AU2016202691B2 - Activin-ActRIIa antagonists and uses for promoting bone growth in cancer patients - Google Patents

Abstract

In certain aspects, the present invention provides compositions and methods for promoting bone growth and increasing bone density, as well as for the treatment of multiple myeloma.

Description

BACKGROUND OF THE INVENTION

Disorders of the bone, ranging from osteoporosis to fractures, represent a set of pathological states for which there are few effective pharmaceutical agents. Treatment instead 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 for the purpose of treating a variety of bone disorders.

Bone growth and mineralization are dependent on the activities of two cell types, osteoclasts and osteoblasts, although chondrocytes and cells of the vasculature also participate in critical aspects of these processes. Developmentally, bone formation occurs through two mechanisms, endochondral ossification and intramembranous ossification, with the former responsible for longitudinal bone formation and the later responsible for the formation of topologically flat bones, such as the bones of the skull. Endochondral ossification requires the sequential formation and degradation of cartilaginous structures in the growth plates that serve as templates for the formation of osteoblasts, osteoclasts, the vasculature and subsequent mineralization. During intramembranous ossification, bone is formed directly in the connective tissues. Both processes require the infiltration of osteoblasts and subsequent matrix deposition.

Fractures and other structural disruptions of bone are healed through a process that, at least superficially, resembles the sequence of developmental events of osteogenesis,

2016202691 27 Apr 2016 including the formation of cartilaginous tissue and subsequent mineralization. The process of fracture healing can occur in two ways. Direct or primary bone healing occurs without callus _

1a

2016202691 27 Apr 2016 formation. Indirect or secondary bone healing occurs with a -callus precursor stage. Primary healing of fractures involves the refbnnation of mechanical continuity across a closely-set disruption. Under suitable conditions, bone-resorbing cells surrounding the disruption show a tunnelling resorptive response and establish pathways for the penetration of blood vessels 5 and subsequent healing. Secondary heating of hones follows a process of inflammation, soft callus formation, callus mineralisation and callus remodelling. In the inflammation stage, haematoma and haemorrhage formation results from, the disruption of periosteal and endosteal blood vessels at the- site of Injury. Inflammatory cells invade the area. In soft callus formation stage, the cells produce new vessels, fibroblasts, intracellular material and 0 supporting cells, forming granulation tissue In the space between the fracture fragments. Clinical union across the disruption is established by fibrous or cartilaginous tissue (soft callus). Osteoblasts are formed and mediate the mineralization of soft callus, which is then replaced by lamellar bone and subjected to the normal remodeling processes.

In addition to fractures and other physical disruptions of bone structure, loss of bone 5 mineral content and bone mass can be caused by a wide variety of conditions and may result in significant medical problems. Changes to bone mass occur in a relatively predictable way over the life of an individual. Up to about age 30, bones of both men and women grow to maximal mass through linear growth of the endochondral growth plates and radial growth. After about age 30 (for trabecular bone, e.g., fiat bones such as the vertebrae and pelvis) and age 40 (for cortical bone, e.g,, long bones found in the limbs), slow bone loss occurs in both men and women. In women, a final phase of substantial hone loss also occurs, probably due to postmenopausal estrogen deficiencies. During this phase, women, may lose an. additional 1.0% of bone mass from the cortical, bone and 25% from the trabecular compartment. Whether progressive bone loss results in a pathological condition such as osteoporosis depends largely on the Initial bone mass of the individual and whether there are exacerbating conditions.

Bone loss is sometimes characterized as an. imbalance in the normal, bone remodeling process. Healthy bone is constantly subject, to remodeling. Remodeling begins with resorption of bone by osteoclasts. The resorbed bone is then replaced by new bone tissue, which is characterized by collagen formation by osteoblasts, and subsequent· calcification. In healthy individuals the rates of resorption and formation are balanced. Osteoporosis is a

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-3chronic, progressive condition, marked by a shift towards resorption, resulting in an overall decrease in bone mass and bone mineralization. Osteoporosis in humans is preceded by clinical osteopenia (bone mineral density that is 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.

Thus, methods for controlling the balance between osteoclast and osteoblast activity can be useful for promoting the healing of fractures and other damage to bone as well as the treatment of disorders, such as osteoporosis, associated with loss of bone mass and bone mineralization.

