Compound for improving muscle performance

Abstract

A compound comprising at least two components, a first component being the nLG3 domain from the C-terminus of human agrin, and at least one second component being selected from a protein or an antagonist antibody that inhibits ActR2B-induced signaling activity in the presence of myostatin, wherein the components are linked by means of a linking element, which compound is an effective treatment for neuromuscular diseases and problems.

Description

Technical Field

[0001] The present disclosure relates to methods for treating pathological disorders or diseases that affect muscle function and compounds for use in such treatments. The methods are particularly suitable for treating, preventing, alleviating, or diagnosing pathological disorders.

[0002] Muscle is a contractile tissue that is responsible for all of the movements of a living organism. Loss of muscle function is more or less invariably harmful. Important components of muscle function are strength, power, and endurance. Muscle strength is the amount of force that a muscle or group of muscles can exert when they contract maximally, generally against an external load. Muscle strength is expressed as the maximum measurable force that can be exerted by a muscle or muscle group to overcome resistance during a single maximal effort. Muscle power is the force that is generated rapidly and combines strength and speed. It is the speed of a performance task. Muscle endurance is the ability of a muscle or group of muscles to hold in an extended fashion or to repeatedly exert force against resistance.

[0003] These performances can be evaluated by the normal assessment of a subject in a test regime such as an exercise machine. The most important is the loss of muscle endurance. For example, improvement in a six-minute walking test or "6MWT" (see, for example, Bautmans et al (BMC Geriatr. 2004 Jul 23;4:6) and Enright (Respir Care. 2003 Aug;48(8):783-5)) is an essential requirement for approval by regulatory authorities such as the US Food and Drug Administration for drugs intended to treat pathological disorders that affect muscle function.

[0004] There are numerous pathological disorders that can lead to the loss of muscle function. As used herein, "pathological disorder" includes, but is not limited to, neuromuscular diseases. Neuromuscular disorders are a very broad term encompassing a number of diseases and conditions that impair muscle function, either directly as a disorder of voluntary muscle or indirectly as a disorder of either the nerve or the neuromuscular junction.

[0005] One pathological disorder that can lead to the loss of muscle function is muscular atrophy. There are numerous causes of muscular atrophy, including the results of treatment with glucocorticoids such as cortisol, dexamethasone, betamethasone, prednisone, methylprednisolone, or prednisolone. Muscular atrophy can also be the result of denervation due to nerve trauma, or the result of degenerative, metabolic, or inflammatory neuropathies (examples include Guillain - Barré syndrome, peripheral neuropathy, or exposure to environmental toxins or drugs).

[0006] Furthermore, muscular atrophy can be the result of myopathies, including myotonia; congenital myopathies including nemaline myopathy, multi / minicore myopathy, and myotubular (centronuclear) myopathy; mitochondrial myopathy; familial periodic paralysis; inflammatory myopathy; metabolic myopathy such as that caused by glycogen or lipid storage diseases; dermatomyositis; polymyositis; inclusion body myositis; myositis ossificans; and myopathies such as rhabdomyolysis and myoglobinuria.

[0007] Myopathy may be caused by muscular dystrophy diseases such as Duchenne type, Becker type, myotonic, facioscapulohumeral type, Emery - Dreifuss type, oculopharyngeal type, scapulohumeral type, limb - girdle type, Fukuyama type, congenital muscular dystrophy, or hereditary distal myopathy. Musculoskeletal disorders can also be osteoporosis, fractures, short stature, or dwarfism.

[0008] Other pathological disorders that can lead to loss of muscle function are adult motor neuron disease, infantile spinal muscular atrophy, amyotrophic lateral sclerosis, juvenile spinal muscular atrophy, autoimmune motor neuropathy with multifocal conductor block, paralysis due to stroke or spinal cord injury, skeletal immobilization due to trauma, prolonged bed rest, voluntary inactivity, involuntary inactivity, metabolic stress or nutritional deficiency, cancer, AIDS, starvation, thyroid disorders, diabetes, benign congenital muscle hypotonia, central core disease, burns, chronic obstructive pulmonary disease, liver diseases (such as fibrosis, cirrhosis examples), sepsis, renal failure, congestive heart failure, aging, time spent in a zero gravity environment, or space travel.

[0009] Examples of age-related conditions that may be treated include sarcopenia, dermal atrophy, muscle wasting, cerebral atrophy, atherosclerosis, arteriosclerosis, emphysema, osteoporosis, osteoarthritis, immunologic incompetence, hypertension, dementia, Huntington's disease, Alzheimer's disease, cataracts, age-related macular degeneration, prostate cancer, stroke, decreased life expectancy, frailty, memory loss, wrinkles, renal dysfunction, and age-related hearing loss; metabolic disorders including type 2 diabetes, metabolic syndrome, hyperglycemia, and obesity. Of course, a patient may be concurrently affected by one or more of these conditions, for example sarcopenia and emphysema, or sarcopenia and renal dysfunction.

[0010] Other conditions considered to be "pathological conditions" as described herein include acute and / or chronic kidney disease or renal failure, liver fibrosis or cirrhosis, cancers such as breast cancer, Parkinson's disease; ALS, cerebral atrophy, or dementia and conditions related to nerve cell death such as anemia. Further, there are losses suffered as a result of age, trauma or inactivity.

[0011] Additional conditions include cachexia, cachexia associated with rheumatoid arthritis, and cachexia associated with cancer.

[0012] To date, few reliable or effective treatments have been developed to treat these disorders. Individual aspects of muscle problems have been addressed. For example, one potential means of treatment for loss of muscle mass is inhibition of myostatin. Myostatin, sometimes referred to as GDF-8 (Growth Differentiation Factor 8), is a member of a family of dimeric growth and differentiation factors that belong to the transforming growth factor beta (TGF-beta) superfamily of structurally related signaling proteins. These proteins signal through a heterodimeric complex of receptor serine kinases that includes at least two type I receptors, ActRIB (ALK4) and ActRIC (ALK7), and two type II receptors, ActRIIA (ACVR2A) and ActR2B (ACVR2B). All of these receptors are transmembrane proteins and are composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with putative serine / threonine specificity. The type I receptor is essential for signaling, while the type II receptors are required for ligand binding and expression of the type I receptor. The type I and type II receptors form a stable complex after ligand binding, resulting in phosphorylation of the type I receptor by the type II receptor.

[0013] Activin receptor IIB (ActR2B) is a receptor for myostatin (GDF8), but many other members of the TGF-beta superfamily, such as activin B, activin AB, inhibin A, inhibin B, GDF3, GDF11, Nodal, BMP2, BMP4, BMP7, BMP9, and BMP10, also bind to and activate ActR2B (see, for example, Tsuchida et al (Endocrine journal 2008 55(1), 11-21)). Blocking the interaction between ActR2B and its ligands can lead to beneficial physiological effects. The interaction between myostatin and this receptor regulates the inhibition of skeletal muscle differentiation via the Smad-dependent pathway. (SMADs are intracellular proteins that transmit extracellular signals from transforming growth factor-beta ligands, such as myostatin, to the nucleus, and these activate downstream gene transcription in the nucleus.) Thus, inhibiting or preventing the binding of myostatin to ActR2B can induce skeletal muscle formation.

[0014] Various groups have investigated this. Bogdanovich et al (Nature, 2002, 420:418-421) reported that an anti-myostatin antibody was able to block myostatin and produced an increase in muscle mass in a mouse model of Duchenne muscular dystrophy. Bradley et al (Cell Mol. Life. Sci. 2008, 65:2119-2124) considered different available approaches for regulating the myostatin / ActR2B interaction, including administering myostatin propeptide to inhibit the release of mature myostatin, administering follistatin to block the myostatin receptor, administering an HDAC inhibitor to induce follistatin production, administering an altered myostatin peptide that prevents myostatin from binding to the receptor, and administering a soluble decoy receptor for myostatin.

[0015] Myostatin acts to inhibit muscle fiber growth and muscle stem cell proliferation. Studies have shown that animals that either lack myostatin or are treated with substances that block the activity of myostatin have significantly larger muscle mass. Furthermore, individuals with mutations in both copies of the myostatin gene have significantly increased muscle mass and, in many cases, are stronger than normal. In certain cases, as described in N Engl J Med 2004; 350:2682-2688 (June 24, 2004), infants born with abnormally developed muscles have been found to have a deficiency of myostatin, presumably due to a defect in the gene responsible for its production.

