if not completely eliminated, thereby significantly improving the quality of life in AIDS patients.
Since the loss of function of GDF8 (an ActRilb ligand) is also associated with fat loss without decreasing nutrient intake, the antagonists of —AcIRIlbem question can be additionally used as a therapeutic agent to delay or prevent development of obesity and type 1 diabetes. This approach is confirmed and supported by the data shown in this patent application, according to which p 51/65 an ActRIlb-Fc protein better demonstrated the metabolic status of obese mice.
The cancer anorexia-cachexia syndrome is among the most life-threatening aspects of cancer. Progressive weight loss in cancer anorexia-cachexia syndrome is a common feature of many types of cancer and is responsible - not only poor quality of life and poor response to chemotherapy, but also a shorter survival time than that found in patients with comparable tumors without weight loss. Associated with anorexia, the loss of fat and muscle tissue, psychological distress and a lower quality of life, cachexia arises from a complex interaction between cancer and the host. It is one of the most common causes of death among cancer patients and is present in 80% of deaths. It is a complex example of metabolic chaos affecting protein, carbohydrate and fat metabolism. Tumors produce direct and indirect abnormalities, resulting in anorexia and weight loss. Currently, there is no treatment to control or to reverse the process. Cancer anorexia-cachexia syndrome affects the production of cytokines, the release of factors that mobilize lipids and induce proteolysis, in addition to changes in intermediate metabolism. Although anorexia is common, a decrease in food intake alone is not able to be responsible for the changes in body composition seen in cancer patients, and the increased intake of nutrients cannot reverse the emaciation syndrome . Cachexia should be suspected in cancer patients if an involuntary weight loss above five percent of the weight existing before morbidity occurs within a six-month period.
Given that systemic overexpression of GDF8 in adult mice has been shown to induce profound muscle and fat loss, analogous to that seen in human cachexia syndromes, the ActRIlb antagonists in question, in the form of pharmaceutical compositions, can be beneficially used to prevent, treat or alleviate the symptoms of cachexia syndrome, in which muscle growth is desired.
In other embodiments, the present invention provides compositions and methods for regulating the body fat content in an animal and for treating or preventing related conditions and, especially, health-compromising conditions related to the body fat content. According to the present invention, regulating (controlling) body weight can refer to reducing or increasing body weight, reducing or increasing the rate of weight gain or increasing or reducing the rate of weight loss, and it additionally includes actively maintaining or not significantly altering body weight (for example, against external or internal influences that might otherwise increase or decrease body weight). One embodiment of the present invention relates to regulating body weight, giving an animal (for example, human) in need of it an ActRilb antagonist.
| 52/65 In a specific embodiment, the present invention relates to methods and compounds to reduce body weight and / or reduce weight gain in an animal and, more especially, to treat or improve obesity in patients in risk of or suffering from obesity. In another specific embodiment, the present invention is directed to methods and compounds for treating an animal that is unable to gain or retain weight (e.g., animal with muscle wasting syndrome). These methods are effective for increasing weight and / or body mass or for reducing weight and / or body mass, or for improving conditions associated with or caused by unfavorably low weight and / or body mass (for example, not healthy).
7. Administration In certain embodiments, the invention provides methods for administering a dose of an ActRIlb polypeptide to a patient. The methods of the invention include administering an ActRIlb protein to a patient on an administration schedule that provides an optimal serum concentration of the ActRIlb protein that is maintained for a desired period of time. A patient can be administered on a schedule that involves a desired combination of the amount of an ActRilb polypeptide administered to the patient, as well as the frequency with which the ActRilb polypeptide is administered to the patient. An ActRilb polypeptide for use in such an administration schedule can be any one of those described in this patent application, as a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 2, 3, 13, 17 or 20, or comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 13 , 17 or 20. An ActRilb polypeptide can include a functional fragment of a natural ActRilb polypeptide, such as that comprising at least 10, 20 or amino acids of a sequence selected from SEQ ID NOs: 1-3 or a sequence sequence of SEQ ID NO: 2, devoid of 10 to 15 amino acids at the C-terminal (the “tail”). In certain embodiments, the ActRIlb polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity with a part of ActRilb, where the N -terminal of the ActRilb polypeptide corresponds to an amino acid residue 30 - selected from amino acids 19 - 25 of SEQ ID NO: 1 and the C-terminal of the ActRilb polypeptide corresponds to an amino acid residue selected from amino acids 109 - 134 of SEQ ID NO: 1. In preferred embodiments, the ActR11b polypeptide is an ActRIlb-Fc fusion protein described in this patent application. A patient can be administered in order to achieve an optimal serum concentration of the ActRIlb polypeptide. An optimal serum concentration can, for example, be sufficient to promote a desired biological effect in vivo. These biological effects include any of the therapeutic effects described in this patent application, including
r 53/65 promote bone growth, increase bone density, treat or prevent a disorder associated with low bone density, treat neuromuscular disorders, adipose tissue disorders, metabolic disorders, neurodegenerative disorders, disorders of muscle mass loss or cancer.