With respect to osteoporosis, estrogen, calcitonin, osteocalcin with vitamin K, or high doses of dietary calcium 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. Such therapeutics, however, are often associated with undesirable side effects.

In one embodiment, the present invention relates to compositions and methods for promoting bone growth and mineralization.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides use of an anti-activin 25 antibody or fragment thereof in the manufacture of a medicament for treating or preventing multiple myeloma in a human patient.

According to a second aspect, the present invention provides a method for treating or preventing multiple myeloma in a human patient, the method comprising administering to the patient an effective amount of an anti-activin antibody or fragment thereof.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

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- 3a In part, the disclosure demonstrates that molecules having activin or ActRlla antagonist activity (“activin antagonists” and “ActRlla antagonists”, collectively “activinActRlla antagonists”) can be used to increase bone density, promote bone growth, and/or increase bone strength. In particular, the disclosure demonstrates that a soluble form of ActRlla acts as an inhibitor of activin-ActRlla signaling and promotes increased bone density, bone growth, and bone strength in vivo. While most pharmaceutical agents that promote bone growth or inhibit bone loss act as either anti-catabolic agents (also commonly referred to as “catabolic agents”) (e.g., bisphosphonates) or anabolic agents (e.g., parathyroid hormone, PTH, when appropriately dosed), the soluble ActRlla protein exhibits dual activity, having both anti-catabolic and anabolic effects. Thus, the disclosure establishes that antagonists of the activin-ActRlla signaling pathway may be used to increase bone density and promote bone growth. While soluble ActRlla may affect bone through a mechanism -₇

2016202691 27 Apr 2016 other than aetivm antagonism, the disclosure nonetheless demonstrates that desirable therapeutic agents may be selected on the basis of an activin-ActRIla antagonist activity. Therefore, in certain embodiments, the disclosure provides methods for using aeriyimAclRUa antagonists, including, for example, actlvin-binding ActRHa polypeptides, anti-aetivin 5 antibodies, anti- ActRHa antibodies, aetivin- or ActR Ila-targeted small molecules and aptamers, and nucleic acids that decrease expression of aetivin and ActRHa, to treat disorders associated with low bone density or low bone strength, such as osteoporosis, or to promote bone growth in patients in need thereof, such as in patients having a bone fracture. The disclosure further demonstrates that aetivin-AoiRHa antagonists are effective in preventing 0 and/or repairing bone damage caused by multiple myeloma tumors and breast tumors, and, additionally, that aedvin-ActRHa antagonists diminish the tumor load in multiple myeloma. Soluble ActRHa polypeptide promotes bone growth without causing a consistently measurable Increase in muscle mass.

In certain aspects, the disclosure provides polypeptides comprising a soluble, aetivin5 binding ActRHa polypeptide that binds to aetivin. ActRHa polypeptides may he formulated as a pharmaceutical preparation comprising the achvin-binding ActRHa polypeptide and a pharmaceutically acceptable carrier. Preferably, the actiyimbinding ActRHa polypeptide hinds to aetivin with a less than 1 micromolar or less than 100, 10 or 1 nanomolar. Optionally, the actiyimbinding ActRHa polypeptide selectively binds arm m versus ODFV1 and/or GDFS, and preferably with a Ko that is at least i O-fold, 20-fold or 50-fold lower with respect to aetivin than with respect to ODF11 and/or GDF8. While not wishing to be bound to a particular mechanism of action, it is expected that this degree of selectivity for aetivin inhibition over GDF'I l/GDFS inhibition accounts for the selective effect on bone without a consistently measurable effect on muscle. In many embodiments, an ActRHa polypeptide25 will be selected lor causing less than 15%, less than 10% or less than 5% increase hi muscle at doses that achieve desirable effects on bone. 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 aetivinbinding ActRHa polypeptide for use in such a preparation may he any of those disclosed herein, such as a polypeptide having an amino acid sequence selected from SEQ ID NOs: 2,

3, 7 or 12, 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, 7,. 12 or 13. An