[0016] A number of recent publications have attempted to utilize this as a treatment, as follows by way of example: - Myostatin-binding proteins such as ActR2B polypeptides (e.g., US7842663 and US8614292), - Follistatin (e.g., US6004937) - Anti-ActR2B antibodies (e.g., US 8,551,482) - Anti-myostatin antibodies (e.g., US7261893, US8063188, US8415159, US8551482 and US8710212) - Anti-activin antibodies (e.g., US 20150037339 A1, WO 2009137075 A1)

[0017] Muscle hypertrophy and / or atrophy are not the whole story. Another factor is denervation. Muscle contraction is caused by action potentials from nerves via the release of electrical and chemical messages. Nerves also provide muscle fibers with a number of trophic factors that are essential for muscle well-being and proper function. The connection between nerves and muscles occurs via a highly complex synaptic structure called the neuromuscular junction (NMJ). Loss of the NMJ results in a decrease in muscle function, independent of the anatomical and biochemical integrity of the muscle itself.

[0018] Some approaches have focused on preventing the loss of the neuromuscular junction. For the proper maintenance of the NMJ, for example, Agrin has been shown by Wu et al (Development. 2010 Apr;137(7):1017-33. doi: 10.1242 / dev.038711) and Tezuka et al (Proc Natl Acad Sci U S A. 2014 Nov 18;111(46):16556-61) to be important for their formation and maintenance of the neuromuscular junction.

[0019] However, it has been found that even the combination of treatments aimed at maintaining increased muscle mass and the NMJ does not prevent the loss of muscle performance (measured by the performance of appropriately treated mice on a treadmill). In short, there are numerous ways to improve the individual aspects of muscle problems, but nothing currently exists that can counter the overall loss of muscle performance.

[0020] A particular group of linked proteins, polypeptides and monoclonal antibodies have now been found to produce exceptional results when improving muscle performance. Accordingly, a compound is provided that comprises at least two components, the first component being human or mouseThe nLG3 domain from the C-terminus of agrin, and at least one second component is selected from a protein or antagonist antibody that inhibits ActR2B-induced signaling activity in the presence of myostatin, and these components are linked by means of a linking entity.

[0021] There is further provided a method for improving muscle performance, which comprises administering an effective amount of a compound as described above herein.

[0022] "Improving muscle performance" means that muscle durability is particularly improved. The compounds as described above herein do not provide much more muscle mass (myostatin) and do not efficiently cluster acetylcholine receptors (at least 100 - 1000 times lower than the fully active agrin fragment), but it is a surprising fact that the overall muscle performance is significantly improved when measured by durability. This can be demonstrated by the experimental methods as described below herein.

[0023] For amino acid sequences suitable for use in the present disclosure and described in the present disclosure, the sequences should be at least 75% identical to the sequences shown below. More specifically, they may be 80%, 85%, 90% or 95% identical, and most specifically 95% identical. In the case of inserts in agrin (described in detail below herein), the inserts should be at least 95% identical. In certain embodiments, all amino acid sequences are at least 95% identical.

[0024] The first component is the nLG3 domain from the C-terminus of agrin. Agrin is a large heparan proteoglycan with a molecular weight of 400 - 600 kDa. (Database accession number NP-940978). The protein core consists of approximately 2000 amino acids and its mass is approximately 225 kDa. It is a multi-domain protein composed of 9 K (Kunitz-type) domains, 2 LE (laminin-EGF-like) domains, 1 SEA (sperm protein, enterokinase, and agrin) domain, 4 EG (epidermal growth factor-like) domains, and 3 LG (laminin globular) domains. Agrin is a very important protein, and agrin-deficient mice die at birth due to respiratory failure. This is caused by the fact that agrin is strictly required for proper innervation of muscle fibers, and these mice are unable to construct proper NMJs.

[0025] Agrin exists in several splice variants and can be expressed as a secreted protein, containing the N-terminal NtA (N-terminal agrin) domain, which is the most abundant form of agrin and the main form expressed in motor neurons. It is produced in the cell body of neurons, transported through axons, and released from the axon terminals of motor nerves into the synaptic cleft of the NMJ. Here, it acts as an agonist of LRP4 and may also serve as a component of the basal lamina. In the CNS, most agrin is expressed as a type II transmembrane protein by alternative splicing at the N-terminus, lacking the N-terminal NtA domain (Bezakova and Ruegg, 2003).

[0026] The serine / threonine (S / T)-rich segment in agrin is responsible for high levels of glycosylation and contains several glycosylation sites and glucosaminoglycan attachment sites that give rise to large-mass proteoglycans. The 75 kDa C-terminal portion of agrin starting from the first EG domain is required for sufficient activity for the clustering activity of the acetylcholine receptor (AChR) in muscle cells, although the most C-terminal 20 kDa fragment is sufficient to induce AChR aggregation (Bezakova and Ruegg, 2003). Several binding sites for interaction partners of agrin, including α-dystroglycan, heparin, several integrins and LRP4, map to the C-terminal region. Large heparan sulfate side chains are binding sites for heparin-binding proteins, such as several growth factors.

[0027] At the C-terminus of human agrin, there are two alternative splicing sites, y and z. At the y site, there may be an insert of 0, 4, 17, or 21 (4 + 17) amino acids, and at the z site, there may be an insert of 0, 8, 11, or 19 (8 + 11) amino acids. The function of the four inserted amino acids at the y site is to create a heparin-binding site. Motor neurons mainly express y4 agrin. The most important agrin splicing site, from the perspective of NMJ maturation, is the z site, which confers the ability to be active as an acetylcholine receptor clustering agent on agrin. In the presence of the 4-amino acid insert at the splicing site y, full-length agrin (y4z8) containing the 8-amino acid insert at the z site produces an agrin variant that has a maximum half-maximal AChR clustering activity of 35 pM in the cultured myotube clustering assay. The 11-amino acid insert results in a maximum half-maximal AChR clustering activity, while the 19-amino acid insert results in a maximum half-maximal AChR clustering activity of 110 pM. Agrin without an insert at this site is not active in clustering acetylcholine receptors on in vitro cultured myotubes (Bezakova and Ruegg, 2003). Thus, the most active form of agrin in the clustering assay is the y4z8 variant, which is expressed by motor neurons.

[0028] The 40 kDa C-terminal fragment of agrin (y4z8) containing the LG2, EG4, and LG3 domains is active in AChR clustering with an EC50 of 130 pM in AchR clustering activity, but shorter fragments were found to have only lower activity. The C-terminal LG3 domain with the z8 insert, the so-called LG3z8 domain, exhibits a maximum half-maximal AchR clustering activity of only 13 nM, which is a factor 100-fold lower than that of the 40 kDa fragment.

[0029] During the development and maturation of the NMJ, agrin is an important molecular player involved in the clustering of acetylcholine receptors. The NMJ is destabilized by the neurotransmitter acetylcholine, but agrin secreted by motor neurons stabilizes and increases the clustering of AChRs via the phosphorylation of MuSK, a membrane-bound receptor tyrosine kinase. The interaction between agrin and MuSK is hypothesized to be mediated via LRP4, a low-density lipoprotein receptor (LDLR)-related protein. Agrin (y4z8) has a 10-fold higher affinity for LRP4 than agrin (y4z0), and has been found to give rise to differential AChR clustering activity of different agrin splicing variants observed in in vitro cultured myotube assays. Upon agrin binding, LRP4 gives rise to autophosphorylation of MuSK, which in turn activates a signaling cascade for the expression and clustering of acetylcholine receptors. The 44kD fragment of agrin has been found to direct the formation of clusters of acetylcholine receptors on the surface of muscle cells (see Hettwer et al (PLOS ONE February 2014. Vol.9, Issue 2, e88739)), which is thought to be the first step in the formation of the NMJ.