According to the description in this patent application, ActRllb polypeptides of the invention demonstrate constant biological efficacy when patients reach and maintain the serum concentration of an ActRilb polypeptide of at least 10 pug / ml. Therefore, in certain embodiments, an ActRllb polypeptide can be administered to a patient on an administration schedule that maintains a serum ActRilb concentration of at least 8 µg / ml, 10 µg / ml, 12.5 µg / ml, 15 µg / ml , 20 µg / ml, 25 µg / ml, 30 µg / ml, 35 µg / ml, 40 µg / ml, 50 µg / ml or at least 70 µg / ml. In certain embodiments, it may be desirable to maintain the serum concentration of the ActRilb polypeptide within an optimum range, such as, for example, within the range of approximately 8 µg / mL to approximately 100 µg / mL (for example, example, 8 - 100 µg / ml, 8 - 70 µg / ml, 8 - 50 µg / ml, 8 - 40 µg / ml, 8-35 µg / ml, 8 - 30 µm / ml, 8 - 25 µg / ml, 8 - 20 µg / ml , 8 - 15 uo / ml, 8 - 12.5 vpg / ml, 8 - 10 pg / ml, 10 - 100 pug / ml, 10 - 70 pa / ml, 10 - 50 ug / ml, 10 - 40 pg / ml, 10 - 35 µg / ml, 10 - 30 µg / ml, 10 - 25 µg / ml, 10 - 20 µg / ml, 10 - 15 µg / ml, 10 - 12.5 µg / ml, 12 - 100 pg / ml, 12 - 70 ug / ml, 12 - 50 ug / ml, 12 - 40 pug / ml, 12 - 35 pg / ml, 12-30 pg / ml, 12- to 25 ug / ml, 12 - 20 pg / ml, 12-15 pg / ml, 15 - 100 ug / ml, 15-70 ug / ml, 15 - 50 ug / ml, 15 - 40 ug / ml, 15-35 upo / ml, 15 - 30 ug / ml, 15 - 25 µg / ml, 15 - 20 µg / ml, 20 - 70 µg / ml, 20 - 50 µg / ml, 20 - 35 µg / ml or 20 - 30 µg / ml).
In certain embodiments, administration schedules of the invention include administering an ActRillb polypeptide in sufficient quantity and at intervals to maintain a desired serum concentration of ActRilb protein for a desired period of time. The desired serum concentration of ActRIlb in a patient will depend on the desired biological effect (eg, treatment of bone disorder). For example, an ActRilb protein can be administered to a patient in sufficient quantity and intervals to maintain the desired serum concentration over a period of approximately one week to a year or more (for example, maintaining the desired serum concentration for 5 days, 7 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 20 days, 25 days, 30 days, one to two weeks, one week to one month, two months, four months, six months, a year or two years or more). Preferably, an AciRilb protein is administered to a patient in an amount sufficient to maintain the desired serum concentration during the course of a therapeutic treatment. In certain circumstances, the administration schedule is sufficient to maintain the desired serum concentration of ActRilb between administration intervals (for example, between administrations of ActRilb).
. 54/65 According to the description in this patent application, the biological effects of ActRIlb polypeptides can be achieved with doses of 0.3 mg / kg or greater. Therefore, in certain modalities, a patient may receive a dose of approximately 0.3 to approximately 15 mg / kg (for example, 0.3; 0.5; 1.0; 1.5; 2.0; 3.0, 4.0, 5.0, 7.0, 10.0, 12.00u 15.0 mg / kg) of an ActRIlb polypeptide. In preferred embodiments, a patient is administered at least 1.0 mg / kg. In addition, the experiments shown below indicate that the serum half-life of an ActRIlb-Fc fusion protein is between about 10 and 16 days. Therefore, in certain aspects, a patient can receive the dose of an ActRilb protein of the invention at least once a month (for example, once every 5-30 days, 10-16 days, 5 days, 7 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days or 16 days; or monthly or every other month).