-42016202691 27 Apr 2016 aetivin-binding. ActRila polypeptide may Include a functional fragment of a natural ActRila polypeptide, such as one comprising at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: I -3 or a sequence of SEQ ID NO; 2, lacking the C-terminal 10 to 15 amino acids (the “ί.&ίΓ), $ A soluble, aetivin-binding ActRila polypeptide may include one or more alterations in the amino acid sequence (e.g., in the ligand-binding domain) relative to a naturally occurring ActRila polypeptide. Examples of altered AciRIb polypeptides are provided in WO 2006/0126.27, pp. 59-60, incorporated by reference herein. The alteration in the amino acid sequence may, for example, alter glycosylation of the polypeptide when produced in a

!) mammalian, insect or other eukaryotic cell or alter proteolytic cleavage of the polypeptide relative to the naturally occurring ActRila polypeptide.

An aetivin-binding ActRila polypeptide may be a fusion protein that has, as one domain, an ActRila polypeptide (e.g., a ligand-binding portion of an ActRila) and one or more additional domains that provide a desirable property, such as improved 5 pharmacokinetics, easier purification, targeting to particular tissues, etc. For example, a domain of a fusion protein may enhance one or more of in vi vo stability, in vivo half life, uptake/administration, tissue localization or distribution, formation of protein complexes, multirnerization of the fusion protein, and/or purification. Dimerization or tnultimerization may provide increased ligand-binding affinity. An activin-bindmg ActRila fusion protein 0 may include an immunoglobulin Fc domain (wild-type or mutant) or a serum albumin or other polypeptide portion (hat provides desirable properties such as improved pharmacokinetics, improved solubility or improved stability, 'Typically, an ActRIb-Fc fusion protein will be produced as a bo.modimeric complex. Optionally, an ActRHa-Fc fusion comprises a relatively unstructured linker positioned between the Fc domain and the 25 extracellular ActRila domain. This unstructured linker may correspond to the roughly 15 ami.no acid unstructured region at the C-terminal cud ofthe extracellular domain of AcfR.Ha r.ho ’ toil?, οι U mas he an aiisficnti sveueme of I. 2, 5, Ί m ?- „cmno aefos o- a length >d between 5 and 15, 20,30, SO or more amino acids that are relatively free of secondary structure, or a mixture of both. A linker may he rich in glycine and proline residues- and may, 30 for example, contain a single sequence of threonine/serine and glycines or repeating sequences of threonine/serine and glycines (e.g.. TG< or SG« singlets or repeats). A fusion

- 5 2016202691 27 Apr 2016 protein may include a -purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidibe sequence, and a GST fusion. Optionally, a soluble ActRIla polypeptide includes, one or more modified amino acid, residues selected from: a glycosylated amino acid, a PEGylated amino acid,, a famesylated amino acid, an acetylated amino acid, a biotinylated 5 amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic deri vatizing agent. A pharmaceutical preparation may 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 pyrogen free, in general, it ts preferable that an ActRIla protein be expressed in a mammalian ceil line that mediates suitably natural 0 ghenw lath'U cl tbe AuRfe p.oto.r so as m dunuush tbe kkehhood of an out womble immune response in a paoent. Human and CHO cell lines have been used successfully, and it is expected that other common mammalian expression systems will be useful.

As described herein. ActRHa proteins designated ActRHa-Fc have desirable properties, including selective binding to aetivm versus GDF8 and/or GDFI1, high affinity ligand binding and serum half life greater than two weeks in animal models, in certain embodiments the invention provides AetRlla-FC. polypeptides and pharmaceutical preparations comprising such polypeptides and a pharmaceutically acceptable excipient.

In certain aspects, the disclosure provides nucleic acids encoding a soluble activinbinding ActRHa polypeptide. An isolated polynucleotide may comprise a coding sequence for a soluble, acti vin-binding ActRIla polypeptide, such as described above. For example, an isolated nucleic acid may include a sequence coding for an extracellular domain (e..g., ligandbinding domain) of an ActRIla and a sequence that would code for part, or all of the transmembrane domain and/or tbe cytoplasmic domain ol an ActRIla, but for a stop codon positioned within the transmembrane domain or the cytoplasmic domain, or positioned between the extracellular domain and the transmembrane domain or cytoplasmic domain.