[0030] As used in this disclosure, the term "LG3" means the 22kDa C-terminal agrin fragment from mouse shown in SEQ ID NO:1 (all sequences are attached to this disclosure and form part thereof). As used in this disclosure, the term "nLG3" means an LG3 fragment that further contains an insertion of 8, 11 or 19 amino acids at the z-site. The sequences inserted at the z-site are ELTNEIPA (z8, SEQ ID NO:2), PETLDSRALFS (z11, SEQ ID NO:3) or ELTNEIPAPETLDSRALFS (z19, SEQ ID NO:4, a combination of SEQ ID NO:2 and SEQ ID NO:3). An example of nLG3 is SEQ ID NO:5. human or

[0031] ​As used herein, the term "(h)LG3" means the 22 kDa C-terminal agrin fragment of human origin shown in SEQ ID NO: 6. As used herein, the term "(h)nLG3" means an (h)LG3 fragment that further contains an insertion of 8, 11, or 19 amino acids at the z-site. The sequences ELANEIPV (z8, SEQ ID NO: 7), PETLDSGALHS (z11, SEQ ID NO: 8), or ELANEIPVPETLDSGALHS (z19, SEQ ID NO: 9, the combination of SEQ ID NO: 7 and SEQ ID NO: 9) inserted at the z-site. A specific example of (h)nLG3 is SEQ ID NO: 10.

[0032] nLG3 may contain additional amino acids at the N-terminus or C-terminus. Such additional amino acids at the N-terminus are present, for example, due to methods of preparation by recombinant synthesis or expression in suitable cells.

[0033] Also included are proteins containing an N-terminal extension by one or more agrin domains up to the natural N-terminus of agrin, as well as protein variants of human agrin that are glycosylated or otherwise enzymatically or chemically modified post-translationally.

[0034] The second component is selected from a protein or an antagonist antibody that inhibits ActR2B-mediated signaling activity in the presence of myostatin. Examples of the second component as used herein refer to proteins or antagonist antibodies such as actR2B (AcvRIIB, actRIIB), or acvRA (actR2, actRII), alk4, alk5. The term ActR refers to the soluble extracellular portion of the mouse ActR2B receptor as defined in SEQ ID NO: 11. This extracellular portion is any part of the transmembrane protein that protrudes into the environment surrounding the cell. The term (h)ActR refers to the extracellular portion of the human ActR2B receptor as defined in SEQ ID NO: 12 (AAC64515.1, GI: 3769443). Another example is follistatin as defined in SEQ ID NO: 25.

[0035] Examples of monoclonal antibodies (mAbs) that inhibit ActR2B-mediated signal transduction activity include ActRmAb (US8551482). Antibodies consist of a light chain (LC) and a heavy chain (HC). A typical example of the LC of ActRmAb (ActRmAb(LC)) is as defined in SEQ ID NO: 29. The HC of ActRmAb (ActRmAb(HC)) is as defined in SEQ ID NO: 28. The (h)nLG3 connected to ActRmAb(HC) (ActRmAb(HC)-(h)nLG3) is as defined in SEQ ID NO: 30. Another example of an antibody is MyomAb (US8063188). This antibody is directed against myostatin and prevents the binding of myostatin to the (h)ActR receptor. A typical example of the LC of MyomAb (MyomAb(LC)) is as defined in SEQ ID NO: 32. The HC of MyomAb (MyomAb(HC)) is as defined in SEQ ID NO: 31. The (h)nLG3 connected to MyomAb(HC) (MyomAb(HC)-(h)nLG3) is as defined in SEQ ID NO: 33.

[0036] This ActR2B-mediated signal transduction inhibitory activity of the mAb may be readily confirmed by means of an assay. Such assays may include, by way of example, a Smad-dependent receptor gene assay, inhibition of myostatin-induced Smad phosphorylation (P-Smad ELISA), and inhibition of myostatin-induced inhibition of skeletal muscle cell differentiation (e.g., by a creatine kinase assay).

[0037] In some embodiments, the second component inhibits myostatin-induced signal transduction when measured in a Smad-dependent receptor gene assay with an IC50 of 10 nM or less, 1 nM or less, or 100 pM or less.

[0038] In some cases, the compounds of the present disclosure can include additional components, meaning that there may be three components joined by two linking elements. The third component acts as a stabilizing component, i.e., it increases the in vitro serum half-life. This may be the result of a decrease in kidney destruction, a decrease in clearance, or other pharmacokinetic effects. "In vivo serum half-life" refers to the half-life of a protein circulating in the blood of an organism. Fusion with the Fc region of an immunoglobulin (IgG molecule) is known to confer desired pharmacokinetic properties and increase the serum half-life of a wide range of proteins. The term "Fc region of an IgG molecule" refers to the Fc domain of an immunoglobulin of isotype IgG, as is well known to those skilled in the art. The Fc region of an IgG molecule is the part of the IgG molecule (IgG1, IgG2, IgG3, and IgG4) responsible for the increase in the in vivo serum half-life of the IgG molecule.

[0039] The third component may also be selected to impart desired properties. As an example, some domains are particularly useful for the isolation of proteins obtained by affinity chromatography. For the purposes of affinity purification, relevant matrices for affinity chromatography are used, such as glutathione, amylase, and nickel or cobalt conjugated resins. A number of such matrices are available in "kit" form, such as the Pharmacia GST purification system and the QIAexpress™ system (Qiagen), which are useful with (His6) fusion partners. As another example, the third domain may be selected to facilitate the detection of the ActR2B polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) as well as "epitope tags", which are typically short peptide sequences for which specific antibodies are available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tag. In some cases, the third domain may have a protease cleavage site, such as factor Xa or thrombin, which allows a relevant protease to partially digest the fusion protein, thereby enabling the recombinant protein to be released therefrom. The released protein can then be isolated from the third domain by subsequent chromatographic separation. In certain preferred embodiments, the ActR domain and the (h)nLG3 domain are linked to a domain that stabilizes the resulting polypeptide in vivo.

[0040] A typical example of the third component is the "Fc" domain, SEQ ID NO: 14. Similarly, fusion to human serum albumin may impart desired properties. Other types of fusion domains that may be selected include multimerization (e.g., dimerization, tetramerization) domains and functional domains, which impart additional biological functions, such as further stimulation of muscle growth.

[0041] The linking element is a short stretch of amino acids that links two protein components. This unconstructed linker may correspond to an unconstructed region of about 15 amino acids at the C-terminus of the extracellular domain of ActR2B ("tail"), or it may be an artificial sequence of 5 and 15, 20, 30, 50 or more amino acids that is relatively unstructured. The linker may be rich in glycine and proline residues and may contain, for example, repetitive sequences of threonine / serine and glycine. Often, multiple repeats of the sequence ggg are used (glycine-glycine-glycine-serine). Glycine imparts mobility and serine is polar. The linkage may be rich in glycine and proline residues and may contain, for example, repetitive sequences of threonine / serine and glycine (e.g., 4 or fewer TG or 4 or fewer SG repeats). A typical linker sequence "L" is as defined in SEQ ID NO: 13. The fusion protein may include an epitope tag, FLAG tag, or purification sub-sequences such as a polyhistidine sequence and GST fusion.

[0042] Examples of ActR domains containing a protein linked to an Fc domain are ActR-Fc (SEQ ID NO: 19), Fc-ActR (SEQ ID NO: 20) and (h)ActR-Fc (SEQ ID NO: 21) (Cadena et al. J Appl Physiol 109: 635-642, 2010). An example of follistatin linked to an Fc domain is as defined in SEQ ID NO: 26. Examples of nLG3 domain-containing proteins linked to an Fc domain are nLG3-Fc (SEQ ID NO: 16), Fc-nLG3 (SEQ ID NO: 15) and Fc-(h)nLG3 (SEQ ID NO: 17). Examples of LG3 domains without the insert Fc-(h)LG3 (SEQ ID NO: 18) were also constructed.