8. Pharmaceutical Compositions In certain embodiments, ActRilb antagonists (e.g., ActRilb polypeptides) of the present invention are formulated with a pharmaceutically acceptable carrier.
For example, an ActRilb polypeptide can be administered alone or as a component of a pharmaceutical formulation (therapeutic composition). The compounds in question can be formulated for administration in any manner convenient for use in human or veterinary medicine.
In certain embodiments, the therapeutic method of the invention includes administering the composition systemically or locally in the form of an implant or device. When administered, the therapeutic composition for use in this invention is in a physiologically acceptable form free of pyrogens. Therapeutically useful agents other than ActRilb antagonists, which can also optionally be included in the composition according to the above description, can be administered simultaneously or in sequence with the compounds in question (for example, ActRilb polypeptides) in methods of the invention.
Typically, ActRilb antagonists will be administered parenterally, and especially intravenously or subcutaneously. Pharmaceutical compositions suitable for parenteral administration may comprise one or more ActRilb polypeptides - combined with one or more aqueous or non-aqueous sterile isotonic dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions shortly before use, which may contain antioxidants, buffers, bacteriostats, solutes that make the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous or non-aqueous vehicles that can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, green oils
. 55/65 minerals, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.
In addition, the composition can be encapsulated or injected into shape to release to a tissue target site (e.g., bone or muscle). In certain embodiments, compositions of the present invention may include a matrix capable of releasing one or more therapeutic compounds (for example, ActRilb polypeptides) to a technical target site (for example, bone or muscle), providing a structure for developing tissue and ideally capable of being reabsorbed into the body. For example, the matrix may allow slow release of ActRilb polypeptides. These matrices can be formed by materials currently in use for other medical implant applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, aesthetic aspect and interface properties. The specific application of the compositions in question will define the appropriate formulation. Possible matrices for the compositions can be biodegradable and chemically defined with calcium sulfate 1, tricalciophosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other possible materials are biodegradable and biologically well defined, such as bone collagen or cardi | tilaginous. Additional matrices are composed of pure proteins or extracellular components of the matrix. Other possible matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates or other ceramics. The matrices can be formed by combinations of any of the types mentioned above, such as lactic acid and hydroxyapatite or collagen and tricalciophosphate. Bioceramics can be altered in composition, such as calcium-aluminate-phosphate, and be processed to alter the size - pore size, particle size, particle shape and biodegradability.
In certain embodiments, methods of the invention may be administered orally, for example, in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, in the form of a solution or suspension in an aqueous or non-aqueous liquid, in the form of a liquid emulsion — oil in water or water in oil, in the form of elixir or syrup, in the form of tablets (using an inert base such as gelatin and glycerin or sucrose and acacia) and / or in the form of mouthwash and the like, each containing a predetermined amount of an agent as an active ingredient. An agent can also be administered as a bolus, eletuary or paste.
In solid dosage forms for oral administration (capsules, tablets, pills, pills, powders, granules and the like), one or more therapeutic compounds of the present invention can be mixed with one or more pharmaceutically acceptable carriers
"56/65 products, such as sodium citrate or dicalcium phosphate and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silic acid; ( 2) aggregates, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate , potato or tapioca starch, alginic acid, certain salicates and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) moisturizing agents, such as ethyl alcohol, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, laurit sulfate sodium and mixtures thereof, and (10) coloring agents, in the case of capsules, tablets and pills , pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type can also be used as filling material for soft and hard filled gelatin capsules, using such excipients as lactose - or milk sugars, as well as high molecular weight polyethylene glycols and the like. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the prior art, such as water or other solvents, emulsifying solubilizing agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate , benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed, peanut, germ, olive, castor and sesame oils), glycerol, alcohol! tetrahydrofuryl, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions may include - adjuvants such as wetting, emulsifying and suspending agents, sweetening, flavoring, coloring, perfume and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents, such as ethoxylated stearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth and mixtures of these. it also contains adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. The prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, sorbic acid and the like. It may also be convenient to include isotonic agents, such as sugars, sodium chloride and the like in the compositions. Additionally, the prolonged absorption of the injectable pharmaceutical form can be caused by the inclusion of agents that delay absorption.
"57/65 tion, such as aluminum monostearate and gelatin.