For example, an isolated polynncleotide may comprise a fell-length ActRIla polynucleotide sequence such as SEQ ID NO: 4 or 5, or a panially truncated version, said isolated 'polynucleotide further comprising a transcription termination codon at. least six hundred nucleotides before the 3 -terminus or otherwise positioned such that translation of the polynucleotide gives rise to an extracellular domain optionally fused to a truncated portion of a full-length ActRIla. A preferred nucleic acid sequence is SEQ ID NO:14 Nucleic acids

-62016202691 27 Apr 2016 disclosed herein may: he operably linked to a promoter for expression, and the disclosure provides cells transformed with such recombinant polynucleotides. Preferably the cell is a mammalian cell such as a CHO ceil.

. In certain aspects, the disclosure provides methods for making a soluble, aetivin5 binding ActRlla polypeptide. Such a method may include expressing any of the nucleic acids (e.g., SEQ ID NO: 4,.5 or 14} disclosed herein in a suitable cell, such as a Chinese hamster ovary (CHO) cell. Such a method may comprise·, a) culturing a ceil under conditions suitable for expression of the soluble ActRlla polypeptide wherein said cell is transformed with a soluble ActRlla expression construct; and b) recovering the soluble ActRlla polypeptide so expressed. Soluble ActRlla polypeptides may be recovered as crude, partially purified or highly purified fractions. Purification may be achieved by a scries of purification steps, including, for example, one, two or three or more of the following, in any order: protein A chromatography, anion exchange chromatography (e.g., Q sepbarose), hydrophobic interaction chromatography (e.g., phenyl sepharose), size exclusion chromatography, and cation ex change chromatography.

In certain aspects, an aetivin-AetRIfe antagonist disclosed herein, such as a soluble, aedvm-bmdtng ActRlla polypeptide, may be used in a method .for promoting bone growth or increasing bone density in a subject,. In certain embodiments, the disclosure provides methods for treating a disorder associated with low bone density, or to promote bone growth, in patients in need thereof. A method may comprise administering to a subject in need thereof an effective amount of acti vin-ActRIIa antagonist. In certain aspects, the disclosure, provides uses of act.Iv.ln~Aet.RHa antagonist .for making a medicament lor the treatment of a disorder or condition as described herein.

In certain aspects, the disclosure provides a method lor identifying an agent that stimulates growth of, or increased mineralization o£ bone. The method comprises; a) identifying a test agent that hinds to aetivin or a ligand-binding domain of an ActRlla polypeptide; and h) evaluating the effect of the agent on growth of, or mineralization of, bone.

BRIEF DESCRIPTION OF THE DRAWINGS

2016202691 27 Apr 2016

Figure 1 shows the purification of ActRIIa-hFc expressed in CHO cells. The protein purifies as a single, well-defined peak.

Figure 2 shows-the binding Of AetRfla-hf c to activin and GDF-l1, as measured by 5 SiaCore^!M assay,

Figure 3 shows a schematic for the A-204 Reporter Gene Assay. The figure shows the Reporter vector: pGL3(CAGA)12 (described in Dennler et al, 1998, EMBO 17: 30913100-) The CAGAI2 motif is present in TGF-Beta responsive genes (PAM gene), so this vector is of general use for factors signaling through Smad 2 and 3,

Figure 4 shows the effects of ActRUa-hFe (diamonds) and ActRUa-mFc (squares) on

GDF-8 signaling in the A-204 Reporter Gene Assay. Both proteins exhibited substantial inhibition ofGDF-8 mediated signaling at plcomolar concentrations.

Figure 5 shows, the effects of three different preparations of ActRfla-hFe on GDF-11 signaling in the A-204 Reporter Gene Assay.

Figure 6 shows examples of DEXA Images of control- and ActRUa-mFc-treated

BALBZc mice, before (top panels) and after (bottom panels) the 12-week treatment period. Pater shading indicates increased bone density.

Figure 7 shows a quantification of the effects of ActRUa-mFc on bone mineral density in BALB/c mice over the 12-week period. Treatments were control (diamonds), 2 mg/fe'g dosing of ActRIfa-mFe (squares), 6 rng/kg dosing of ActRUa-mFc (triangles) and 10 mg/fcg dosing of ActRUa-mFc (circles), .