[0043] Certain embodiments include the following components. A-L-B B-L-A A-L-C-L-B B-L-C-L-A C-L-B-L-A C-L-A-L-B B-L-A-L-C A-L-B-L-C A-L-D D-L-A A-L-E E-L-A (wherein, A represents an Agrin nLG3 domain-containing protein, B represents the extracellular domains of ActRIB, ActRIC, ActRIIA, ActR2B receptor proteins and follistatin, C represents a "stabilizing" domain, D represents an ActR2B-mediated signal transduction inhibitory mAb against ActRIB, ActRIC, ActRIIA, and ActR2B receptor proteins, E represents an ActR2B-mediated signal transduction inhibitory mAb against members of the TGF-beta superfamily, L represents a linking element.) Specific combinations are B-L-C-L-A, D-L-A and E-L-A

[0044] Examples of these specific combinations are ActR-Fc-nLG3 as defined in SEQ ID NO: 22, (h)ActR-Fc-(h)nLG3 as defined in SEQ ID NO: 23, Fol-Fc-nLG3 as defined in SEQ ID NO: 27. The ActRmAb(HC) linked to (h)nLG3 (ActRmAb(HC)-(h)nLG3) is as defined in SEQ ID NO: 30. MyomAb((h)nLG3 linked HC) (MyomAb(HC)-(h)nLG3) is as defined in SEQ ID NO: 33. The corresponding light chain of the antibody needs to be expressed simultaneously with the heavy chain to produce a fully functional antibody.

[0045] The heavy and light chains of the antibodies of the present disclosure may be expressed as a continuous single-chain protein together with first and second components joined by a linking element (see, for example, Bird et al., 1988 Science 242:423-426; Huston et al., 1988 Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature 348:552-554). The continuous single-chain protein may be linked to (h)nLG3.

[0046] The compounds of the present disclosure may be made by known methods. For example, the DNA encoding the compound is expressed in a suitable expression system and the resulting protein is subsequently purified. Several prokaryotic and eukaryotic expression systems are suitable for the production and secretion of the compounds of the present disclosure. Prokaryotic expression systems include, but are not limited to, expression in E. coli. Eukaryotic expression systems include expression in mouse myeloma cells, baculovirus-mediated expression in insect cells, and expression in human embryonic kidney (HEK) cells, transient expression in Chinese hamster ovary (CHO) cells, and stable expression in Pichia pastoris. These systems have the advantage that they can be easily adapted to serum-free conditions to reduce the amount of contaminating protein in the supernatant and can be adapted for large-scale production. Furthermore, various cell lines may be used, including HEK293T and HEK293 cells, COS cells, CHO cells, HeLa cells, H9 cells, Jurkat cells, NIH3T3 cells, C127 cells, CV1 cells, CAP cells, or SF cells.

[0047] Since mammalian, yeast, insect, and plant cells can all introduce different glycosylation patterns that can be affected by the amino acid sequence of the peptide, the sequence of the components may be appropriately adjusted depending on the type of expression system used. In general, proteins for use in humans are expressed in mammalian cell lines that provide appropriate glycosylation, such as the HEK293 or CHO cell lines, although other mammalian expression cell lines are also predicted to be useful.

[0048] The compounds of the present disclosure may be purified by standard protein purification techniques. Immunoglobulin G may be purified using protein A or G. His-tagged proteins can be purified using IMAC, but ion exchange chromatography using a heparin column or affinity purification can also be used. Purification via an antibody raised against the C-terminal portion of agrin can also be used. The eluted protein can then be further purified using a hydroxyapatite column or by gel filtration.

[0049] The compounds of the present disclosure are useful in pharmaceutical compositions. Accordingly, the present disclosure provides a pharmaceutical composition comprising at least one compound as described above formulated with a pharmaceutically acceptable carrier.

[0050] The pharmaceutical compositions of the present disclosure are particularly useful for the treatment of pathological conditions leading to loss of muscle function. Non-limiting examples of such conditions include: - Muscle atrophy as a result of treatment with glucocorticoids such as cortisol, dexamethasone, betamethasone, prednisone, methylprednisolone, or prednisolone, - Muscle atrophy as a result of denervation due to nerve injury or as a result of neurodegenerative, metabolic, or inflammatory neuropathies (examples include Guillain-Barré syndrome, peripheral neuropathy, or exposure to environmental toxins or drugs), - Myotonia; congenital myopathies including nemaline myopathy, multi / minicore myopathy, and myotubular (centronuclear) myopathy; mitochondrial myopathy; familial periodic paralysis; inflammatory myopathy; metabolic myopathy such as caused by glycogen or lipid storage diseases; dermatomyositis; polymyositis; inclusion body myositis; myositis ossificans; muscle atrophy as a result of myopathies such as rhabdomyolysis and myoglobinuria, - Myopathies caused by muscular dystrophy diseases such as Duchenne, Becker, myotonic, facioscapulohumeral, Emery-Dreifuss, oculopharyngeal, scapulohumeral, limb-girdle, Fukuyama, congenital muscular dystrophy, or hereditary distal myopathy, - Skeletal disorders such as osteoporosis, fractures, short stature, or dwarfism;

[0051] - Adult motor neuron disease, infantile spinal muscular atrophy, amyotrophic lateral sclerosis, juvenile spinal muscular atrophy, autoimmune motor neuropathy with multifocal conductor block, paralysis due to stroke or spinal cord injury, skeletal immobilization due to trauma, prolonged bed rest, voluntary inactivity, involuntary inactivity, metabolic stress or nutritional deficiency, cancer, AIDS, starvation, thyroid disorders, diabetes, benign congenital hypotonia, central core disease, burns, chronic obstructive pulmonary disease, liver diseases (examples such as fibrosis, cirrhosis), sepsis, renal failure, congestive heart failure, aging, time spent in space travel or a zero gravity environment.

[0052] - Age-related conditions such as sarcopenia, skin atrophy, muscle wasting, brain atrophy, atherosclerosis, arteriosclerosis, emphysema, osteoporosis, osteoarthritis, immunologic incompetence, hypertension, dementia, Huntington's disease, Alzheimer's disease, cataracts, age-related macular degeneration, prostate cancer, stroke, decreased life expectancy, frailty, memory loss, wrinkles, kidney dysfunction, and age-related hearing loss; metabolic disorders including type 2 diabetes, metabolic syndrome, hyperglycemia, and obesity. Of course, the patient may be simultaneously affected by one or more of these conditions, by way of example sarcopenia and emphysema, or sarcopenia and kidney dysfunction.

[0053] - Acute and / or chronic kidney disease or renal insufficiency, liver fibrosis or cirrhosis, cancers such as breast cancer, pathological disorders such as Parkinson's disease; conditions related to neuronal cell death such as ALS, brain atrophy, or dementia and anemia. Further, there are losses suffered as a result of age, trauma, or inactivity. - Further conditions such as cachexia, cachexia related to rheumatoid arthritis, and cachexia related to cancer.

[0054] The pharmaceutical compounds of the present disclosure may also be administered in combination therapy, i.e., in combination with other agents. By way of example, the combination therapy may include the anti-ActR2B antibody of the present disclosure in combination with at least one other muscle mass / strength increasing agent, by way of example IGF-1, IGF-2 or variants of IGF-1 or IGF-2, anti-myostatin antibodies, myostatin propeptide, myostatin decoy protein that binds but does not activate ActR2B, beta2 agonists, ghrelin agonists, SARMs, GH agonists / mimetics or follistatin. The pharmaceutical compounds of the present disclosure may also be administered in combination therapy with Nusinersen or similar compounds. Nusinersen, an antisense oligonucleotide that modulates alternative splicing of the SMN2 gene and functionally converts it to the SMN1 gene, is an investigational drug for spinal muscular atrophy.

[0055] As used herein, "pharmaceutically acceptable carrier" includes physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epithelial administration (by way of example, injection or infusion). Depending on the route of administration, the compounds of the present disclosure may be coated with a material to protect the compounds from the action of acids and other natural conditions that may inactivate the compounds.

[0056] The compounds of the present disclosure may be in the form of pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refer to salts that retain the desired biological activity of the parent compound but do not impart any undesired toxicological effects (see, for example, Berge, S. M., et al., 1977 J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and non-toxic organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, fatty acids and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0057] The pharmaceutical compositions of the present disclosure may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0058] Examples of suitable water-soluble and water-insoluble carriers that may be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0059] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms may be ensured by both the above-described sterilization procedures and the inclusion of various antibacterial and antifungal agents (examples include parabens, chlorobutanol, phenolsorbic acid, etc.). It may also be desirable to include in the composition isotonic agents such as sugar, sodium chloride, and the like. Furthermore, sustained absorption of injectable pharmaceutical forms may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0060] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in the pharmaceutical compounds of the present disclosure is contemplated, except in cases where any convenient medium or agent is incompatible with the active compound. Auxiliary active compounds can also be incorporated into the compounds.