It is understood that the dose schedule will be determined by the responsible physician, considering several factors that modify the action of the compounds in question of the invention (for example, ActRIlb antagonists). The various factors include, but are not limited to, the amount of bone weight desired to be formed, the degree of loss of bone density, the location of bone damage, the condition of the damaged bone, the age, sex and diet of the patient, gravity - the severity of any disease that may be contributing to bone loss, the time of administration and other clinical factors. Optionally, the dose can vary with the type of matrix used in the reconstruction and the types of compounds in the composition. The addition of other known growth factors to the final composition can also affect the dose. Progress can be monitored by periodic growth assessment and / or bone repair, for example, by radiological examinations (including DEXA), histomorphometric determinations and by tetracycline staining.
Experiments shown below demonstrate that the effects of ActRilb-Fc on bone can be achieved with a single dose of 1 mg / kg or greater. The observed serum half-life is between approximately 10 and 16 days. Therefore, a sustained effective serum level can be achieved, for example, by administering approximately 0.5 to 5 mg / kg weekly or every two weeks. For example, doses of 0.3; 0.5; 1, 2, 3: or 5 mg / kg, or intermediate values, could be used once every 7 days, once every 10 days, once every 15 days or monthly or every two months. Other exemplary administration schemes are provided below.
In certain embodiments, the present invention also provides gene therapy for the in vivo production of ActRilb polypeptides. This therapy would achieve its therapeutic effect by introducing ActRilb polynucleotide sequences into cells or tissues having the disorders listed above. ActRIlb polynucleotide sequences can be released using a recombinant expression vector, such as a chimeric virus or a colloidal dispersion system. The preferred method for the therapeutic release of ActRilb polynucleotide sequences is the use of targeted liposomes.
Several viral vectors that can be used for gene therapy as taught in this specification include adenovirus, herpes virus, Vaccinia virus or, preferably, an RNA virus like retrovirus. Preferably, the retroviral vector is derived from a murine or avian retrovirus. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMUuLV), Harvey murine sarcoma virus (HaMuSV), murine breast tumor virus - (MUuMTV) and sarcoma virus of Rous (RSV). Some additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or embed a seal to a selectable marker, so that transducid cells can be identified and
, 58/65 generated. The retroviral vectors produced for a specific target by fixing, for example, sugar, glycolipid or protein. The preferred target is achieved using an antibody. Those skilled in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to make it possible to target the specific release of the retroviral vector containing the ActRilb polynucleotide. In a specific modality, the vector is directed to bone or cartilage.
Alternatively, tissue culture cells can be directly transfected with plasmids encoding the structural retroviral genes gag, pol and env, by conventional transfection with calcium phosphate. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
Another release system targeted to ActRilb polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include complexes of macromolecules, nanocapsules, microspheres, spheres and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system of this invention is liposome. Liposomes are artificial membranous vesicles that are useful as a vehicle for in vitro and in vivo release. Intact RNA, DNA and virions can be encapsulated inside the aqueous interior and released to cells in a biologically active form (see, for example, Fraley et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfer using a liposome-type vehicle are known in the art, see, for example, Mannino et a., Biotechniques, 6: 682, 1988. The composition of the liposome is usually a combination of phospholipids, usually combined with steroids, especially cholesterol. Other phospholipids or other lipids - can be used as well. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
Examples of lipids useful in the production of liposomes include phosphatidyl compounds, such as phosphatidyldiglycerol !, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. The targeting of liposomes is also possible based on, for example, organ specificity, cell specificity and organelle specificity, and is known in the prior art.
Exemplification The invention in the process of general description will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain modalities and modalities of the present invention and are not intended to limit the invention.
"59/65 Example 1: ActRlIlb-Fc fusion proteins Applicants have constructed a soluble ActRIlb fusion protein that has the extracellular domain of human ActRIlb fused to mouse Fc domain with a | minimum (three glycine amino acids) medium The constructs are called ActRlilb-hFc and AcciRllb-mFc, respectively.
ActRilb-hFc is shown below as purified from CHO cell lines (SEQ ID NO: 13): GRGEAETRECIYYNANWELERTNQOSGLERCEGEQDKRLHCYASWRNSSGTIELV KKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAP TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPVPIEKTISKAK GOPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The ActRIlb-hFc and ActRIlb-mFc proteins were expressed in CHO cell lines. Three different leading sequences were considered: (i) Bee honey melitin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: ã 14) (ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP i (SEQ ID NO: 15) (iii ) Native: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 16).