Figure 8 shows a quantification of the effects of ActRffa-mFe on bone mineral content in BALBZc mice over the id-week period. Treatments were control (diamonds), 2 rngdeg dosing of ActRUa-mFc (squares), 6 mg/kg dosing of ActRUa-mFc (triangles) and 10 mg/fcg dosing of ActRUa-mFc (circles) .

Figure 9 shows a quantification of the effects of ActRUa-mFc on bone mineral density of the trabecular bone in ovariectomteed (OVX) or sham operated (SHAM) CS7BL6 mice over after a 6-week period. Treatments were control (PBS) or 10 mg/kg dosing of AetR I l a-m Fe (A et R II a )-82016202691 27 Apr 2016

Figure 10 shows a quantification of the effects of ActRIIa-mFc on the trabecular hone in ovarieetomized (OVX) C57BL6 .mice o ver a 12-week period. Treatments were control (PBS; pale fears) or 10 rng/kg dosing of AotRHa-mFc (AetRIla; dark fears).

Figure 1' 1 shows a quantification of the effects of ActRIlawnPe on the trabecular bone 5 in sham operated C578L6 mice after 6 or 1 2 weeks of treatment period. Treatments were control (PBS; pale fears) or 10 mg/kg dosing of ActRIIa-mFc (ActRlla; dark bars).

Figure 12 shows the results of pQCT analysis ofbone density in ovariectemteed mice over 12 weeks of treatment. Treatments were control (PBS; pale fears) or ActRIIa-wPc (dark fears), ymxis; mg/ecm

Figure 1.3 depicts the results of pQCT analysts of hone density in sham operated mice over 12 weeks of treatment. Treatments were control (PBS; pale bars) or ActRlia-mFe (dark bars). y-axis; rng/eem

Figures 14A and 14B show whole body DBXA analysis after 12 weeks of treatment (A) and ex ww analysis of femurs (8). Light areas depict areas of high bone density,

Figure 15 shows ex v/yo pQCT analysis of the femoral midshaft after twelve weeks of treatment. Treatments were vehicle control (PBS., dark bars) and ActROa-aiFe (pale bars). The four bars to the left show total bone density while the tour bars to the rigid show cortical bone density, The first pair of bars in each set of four fears represent data from ovariectornized mice, while the second pair of fears represent data ftorn sham operated mice,

Figure I d shows ex vrn? pQCT analysis and diaphyseal bone content of the femoral midshaft ailci twelve weeks >d neaftnent. ficahncnta wvre vehicle ci-nVol tPBS. dark b,n\; or ActRfhwmFe (pale fears). The tour bars to the left show total bone content while the four bars to the right show cortical bone content. The first pair of bars in each set of four bars .represent data from ovariectornized mice while the second pair of bars represen t data from sham operated mice.

Figure I? shows ex vrw pQCT analysis of'the femoral midshaft and femoral cortical thickness. Treatments were control (PBS, dark bars) and AetRlla-rnFc (pale bars). The four fears to the left show endosteal circumference while the four bars to the right show periosteal circumference. The first pair of bars in each set of four bars represent data from ovariectornized mice while the second pair of fears represent data from sham operated mice.

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Figure 18 depicts the results of mechanical testing of femurs after twelve weeks of treatment. Treatments were control (PBS, dark bars) and AetRlla~mFe (pale bars). The two bars to the left represent data from ovariectomized mice while the last two bars represent data from sham operated mice,

Figure 19 shows the effects of Aetdla-mFe on trabecular bone volume.

Figure 20 shows the effects of Aetrlla-mFc on trabecular architecture in the distal lemur.

Figure 21 shows the effects of ActrOa-mFc on cortical bone.

Figure 22 shows the effects of AetrfIa~mFe on the mechanical strength of bone,

Figure 23 show's the effects of different doses of AetRIla-mFc on bone characteristics at three different dosages.

Figure 24 shows bone histomorphometry indicating that ActRIIa-mFc has dual anabolic ami anti-resorptive activity.

Figure 25 shows additional histomorphometrie data.