[0061] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compounds can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, by way of example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, the compounds can contain isotonic agents, such as sugars, polyhydric alcohols like mannitol, sorbitol, or sodium chloride, etc. Sustained absorption of injectable compounds can be brought about by including in the compound an agent that delays absorption, such as monostearates and gelatin.

[0062] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the agents enumerated above, and, if necessary, subsequently by sterile microfiltration methods. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing the basic dispersion medium and the other necessary agents from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying (lyophilization), which yield the powder of the active agent and any further desired agents from its previously sterile-filtered solution.

[0063] The amount of compound that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active agent that can be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect. Generally, this amount will vary from about 0.01 percent to about 99 percent, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of the active agent, out of 100 percent, when combined with a pharmaceutically acceptable carrier.

[0064] The dosing regimen is adjusted to provide the optimal desired response (such as, for example, a therapeutic response). By way of example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally decreased or increased as indicated by the exigencies of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compounds in dosage unit form. As used herein, a dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure are dictated by and directly dependent on the unique characteristics of the active compound, the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such active compounds for the treatment of individual susceptibilities.

[0065] The dosage for administration of the compound varies from about 0.0001 to 100 mg / kg of the host's body weight, and more usually from 0.01 to 5 mg / kg. By way of example, the dosage can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight, or can be in the range of 1 to 10 mg / kg or 3 to 7 mg / kg. Examples of treatment regimens involve administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Alternatively, the compound may be administered about once a year or only once. Such administration may be by intravenous or subcutaneous means. The dosing regimen for the compounds of the present disclosure includes 1 mg / kg body weight or 3 mg / kg body weight by intravenous administration, and the antibody is given using one of the following dosing schedules: every four weeks for six doses, then every three months; every three weeks; 3 mg / kg body weight once, then 1 mg / kg body weight every three months.

[0066] The dosage should be one that causes enhancement of muscle performance. In various embodiments, the effect is on skeletal muscle. In various embodiments, the dosage causes muscle hypertrophy that does not exceed a proportional increase in the size of internal organs (by way of example, the heart, lungs, liver, kidneys). Such proportional increase may be compared by measuring either mass or volume.

[0067] In some methods, two or more compounds of the present disclosure having different binding properties may be administered simultaneously, in which case the dosage of each compound administered is within the indicated range. Compounds are usually administered on a number of occasions. The interval between single administrations can be, by way of example, weekly, monthly, every three months, every six months, or annually. The interval can also be irregular if indicated by measuring the blood level of the compound against the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1 to 1000 μg / ml, and in some methods is about 25 to 300 μg / ml.

[0068] Alternatively, the compound can be administered as a sustained release formulation, in which case less frequent dosing is required. The dosage and frequency will vary depending on the half-life of the compound in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies in that order. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. Some patients will continue to receive treatment for the remainder of their lives. In therapeutic use, relatively high dosages at relatively short intervals are sometimes required until the progression of the disease is reduced or halted, or until the patient shows a partial or complete remission of the symptoms of the disease. Thereafter, the patient can be administered a prophylactic regimen.

[0069] The actual dosage level of the compound in the pharmaceutical composition of the present disclosure may vary so as to obtain an amount of the compound effective to achieve the desired therapeutic response for a particular patient, compound, and mode of administration without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors including the activity of the particular compound of the present disclosure being used, or an ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and / or materials being used in combination with the particular compound being used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, as well as similar factors well known in the medical arts.

[0070] A "pharmaceutically effective dosage" of the compounds of the present disclosure can result in a decrease in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or prevention of impairment or disability resulting from the disease (i.e., an increase in muscle mass and / or strength).

[0071] The compounds of the present disclosure can be administered by one or more routes of administration using one or more of the various methods known in the art. As will be appreciated by those skilled in the art, the route of administration and / or mode will vary depending on the desired result. Routes of administration of the antibodies of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, or other parenteral routes of administration, including, by way of example, injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intratendinous, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcorneal, intraarticular, subcapsular, subdural, intraspinal, epidural, and intrasternal injection and infusion. In one embodiment, the antibody is administered intravenously. In another embodiment, the antibody is administered subcutaneously.

[0072] Alternatively, the compounds of the present disclosure can be administered by parenteral routes such as topical, epidermal, or mucosal routes of administration, including, by way of example, intranasal, oral, intravaginal, rectal, sublingual, or topical administration.

[0073] The active compounds can be prepared with carriers that will protect the compound against rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Numerous methods for the manufacture of such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0074] The compounds can be administered using medical devices known in the art. By way of example, in one aspect, the compounds of the present disclosure can be administered using a needleless subcutaneous injection device, such as the devices shown in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Examples of well-known implants and modules useful in the present disclosure include: U.S. Pat. No. 4,487,603 showing an implantable micro-infusion pump for dispensing a medicament at a controlled rate; U.S. Pat. No. 4,486,194 showing a therapeutic device for administering a medicament through the skin; U.S. Pat. No. 4,447,233 showing a medicament infusion pump for delivering a medicament at an accurate infusion rate; U.S. Pat. No. 4,447,224 showing a variable flow implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196 showing an osmotic drug delivery system having a plurality of chamber compartments; and U.S. Pat. No. 4,475,196 showing an osmotic drug delivery system. Numerous other such implants, delivery systems, and modules are known to those of skill in the art and include those made by MicroCHIPS™ (Bedford, Mass.).

[0075] In certain embodiments, the compounds of the disclosure can be formulated to ensure proper dispersion in vivo. As an example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the compounds of the disclosure pass through the BBB (if desired), these can be formulated, for example, in liposomes. Methods for manufacturing liposomes can be found, for example, in U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes contain one or more components that are selectively transported to specific cells or organs and can thus enhance targeted drug delivery (see, for example, V. V. Ranade, 1989 J. Clin Pharmacol. 29:685). Examples of targeting components include folic acid or biotin (see, for example, U.S. Pat. No. 5,416,016); mannose (Umezawa et al., 1988 Biochem. Biophys. Res. Commun 153:1038); antibodies (P. G. Bloeman et al., 1995 FEBS Lett. 357:140; M. Owais et al., 1995 Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al., 1995 Am. J. Physiol. 1233:134); p120 (Schreier et al., 1994 J. Biol. Chem. 269:9090); K. Keinanen; M. L. Laukkanen, 1994 FEBS Lett. 346:123; J. J. Killion; I. J. Fidler, 1994 Imrnunomethods 4:273 are also referred to).

[0076] A further surprising and advantageous effect of the compounds of the disclosure is that they are myostatin inhibitors that are far more specific than those known in the art. Myostatin blockers are known to have multiple effects not only on muscle fibers but also on satellite cells. Satellite cells are a heterogeneous population of stem and progenitor cells required for the growth, maintenance, and regeneration of skeletal muscle. Myostatin blockers switch the growth and differentiation of these muscle stem cells (McCroskery et al. (2003) J. Cell Biol. 162, 1135-1147).

[0077] Myostatin blockers have different effects on muscle fibers; they switch off the proteolysis of muscle filaments (responsible for muscle contraction) and turn on the protein synthesis of muscle filaments (Curr Opin Support Palliat Care. 2011 Dec; 5(4): 334-341). Myosin is the name of a family of muscle protein filaments whose role in muscle contraction is known. These include a family of ATP-dependent motor proteins whose role in muscle contraction is known. During muscle contraction, muscle filaments such as myosin can be damaged and need to be broken down and replaced by new filaments. Human mutations in muscle proteolysis are known to lead to proximal muscle weakness and hypertrophic cardiomyopathy. In a paper by Olive et al (Human Molecular Genetics, 2015, 1-13), it was demonstrated that the muscle fibers of patients contained inclusions formed by myosin and myosin-related proteins.

[0078] Myostatin blockers can also block the proteolysis of muscle filaments and thus, due to prolonged exposure to myostatin inhibitors, can lead to the accumulation of damaged muscle filaments. This can be an undesirable side effect of myostatin blockers such as ActR-Fc, ActRmAb, and MyomAb. This may also explain the relatively low activity of these proteins in performance assays such as the treadmill.