The selected form employs the leading TPA sequence and has the following unprocessed amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQSGLER CEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGN —FCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQOQGNVESCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 17) This polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO: 18): ATGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCT GTGTGGAGCA GTCTTCGTIT CGCCCGGCGC CTCTGGGCEGT GGGGAGGCTG AGACACGGGA GTGCATCTAC. TACAACGCCA ACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCG CTGCGAAGGC GAGCAGGACA AGCGGCTGCA CTGCTACGCC TCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGA AGGGCTGCTG GCTAGATGAC TTCAACTGCT ACGATAGGCA GGAGTGTGTG GCCACTGAGG
- 60/65 AGAACCCCCA “GGTGTACTTC TGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTC ATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCC ACCCCCGACA “GCCCCCACCG GTGGTGGAAC TCACACATGC CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAG TCTTCCTCTT ccCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC “AAGGAGTACA AGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGA GAAAACCATC TCCAAMAGCCA AAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACT CCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA - CAANGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC GTGATGCATG
AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT AMATGA 1 The N-terminal sequencing of the material produced in CHO cells revealed an important -GRGEAE sequence (SEQ ID NO: 19). In particular, other constructs reported in the literature start with a sequence -SGR ....
Purification could be carried out by a series of column chromatography steps, including, for example, three or more of the following in any order: chromatography with protein A, chromatography with Q sepharose, chromatography with phenyl sepharose, chromatography of size exclusion and cation exchange chromatography. The chromatography could be completed with viral filtration and buffer exchange.
ActRIlb-Fc fusion proteins were also expressed in HEK293 cells and COS cells. Although the material of all cell lines and reasonable culture conditions provided protein with muscle building activity in vivo, variability in potency was observed, perhaps related to the selection of cell line and / or culture conditions. Purification was carried out as presented in the specification above.
Example 2: Generation of mutant ActRIlb-Fc Applicants generated a series of mutations in the extracellular domain of ActRI-lb and produced these mutant proteins as soluble fusion proteins between extracellular ActRilb and an Fc domain. The basic ActRIlb-Fc fusion has the sequence (underlined Fc portion) (SEQ ID NO: 20): SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIEL VKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTA PTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
- 61/65 VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK Several mutations, including N and C-terminal truncation, have been introduced in the basic ActRIlb-Fc protein. Based on the data presented in Example 1, it is predicted that these constructs, if expressed with a TPA leader sequence, will be devoid of the N-terminal serine. Mutations were generated in the extracellular domain of ActRilb by PCR mutagenesis. After PCR, the fragments were purified through a Qiagen column, digested with Sfol and Agel and purified on gel. These fragments were ligated into the —pAIDA4 expression vector (see WO2006 / 012627), such that, upon ligation, a human IgG1 fusion chimera was created. Upon transformation into E. coli DH5 alpha, colonies were collected and DNAs were isolated. For mouse constructs (mFc), a murine IgG2a has been substituted for human I9G1. The sequences of all mutants were verified. All mutants were produced in HEK293T cells by transient transfection. Briefly, in a 500 ml spinner flask, HEK293T cells were established at 6x10º cells / mlL in Freestyle medium (Invitrogen) in 250 ml volume and grown overnight. The following day, these cells were treated with E DNA: PEI complex (1: 1) at 0.5 µg / ml final DNA concentration. After 4 hours, 250 ml of meiofor was added, and the cells were cultured for 7 days. Conditioned medium was collected by reducing cell rotation and was concentrated. The mutants were purified using a variety of techniques, including, for example, protein A column, and eluted with low pH (3.0) glycine buffer. After neutralization, they were dialyzed against PBS. Mutants were also produced in CHO cells by a similar methodology. The mutants were tested in binding assays and / or bioassays described below. In some situations, the tests were carried out with conditioned medium, instead of purified proteins. Example 3. GDF-11 and activin-mediated signaling bioassay An A-204 reporter gene assay was used to evaluate the effects of ActRllb-Fc proteins on GDF-11 and Activivin A signaling. Cell line: Human rhabdomyosarcoma (derived from muscle). Reporter vector: PGL3 (CAGA) 12 (Described in Dennler et al, 1998, EMBO 17: 3091-3100). See Figure 1. The CAGA12 motif is present in genes that respond to TGF-Beta (PAI-1 gene), so this vector is in use — general for signaling factors through Smad2 and 3. Day 1: Sharing A-204 cells in a 48-well plate. Day 2: A-204 cells transfected with pGL3 (CAGA) 12, 10 µg, or pGL3 (CAGA) 12
. 62/65 (10 ug) + pRLOMV (1 ug) and Fugenium.
Day 3: Add factors (diluted in medium + 0.1% BSA). Inhibitors need to be pre-incubated with Factors for 1 hour before being added to the cells. Six hours later, cells washed with PBS, and cell lysis.