[0079] The coupling of nLG3 described in this disclosure to ActR-Fc, ActRmAb, and MyomAb has resulted in novel compounds that are more specific in their mode of action. Proteins such as ActR-Fc-nLG3, ActRmAb-nLG3, and MyomAb-nLG3 activate only satellite cells and have no direct effect on muscle fibers. Currently, it is unclear how such proteins achieve this new level of specificity in their mode of action. One explanation, which does not limit the scope of this disclosure in any way, could be that the Act RIIB receptor also uses the LRP protein as a common receptor. nLG3 is very well known to bind to LRP4. Binding to LRP4 is very important. The protein ActR-Fc-LG3 (note the difference between LG3 and n LG3) used as a control leads to a similar weight and muscle increase as ActR-Fc, and thus the addition of the LG3 domain is not sufficient for this new activity (see Figure 12). The LG3 domain must have the appropriate insert that allows it to bind to LRP4. This result also indicates that the bulky residues at the C-terminus of ActR-Fc are probably not the reason for the novel effect of ActR-Fc-nLG3.

[0080] Accordingly, this disclosure also provides a method for specifically activating skeletal muscle satellite cells in the absence of a direct effect on muscle fibers, the method comprising treating the muscle with a compound as described above in this specification. This disclosure is further described with reference to the following examples and the associated drawings, which describe specific embodiments and are not limiting in any way. A more detailed explanation of Figures 1-11 is provided below, but the basic details are as follows.

Brief Description of the Drawings

[0081]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

DETAILED DESCRIPTION OF THE INVENTION

[0082] Protein synthesis The cDNA was obtained commercially. The cDNA was cloned into the mammalian gene expression vector pEvi3 (evitria AG, Switzerland) via the restriction enzymes NotI and HindIII. Plasmid DNA was prepared under low endotoxin conditions using a commercially available DNA purification kit (Macherey Nagel, Germany). The protein Fc-nLG3 was obtained using SEQ ID NO: 34. The protein nLG3-Fc was constructed using SEQ ID NO: 35. Fc-(h)nLG3 was constructed using SEQ ID NO: 36. Fc-(h)LG3 was constructed using SEQ ID NO: 37. Fc-ActR was constructed using SEQ ID NO: 38. ActR-Fc was constructed using SEQ ID NO: 39. (h)ActR-Fc was constructed using SEQ ID NO: 40. ActR-Fc-nLG3 was constructed using SEQ ID NO: 41.

[0083] (h)ActR-Fc-(h)nLG3 was constructed using SEQ ID NO: 42, (h)ActR-Fc-(h)LG3 was constructed using SEQ ID NO: 43, ActRmAb(LC) was constructed using SEQ ID NO: 44, and ActRmAb(HC) was constructed using SEQ ID NO: 45. ActRmAb(HC)-(h)nLG3 was constructed using SEQ ID NO: 46. MyomAb(LC) was constructed using SEQ ID NO: 47, and MyomAb(HC) was constructed using SEQ ID NO: 48. MyomAb(HC)-(h)nLG3 was constructed using SEQ ID NO: 49. Fol-Fc and Fol-Fc-nLG3 were constructed using SEQ ID NO: 50 and SEQ ID NO: 51, respectively.

[0084] Protein production and purification All proteins were produced by CHO K1 cells. The seed was grown in eviGrow™ medium (evitria AG, Switzerland), a serum-free medium that does not contain chemically defined animal components. Transfection and production were carried out at 37 °C and 5% CO2 in eviMake™ (evitria AG, Switzerland), a serum-free medium that does not contain animal components. ActRmAb and MyomAb were produced by co-transfection with IgG heavy and light chain expression vector DNA. The resulting antibodies were named ActmAb and MyomAb, respectively. ActRmAb-(h)nLG3 was created by co-transfection of vector DNA prepared using ActRmAb(HC)-(h)nLG3 and ActRmAb(LC). The resulting antibody was named ActmAb-(h)nLG3. MyomAb-(h)nLG3 was produced by co-transfection of vector DNA produced using MyomAb(HC)-(h)nLG3 and MyomAb(LC). The resulting antibody was named MyomAb-(h)nLG3.

[0085] The supernatant was harvested by centrifugation and sterile filtration (0.2 μm) on the 8th day after transfection. The target protein was subsequently purified via protein A affinity chromatography on a Bio-Rad BioLogic DuoFlow FPLC system using PBS as the wash buffer, 0.1 mol / l glycine pH 3.0 as the elution buffer, and 1 mol / l TRIS pH 10 as the neutralization buffer.

[0086] Identification of proteins by SDS-PAGE gel electrophoresis Each compound was eluted in 4X LDS Sample Buffer (Invitrogen) and 10X reducing agent (Invitrogen) to reach a concentration of 1 μg. The samples were heated at 70 °C for 10 minutes and subsequently run on a 4–12% Bis-Tris Plus gel (Invitrogen). The gel was run at a voltage of 200 V for 35 minutes. The target protein fraction was identified by Coomassie staining of the gel. The gel was left in Coomassie staining solution (0.26% Coomassie blue, 10% acetic acid, 25% methanol) for 4 hours. After removing the Coomassie solution, the gel was incubated overnight in destaining solution (10% acetic acid, 25% methanol) to remove excess dye. The gel was scanned and images were taken using a densitometer (BioRad).

[0087] Clustering of acetylcholine receptors in C2C12 mouse cells C2C12 mouse muscle cells were cultured skeletal myoblasts from ATCC (ATCC-LGC Standards S.r.l., Italy), cultured in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma, Italy) with 10% FBS (Sigma) containing 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin (all purchased from Invitrogen-Gibco). These were cultured in the previous medium on 8-well chamber slides for 2–3 days and then replaced with DMEM and 3% FBS to obtain myotubes. The myotubes were incubated with Agrin constructs at 10 μM (micromolar) for 24 hours and fixed at RT with 2% paraformaldehyde for 20 minutes. Samples were stained for AChR by incubating the cells with Alexafluor 555-conjugated α-Bungarotoxin (1:500; Invitrogen, Itally) at RT for 1 h. The cells were then rinsed and the coverslips were mounted by dropping PB 0.1M. The level of AChR clustering was compared by determining the average number of AChR clusters at established positions at a magnification of 40X using a fluorescence microscope.

[0088] Animal research Ethical statement All procedures involving the use of laboratory animals were conducted in accordance with the Italian law (DL n. 116, G.U., Supp. 40, February 18, 1992; permit number 17 / 2010-B, June 30, 2010) and the European Communities Council Directive of 24 November 1986 (86 / 609 / EEC).

[0089] Animal 9-week-old animals In one experiment, 9-week-old male C57BL / 6 mice (n = 5 per group, Harlan, Italy) were randomized by weight and then treated subcutaneously with protein. The proteins used are shown in the figure. Phosphate-buffered saline (PBS), pH 7.4, was used as vehicle control. The dosage was 10 mg / kg, administered three times a week for 2 weeks on days 1, 3, 5, 8, 10, and 12. The total dosage for the mixture was 20 mg / kg, consisting of a 1:1 mixture of ActR-Fc and Fc-nLG3 such that each protein was given at 10 mg / kg. Body weight was measured three times a week before dosing, 25 days after the start of dosing, and the mice were euthanized with CO2. Gastrocnemius, quadriceps, and triceps brachii muscles were harvested and weighed.

[0090] 22-month-old animals In the experiments with aged mice, 24 male mice of the C57 / BL6 strain (purchased from Charles River, France) were used. At the start of the experimental procedure, the mice were 22 months old. The body weight of the animals was measured and ear punches were taken; the mice were maintained in normal cages, five per cage, under a 12 / 12 h light / dark cycle, and given food and water ad libitum. Injections were administered subcutaneously (10 mg / kg) three times a week for five consecutive weeks. For the five-week treatment, the compound was injected on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26, 29, 31, 33. The animals were randomly divided into three experimental groups: a control group receiving an injection of PBS (PBS), a treatment group AcrR-Fc-nLG3, and ActR-Fc.

[0091] Body weight The body weight of the mice was measured five times a week throughout the experiment.

[0092] Rotarod Rotarod measurements were performed on the 7650 acceleration model of the Rotarod™ device (Ugo Basile, Italy). The mice were placed on the rod of the Rotarod. The rod accelerates slowly from 4 to 32 rpm. The time the mouse stays on the rod is recorded, and the test is terminated when the mouse can no longer remain on the rod. The maximum test period in the standard test is 5 minutes. In the extended test, the maximum test period is 30 minutes. The Rotarod performance of different treatment groups is shown. The standard deviation is shown as error bars. (N = 5 for each group). The standard test was performed on days 18 and 21 of treatment, after two motor tests. The data are the average of four on two days. The extended test is performed on day 21 of treatment.

[0093] Treadmill exercise Mice were trained on a treadmill apparatus (Panlab, Harvard Apparatus) three times per week in the afternoon. The device has the ability to simultaneously exercise up to five mice in individual lanes. Mice were trained on the treadmill for three weeks before starting compound injection and then for three weeks during compound / PBS administration. Each mouse was tested using an accelerating treadmill protocol. Briefly, mice were appropriately acclimated to the treadmill before any experiment. A few days before the experimental run, the mice were placed on the treadmill in their respective lanes with the shocking grid removed and the belt moving, and the device was allowed to explore them for several minutes. During the experiment, the mice were warmed up before the run. For this, the belt was started at a low speed (16 cm / sec) and the shocking grid was gradually turned on to 0.2 mA. The warm-up period was 2 minutes. After the warm-up period, the mice were tested for their running performance. The treadmill speed started at 16 cm / sec and was accelerated by 1 cm per minute. The acceleration continued until the mice reached a state of exhaustion. If a mouse received more than 10 shocks per minute, this level was considered a state of exhaustion and the experiment was stopped for that particular mouse. After exhaustion, the shocking grid was inactivated and the mice were returned to their cages. The running distance, the number of shocks received per minute, and the total number of shocks were evaluated for each mouse.

[0094] Grip strength test Forelimb grip strength was measured using a Grip Strength Meter (Ugo Basile, Varese, Italy). Control and treated mice were tested twice a week during the first 6 weeks of the experiment and 5 times a week during the last 2 weeks of the experiment. The mice were held by the tail and made to grasp a T-bar with their forelimbs. Once the mice grasped the bar with both limbs, they were pulled away from the bar until they released it. The digital meter displayed the level of tension (in grams) exerted on the bar by the mice. Each animal was given 5 consecutive tests, and the lowest and highest values were excluded by analysis and the average value was taken.

[0095] Muscle Isolation and Storage Mice were sacrificed by cervical dislocation. After dislocation, fresh skeletal muscles (triceps, quadriceps, and gastrocnemius) were rapidly dissected from the skin and bone using forceps and scissors. Muscle wet weight was measured immediately after isolation. Next, the muscles (three per mouse) were placed in a Peel-A-Way embedding mold (Sigma-Aldrich; E6032-1cs) containing Killik (Bio-Optica, Milan; 05-9801), an embedding agent for cryostat neutral, for cryosections. The minimum possible amount of Killik was used to cover the muscles to allow for rapid freezing. The mold was then quickly transferred for 20 - 40 seconds to a beaker filled with isopentane (1-methylbutane; Sigma-Aldrich; M32631) and dry ice (-80 °C) (a longer contact time can cause the formation of cracks in the sample; insufficient time can cause freezing artifacts), and then the muscle samples were transferred to dry ice. For long-term storage, the samples were maintained at -80 °C. The other three muscles per mouse were quickly placed in tubes and covered with at least 1 ml of RNAlater (Sigma-Aldrich) to stabilize and protect the RNA with rapid RNase inactivation. The samples were maintained at 4 °C for 24 hours, and then the RNAlater was removed from the tubes and stored at -80 °C until use.

[0096] Cryosectioning Before cryosectioning, the samples were placed in the cryostat for at least 20 minutes before further processing. The samples were mounted on the round metallic mount of the cryostat using the Killik embedding agent. Cross-sections with a thickness of 20 μm were prepared and collected on warm (RT) gelatinous Superfrost slides (ThermoScientific Menzel Glaser (217655)). The sections were dried at RT for 1 hour and then stored at -20 °C.

[0097] Morphometric analysis of muscle Cross-sections of mouse muscles were stained with hematoxylin Gill No2 (Sigma-Aldrich (GHS232)) and 1% eosin Y aqueous solution (see H / E staining procedure). Morphometric analysis was performed on three cross-sections for each experimental group. The following parameters were evaluated: 1) the area and perimeter of peripheral and central nucleated fibers, 2) the total number of nuclei relative to the number of fibers, 3) the percentage of central nuclei relative to the total number.

[0098] Data analysis and statistics Data are presented as mean ± S.D. (standard deviation of the mean). An independent Student's t-test was used to determine the significant differences between experimental groups. * Values of p < 0.05 are significant, ** p < 0.01 is very significant, and, *** p < 0.001 is considered particularly significant. The results obtained are described with reference to the figures.

[0099] Figure 1 shows the formation of acetylcholine receptor clusters (dots). 1A, control treated with vehicle; 1B, Fc-nLG3; 1C, nLG3-Fc; 1D, ActR-Fc-nLG3. As predicted for high concentrations (10 μM) of Fc-agrin (1B) and agrin-Fc (1C), AChR clusters were visible. However, when ActR-Fc-nLG3 (1D) and PBS (1A) were used, distinct clusters were not visible. Only occasional, probably spontaneous, clusters were visible. Furthermore, nLG3-Fc-ActR-treated cells also did not show clusters on C2C12-treated cells (results not shown). As predicted, AChR clusters appeared only at high concentrations on nLG3-Fc- and Fc-nLG3-treated C2C12 cells. At 1 μM, clusters were not visible. Why ActR-Fc-nLG3 did not show AChR clusters was not clear. The "ActR" portion of ActR-Fc-nLG3 may inhibit cluster formation. This may be caused by steric hindrance, making it impossible for proper agrin binding, or the agrin and myostatin signaling pathways may interfere.

[0100] Figure 2 shows Coomassie-stained SDS-PAGE of Fc-nLG3 (lane 1); nLG3-Fc (lane 2); Fc-ActR (lane 3); ActR-Fc (lane 4); ActR-Fc-nLG3 (lane 5); (h)ActR-Fc (lane 6); Fc-(h)nLG3 (lane 7); (h)ActR-Fc-(h)nLG3 (lane 8); (h)ActR-Fc-(h)LG3 (lane 9); ActRmAb (lane 10); ActRmAb-(h)nLG3 (lane 11); MyomAb (lane 12); MyomAb-(h)nLG3 (lane 13). All observed protein bands are as predicted. The protein bands for ActR and (h)ActR derivatives are fuzzy because this protein is glycosylated and the degree of glycosylation generates multiple bands of the same protein.

[0101] Figure 3 shows that relative body weight increases over time. Nine-week-old mice were treated with vehicle, ActR-Fc, Fc-nLG3, ActR-Fc-nLG3, and a 1:1 mixture of ActR-Fc and Fc-nLG3 (Figure 2A); vehicle, (h)ActR-Fc, (h)Fc-(h)nLG3, and (h)ActR-Fc-(h)nLG3 (Figure 2B); vehicle, ActmAb and ActmAb-nLG3 (Figure 3C); vehicle, MyomAb and MyomAB-(h)nLG3 (Figure 2D). As predicted, mice treated with ActR-Fc, ActR-Fc-nLG3, and the ActR-Fc+Fc-nLG3 mixture had significantly increased body weight on day 15 compared to vehicle-treated mice. Surprisingly, ActR-Fc-nLG3-treated mice had significantly less body weight compared to ActR-Fc, ActR-Fc-nLG3, and the ActR-Fc+Fc-nLG3 mixture. Also, (h)Fc-(h)nLG3, ActmAb-nLG3 MyomAb-(h)nLG3 had significantly less body weight compared to their relative control compounds, (h)ActR-Fc, ActmAb and MyomAb.