This is followed by a luciferase assay. In the absence of any inhibitors, Ativin A showed stimulation of 10-fold reporter gene expression and EDs5, - 2 ng / ml. GDF-11: 16-fold stimulation, EDs9: - 1.5 ng / ml.
ACtRIID (R64, 20-134) is a potent inhibitor of activin, GDF-8 and GDF-11 activity in this assay. Variants were also tested in this trial.
Example 4: Single ascending human dose clinical study The ActRIIb (20-134) -hFc protein described in Example 1 was administered to human patients in a randomized, double-blind, placebo-controlled study that was conducted to assess primarily the protein safety in healthy postmenopausal women. Forty-eight participants were randomized to six cohorts of 8 each to receive a single dose of ActRIlb-hFc or placebo (6 for active agent: 2 for placebo). Dose levels ranged from 0.02 to 3.0 mg / kg, administered subcutaneously (SC). All participants were followed for 57 days. Participants were excluded from participation in the study if they took medications that affected bone metabolism within 6 months of inclusion in the study. Safety assessments were conducted, following each cohort, to determine the gradual increase in dose. In addition to pharmacokinetic analyzes (PK), the biological activity of ActRilb-hFc was also assessed by measuring biochemical markers of bone formation and resorption and FSH levels.
No serious adverse events were reported in this study.
The PK analysis of ActRilb-hFc showed a linear profile with the dose and mean half-life of approximately 10 - 16 days (see Figure 2). The area under the curve (AUC) for ActRI-Ib-hFc was linearly related to the dose, and absorption after SC administration was essentially complete. At higher doses, ActRIlb-hFc caused an increase in lean body mass (LBM), as demonstrated using dual energy X-ray absorptiometry (DXA) tests (see Figure 3). The average increase in LBM, when compared to baseline, was 2.6% (0.92 kg) in two months after the dose at 3 mg / kg. The placebo-treated group decreased by 0.2%. More participants at the higher doses (1 and 3 mg / kg) showed at least a 0.5 kg increase in LBM as early as 15 days after the administration of ActRilb-hFc, which was sustained until Day 57, when compared placebo and lower doses (see Figure 4). In patients in whom doses of AcTRilb-hFoa1e3 mgkg were administered, significant dose-dependent changes were measured in serum biomarkers of fat metabolism (increased adiponectin and reduced leptin) (see Figures 5 and 6). ActRilb-hFc caused a rapid sustained increase due to
. 63/65 dose pending in serum levels of specific bone alkaline phosphatase (BSAP), which is a marker of anabolic bone growth, and dose-dependent decrease in type 1 collagen C-terminal telopeptide (CTX), which is a marker for bone resorption (see Figures 7 and 8). BSAP levels revealed effects close to saturation at the highest dose of the drug, indicating that half of the maximum effects on this bone anabolic biomarker could be achieved at a dose of 1 mg / kg or lower. These changes in bone biomarkers were sustained for approximately 57 days at the highest dose tested levels. There was also a dose-dependent decrease in serum FSH levels, compatible with activin inhibition (see Figure 9).
Increases in thigh muscle volume were observed by magnetic resonance imaging (MRI) at 29 days after single doses of ActRilb-hFc at 1 and 3 mg / kg, with a significant increase at the highest dose (3 mg / kg), when compared to placebo (see Figure 10). In particular, in 29 days after the administration of a single dose of 3 mg / kg of ActRllb-hFc, a 16.4% increase in muscle area, 11.5% increase in muscle volume and 2.2% decrease in subcutaneous fat. The mean percentage change from baseline in thigh muscle volume at Day 29 with dose levels of 1 and 3 mg / kg, compared to placebo, is shown in Figure 11, A single dose of ActRllb-hFc delivered to healthy women in post-menopause was safe and well tolerated for the range of tested dose levels. This clinical study demonstrates that, in humans, ActRilb-hFc is an osteoanabolic agent with biological evidence of increased bone formation and decreased bone resorption. ActRilb-hFc also increases muscle size and function.
Example 5: Human clinical study of multiple ascending doses A randomized, double-blind, placebo-controlled, multiple dose, gradual increase study was conducted to primarily assess the safety, tolerance, PK and PD effects of ActRllb- hFc in healthy postmenopausal women. The study included six cohorts of 10 participants each. Participants in each cohort were ranked to receive ActRilb-hFc or placebo (8 participants for active agent and 2 for placebo per cohort).