[0102] Figure 4 shows the relative muscle weights of mice treated with vehicle, ActR-Fc, Fc-nLG3, ActR-Fc-nLG3, a 1:1 mixture of ActR-Fc and Fc-nLG3, (h)ActR-Fc, Fc-(h)nLG3, (h)ActR-Fc-(h)nLG3, ActmAb, ActmAb-nLG3, MyomAb, and MyomAB-(h)nLG3. The relative average muscle weights for the gastrocnemius, quadriceps, and triceps muscles were calculated and compared to the muscles of vehicle-treated mice. The results for relative muscle weight were similar to those for total body weight. As expected, all compounds except Fc-nLG3 had significantly increased relative muscle weights. Surprisingly, compounds further carrying nLG3, or the human version of nLG3, (h)nLG3, ActR-Fc-nLG3, (h)ActR-Fc-(h)nLG3, ActmAb-(h)nLG3, MyomAb-(h)nLG3 had significantly less body weight compared to their control compounds.

[0103] Figure 5 shows the rotarod performance of mice. The performance of ActR-Fc, Fc-nLG3, a 1:1 mixture of ActR-Fc and Fc-nLG3, ActmAb, and MyomAb-treated mice did not increase significantly. Surprisingly, the performance of the nLG3 or (h)nLG3-containing compounds ActR-Fc-nLG3, ActmAb-nLG3, and MyomAB-(h)nLG3 was significantly increased compared to their control compounds ActR-Fc, ActmAb, and MyomAb.

[0104] Figure 6 shows that relative body weight increases over time. The relative average body weight was calculated weekly. All 22-week-old mice were treated with vehicle (PBS) for the first 3 weeks of the experiment. In the following 5 weeks, the aged mice were treated with vehicle, ActR-Fc, and ActR-Fc-nLG3. After 3 weeks, ActR-Fc-administered animals reached significantly higher levels of increased body weight compared to vehicle. After 3 weeks, ActR-Fc-nLG3-administered animals had significantly less body weight than that of ActR-Fc but had significantly increased body weight compared to vehicle.

[0105] Figure 7 shows the relative muscle wet weight. The relative average muscle weight for the gastrocnemius, quadriceps, and triceps muscles was calculated compared to the muscles of vehicle-treated mice. ActR-Fc-administered animals had increased, highly significant levels of muscle weight compared to the vehicle. ActR-Fc-nLG3-administered animals had a significantly increased muscle weight compared to the vehicle, but significantly less than that of ActR-Fc.

[0106] Figure 8 shows the treadmill performance of aged mice. Figure 8A shows the treadmill performance during the 3rd week of vehicle administration (before treatment). At this point, the performance of all groups was very similar. Figure 8B shows the average treadmill performance during weeks 5 and 6 (after treatment). The performance of ActR-Fc- and vehicle-treated mice was lower (not significant) after treatment compared to before treatment. As the mice aged, their treadmill performance appeared to decrease. Surprisingly, the performance of ActR-Fc-nLG3-treated mice improved (p = X) after treatment compared to before treatment. This indicates that the treadmill performance improved despite the mice aging. The performance of ActR-Fc-nLG3 in the treated mice was significantly higher than that of vehicle- and ActR-Fc-treated mice. This indicates that treatment with ActR-Fc-nLG3 improves the muscle durability of aged mice.

[0107] Figure 9 shows the average number of motivation electrical pulses per minute during treadmill runs in weeks 5 and 6. Mice require more electrical pulses when fatigued. ActR-Fc and vehicle-treated mice required more pulses than ActR-Fc-nLG3-treated mice. This was highly significant (p < 0.001). Interestingly, during the first 9 minutes, all three groups of mice required approximately the same number of pulses with no statistical difference. As the time on the treadmill increased, the performance of ActR-Fc-nLG3-treated mice was far superior, and the mice required fewer pulses than vehicle- and ActR-Fc-treated mice. This also clearly shows that mice treated with ActR-Fc-nLG3 have improved muscle endurance.

[0108] Figure 10 shows the average grip strength (GS) performance of mice during week 3 of vehicle administration (GS before treatment). At this point, the performance of all groups was very similar. Figure 7B shows the average grip strength (GS) performance between weeks 5 and 6 (GS after treatment). The performance of vehicle-treated mice was lower (significant) after treatment compared to before treatment. The performance of ActR-Fc and ActR-Fc-nLG3-treated mice was higher (significant) after treatment compared to before treatment. The GS performance of ActR-Fc-nLG3 and ActR-Fc-treated mice was very similar after treatment, and both were significantly increased compared to vehicle-treated mice. Thus, administration of the compound ActR-Fc-nLG3 has maintained increased muscle strength performance similar to ActR-Fc.

[0109] Figure 11 Summary of muscle pathology. Cross-sectional area (CSA), and the number of nuclei per muscle fiber were determined for vehicle, ActR-Fc and ActR-Fc-nLG3 treated mice. From these results, the number of nuclei per CSA was calculated. In Figure 11, relative values for CSA, and the number of nuclei per nucleus per fiber, and the number of nuclei per CSA are described. ActR-Fc (p<0.001) and ActR-Fc-nLG3 (p<0.05) treated mice have statistically significantly increased CSA and number of nuclei per fiber compared to vehicle treated mice. Furthermore, ActR-Fc treated mice have significantly increased CSA (p<0.01) and number of nuclei (p<0.05) compared to ActR-Fc-nLG3. However, ActR-Fc treated mice have a significantly decreased number of nuclei per fiber area (p<0.05) compared to vehicle or ActR-Fc-nLG3. Nucleation is promoted by the activity of satellite cells. As satellite cells proliferate and differentiate, they will fuse with existing muscle fibers and lead to more nuclei in the muscle fibers.

[0110] Figure 12 shows that relative body weight increases over time. Nine-week-old mice were treated with vehicle, (h)ActR-Fc, (h)ActR-Fc-(h)nLG3, and (h)ActR-Fc-(h)LG3. On day 19, ActR-Fc, (h)ActR-Fc-(h)nLG3, and (h)ActR-Fc-(h)LG3-treated mice had significantly increased relative body weights compared to vehicle-treated mice (p < 0.001, p < 0.05, p < 0.001, respectively). There was no significant difference in relative body weight between (h)ActR-Fc-(h)LG3 and (h)ActR-Fc. Notably, (h)ActR-Fc-(h)LG3-treated mice had significantly (p > 0.01) greater body weights compared to (h)ActR-Fc-(h)nLG3. The two proteins differ only by the insertion of an 8-amino acid sequence in (h)nLG3. Since this insert is responsible for binding to the LRP4 receptor, (h)nLG3 binds to the LRP4 receptor while (h)LG3 does not. Mice treated with (h)ActR-Fc-(h)nLG3 showed a growth curve similar to ActR-Fc-nLG3 (Figure 3).

[0111] From these results, perhaps the muscle growth of ActR-Fc-nLG3 is only caused by the proliferation of muscle stem cells (i.e., satellite cells) that fuse with muscle fibers to lead to more nuclei. More nuclei would lead to more protein synthesis in muscle fibers, resulting in a moderate increase in muscle and body weight in ActR-Fc-nLG3, (h)ActR-Fc-(h)nLG3, ActRmAb-nLG3, and MyomAb-nLG3-treated animals. Treatment with ActR-Fc also leads to more nuclei, but fiber proliferation is over proportional and actually leads to a smaller nuclear density.

Claims

1. A compound comprising at least two components, wherein the first component is the nLG3 domain from the C-terminus of human or mouse Agrin, and at least one second component is selected from a protein or an antagonist antibody that inhibits ActR2B-induced signaling activity in the presence of myostatin, and these components are linked by means of a linking element, the nLG3 domain from the C-terminus of human or mouse Agrin consists of a polypeptide represented by an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 10 or SEQ ID NO: 5, respectively, the protein that inhibits ActR2B-induced signaling activity in the presence of myostatin consists of a protein represented by an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 25, the antagonist antibody that inhibits ActR2B-induced signaling activity in the presence of myostatin is an antibody comprising a light chain (LC) represented by SEQ ID NO: 29 and a heavy chain (HC) represented by SEQ ID NO: 28, or an antibody comprising a light chain (LC) represented by SEQ ID NO: 32 and a heavy chain (HC) represented by SEQ ID NO: 31, said compound.

2. A composition for improving muscle performance, comprising an effective amount of the compound according to Claim 1.

3. A pharmaceutical composition comprising at least one compound according to Claim 1 or 2, formulated with a pharmaceutically acceptable carrier.

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