The cohorts were administered as follows: Cohort 1 (0.1 mg / kg), Cohort 2 (0.3 mg / kg) and Cohort 3 (1 mg / kg) received ActRIlb-hFc subcutaneously (SC) every 14 days, totaling 3 doses on Days 1, 15 and 29. Cohort 4 (1 mg / kg), Cohort 5 (2 mg / kg) and Cohort 6 (3 mg / kg) received ActRllb-hFc SC every 28 days, totaling 2 doses on Days 1 and 29.
The following data were evaluated: adverse events, laboratory tests (hematology, biochemistry, urinalysis, endocrine function tests, adrenal function tests, ACTH stimulation tests), vital signs including supine blood pressure (de-
. 64/65 after at least 5 minutes) and standing (after 2 minutes + 30 seconds), ECG, echocardiogram (ECHO), physical exams and anti-drug antibodies. Pharmacokinetics: area under the ActRilb-hFc serum concentration curve (AUC), peak concentration (Cmax), time to peak concentration (Tmax), elimination half-life (t1 / 2), clearance (CI / F) and volume of distribution (VZ / F). Pharmacodynamics: FSH, lean body mass, measured by DXA examinations of total body, bone mineral density (BMD), measured by DXA examinations of the lumbar spine, hips and total body, muscle size, measured by MRI examinations in Cohorts 3 -6, and other markers of pharmacodynamic effect. As shown in Figure 14, serum concentrations of ActRllb-hFc were related to the amount and frequency of the dose. Notably, the doses of 2 mg / kg and 3 mg / kg, delivered every 28 days, reached high concentration peaks with concentrated troughs. The dose of 1 mg / kg delivered every 14 days reached an increasing concentration with smaller valleys. After the second dose, the 1 mg / kg dose, delivered every 14 days, maintained an average serum concentration in patients above approximately 12 micrograms-mL / mL, while the doses of 2 mg / kg and 3 mg / kg , supplied every 28 days, showed vouchers for an average of approximately 8 and 10 micrograms / mL, respectively. As shown: in Figure 15, the valleys in serum concentration were reflected in effects on FSH. FSH production is believed to be stimulated by activins and therefore the use of an activin antagonist, such as ActRilb-hFc, is expected to inhibit FSH production. In fact, all doses of ActRIlb-hFc exhibited some inhibition of FSH. This is shown by the dashes falling below the X axis in Figure 15. Interestingly, the dose of 1 mg / kg, delivered every 14 days, did not show an average decrease in FSH inhibition during the administration period, while the doses of 2 mg / kg and 3 mg / kg, supplied every 28 days, exhibited a substantial increase in FSH production (consequently, decrease in FSH suppression) which corresponded to the valley in serum concentration (approximately between Days 15 and 36 of the study). We conclude that the administration of ActRIlb-hFc proteins in a way that allows the serum concentration to drop by up to 8 or 10 micrograms / mL causes a transient release in the suppression of activin signaling by the drug and, perhaps, a transient release in the suppression of other binders, ok! like myostatin or GDF-
11. Namely, the dose of 1 mg / kg, delivered every 14 days seemed to cause a greater increase in muscle mass, as measured by total lean body mass (see Figure 16), than less frequent doses of 2 mg / kg and 3 mg / kg. While patients at doses of 2 mg / kg and 3 mg / kg, delivered every 28 days, received a total of 4 or 6 mg / kg of drug during the study, patients at a dose of 1 mg / kg, delivered every 14 days days, they received a total of only 3 mg / kg of drug during the study. However, the lowest and most frequent dose provided a greater apparent effect on muscle mass (shown in this study as a trend without reaching statistical significance). It is expected that
. 65/65 that higher efficacy at a lower dose is due to the more sustained suppression of ligands that occurs with the most frequent administration schedule, as demonstrated by the effects on FSH.
As a consequence, it is anticipated that an administration regimen that maintains a serum concentration above approximately 8, 10 or 12 micrograms per ml will provide optimal efficacy for drugs based on the mode of action of AcRIRI-hFc.
This conclusion should apply to a wide range of ActRilb-hFc proteins, regardless of serum half-life, as long as those proteins have an affinity for activins and myostatin / GDF-11 that are in the same range as for wild-type ActRllb-hFc.
Incorporation by reference All publications and patents cited in this patent application are incorporated here, in their entirety, as they would be if each publication or patent had been individually and specifically indicated to be incorporated by reference in this patent application.
Although object-specific modalities have been discussed, the above descriptive report is illustrative and not restrictive.
Many variations will become evident to those versed in the state of the art when reviewing this specification and the claims below.
The full scope of the invention must be determined by reference to the E claims, together with the full scope of their equivalents, and the specification, together with such variations.

权利要求:
Claims (14)
[1]
1. Use of an ActRllb-Fc fusion protein, CHARACTERIZED because it is in the manufacture of a medicine to maintain the serum concentration of the ActRllb-Fc fusion protein at least 8 µg / ml. in a patient.
[2]
2. Use, according to claim 1, CHARACTERIZED by the fact that the serum concentration of the ActRIlb-Fc fusion protein is maintained at a concentration of between 8 and 70 µg / mL.
[3]
3.Use, according to claim 1, CHARACTERIZED by the fact that the medication is administered in at least 0.3 - 5 mg / kg of ActRilb-Fc fusion protein to the patient.
[4]
4. Use, according to any one of claims 1 to 3, CHARACTERIZED by the fact that the drug is administered to the patient once every 5 to 30 days.
[5]
5. Use according to any of the claims of | to 4, CHARACTERIZED by the fact that the patient is suffering from a disorder related to the skeletal system.
[6]
6. Use, according to any one of claims 1 to 4, CHARACTERIZED by the fact that the patient suffers from cancer.
[7]
7. Use, according to claim 6, CHARACTERIZED by the fact that the patient is suffering from breast cancer.
[8]
8. Use, according to any one of claims 1 to 4, CHARACTERIZED by the fact that the patient is suffering from a disorder associated with muscle loss caused by insufficient muscle growth.
[9]
9. Use according to any one of claims 1 to 8, CHARACTERIZED by the fact that the ActRllb-Fc fusion protein is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, 3 or 13; b) a polypeptide comprising an amino acid sequence at least 95% identical to SEQIDNO: 2.3 or 13; c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 3 or 13; d) a polypeptide comprising at least 50 consecutive amino acids of SEQIDNO: 2; and e) a polypeptide comprising an amino acid sequence at least 90% identical to a part of ActRilb, where the N-terminal corresponds to an amino acid residue selected from amino acids 19 - 25 of SEQ ID NO: 1 and where the C-terminal corresponds to an amino acid residue selected from amino acids 109 - 134 of SEQ ID NO: 1; f) a polypeptide comprising an amino acid sequence at least 95% identical to a part of ActRllb, where the N-terminus corresponds to an amino acid residue selected from amino acids 19 - 25 of SEQ ID NO: 1 and where the C-terminal corresponds to an amino acid residue selected from amino acids 109-134 of SEQIDNO: 1; eg) a polypeptide comprising a part of ActRilb, where the N-terminal corresponds to an amino acid residue selected from amino acids 19 - 25 of SEQ IDNO1e where the C-terminal corresponds to an amino acid residue selected from amino acids 109-134 of SEQ ID NO: 1.
[10]
10. Use according to any one of claims 1 to 9, CHARACTERIZED by the fact that the ActRllb-Fc fusion protein has one or more of the following characteristics: Bind to an ActRIlb ligand with a Kp of at least 10 M; and Inhibit ActRIilb signaling in a cell.
[11]
11. Use according to any one of claims 1 to 10, CHARACTERIZED by the fact that the ActRIlb-Fc fusion protein includes one or more modified amino acid residues, selected from: glycosylated amino acid, PE-Guided amino acid, farnesylated amino acid, acetylated amino acid, biotinylated amino acid, amino acid conjugated to a lipid group and amino acid conjugated to an organic derivatizing agent.
[12]
12. Use according to any one of claims 1 to 11, CHARACTERIZED by the fact that the ActRIlb-Fc fusion protein has a serum half-life between 10 € and 16 days in normal, healthy humans.
[13]
13. Use, according to claim 1, CHARACTERIZED by the fact that the medication is administered to the patient with 0.3 to 5 mg / kg of ActRllb-Fc once every 5 to days.
[14]
14. Use of an ActRIlb-Fc fusion protein CHARACTERIZED because it is in the manufacture of a medicine to: (i) promote anabolic bone growth or (ii) treat bone tumor.

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法律状态:
2020-09-15| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-10-27| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-02-23| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-04-20| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-10-05| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2022-02-01| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US27628709P| true| 2009-09-09|2009-09-09|

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US61/276.287|2009-09-09|

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PCT/US2010/048322|WO2011031901A1|2009-09-09|2010-09-09|Actriib antagonists and dosing and uses thereof|

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