Figure 26 shows unages of mouse femurs from naive and tumor-carrying mice, and the effects of AeiRl.ia~rnFc treatment on bone morphology in the multiple myeloma model. Mice carrying multiple myeloma tumors (5T2) show marked pitting and degradation la the hone relative to normal once ffrasve). Treatment with ActRIIa-mFc eliminates this effect.

Figure 27 shows results from the human clinical trial described in Example 5, where 0 the area-under-curve (AUC) and administered dose of AetRIIa-hFe have a linear correlation, regardless of whether ActRUa-hFc was administered intravenously (IV) or subcnumeoosly (SC).

Figure 28 shows a comparison of serum levels of ActRIla-hFe in patients administered IV or SC,

Figure 29 shows bone: alkaline phosphatase (BAP) levels in response to different dose levels of ActRIla-bFc BAP is a marker for anabolic hone growth.

Figure.30 shows the cooperati ve effects of ActRI la-mFc (RA P-011) and a bisphosphonate agent (zoledronate) in mice.

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DETAILED DESCRIPTION OF THE INVENTION

T Oyeryiew

The transforming growth factor-beta (TGF-bcta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins 5 are known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, ehondrogenesis, eardiogenesis, hematopoiesis, neurogenesis, and epithelial eel; differentiation. The family is 0 divided into two general branches: the BMP/GDF and the TGF-betaZActivin/ΒΜΡ10 branches, whose members have diverse, often eomplementary effects. By manipulating the activity of a member of the TGF-heta family, it is often possible to cause significant physiological changes in an organism, For example, the Piedmontese .and Belgian Blue cattle breeds carry a loss-of-funetlon mutation in the GDPS (also called myostatm) gene that causes 5 a marked increase in muscle mass, Grobet et ah, Nat Genet. 199?, 17(1):71-4, Furthermore, in humans, inactive alleles of GDPS are associated with increased muscle mass -and, reportedly, exceptional strength, Sehuelke et ah, N Engl .1 Med 2004» 350:2682-8.

Activins are dimeric polypeptide growth factors that belong to fheTGE-beta superfamily, There are three principle activin forms (A. B, and AB) that are honm/beterodimers of two closely related β subunits $_a|$a, βηβη, and β.4Μ· The human genome also encodes an activin C and an activin E, which are primarily expressed in the fiver. In the TGP-beta superfamily, activins are unique and multifunctional factors that can stimulate hormone production in ovarian and placental cells, support neuronal ceil survival, influence cell-cycle progress positively or negatively depending on cell type, and induce25 mesodermal differentiation at least in amphibian embryos (DePaolo et al., 1991, Proc See Ep Biol Med, 198:500-512; Dyson et al,, 1997, Cum Biot 7:81-84; Woodruff, i998, Biochem Pharmacol. 55:953-963), Moreover, erythroid differentiation factor (PDF) isolated from the stimulated human monocytic leukemic cells was found to be identical to activin A (Murata et ah, 1988, ENAS, 85:2434), ft 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 heterodimer, inbibin. For example, during the release of follicle- U 2016202691 27 Apr 2016 stimulating hormone (FSH) from the pituitary, activin promotes FSH secretion and synthesis, while inhibin prevents FSH secretion and synthesis. Other proteins that may regulate activin bioactivity and/or bind to activin include follistatin (FS), follistatin-related protein (FSRP), and «2-macroglobuJin.

TGP-β signals are mediated by heteromeric complexes of type 1 and type- U serine/ threonine kinase receptors, which phosphoryiate and activate downstream Smad proteins upon ligand stimulation (Massague, 2000, Nat. Rev. Mol. Cell Biot 1:169-178). These type I and type 11 receptors are transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specificity, Type I receptors are essential for signaling; and type II receptors are required lor binding ligands and tor expression of type I receptors. Type I and II activin receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type ff receptors.

Two related type II receptors, ActRlla and AetRlib, have been identified as the type Π receptors for aetivins. (Mathews and Vale, 1991, Cell 65:973-982; Attisanoet al., 1992, Cell 68; 97-108). Besides aetivins, AetROa and ActRllb can biochemically interact with several other TGF-β family proteins, including BMP7, Nodal, GDF8, and GDF'l I (Yamashlta et ah, 1995,1. Ceil Biol. I30:217-226; Lee and MePherron, 2001, Proc. Nad. Acad, Sci, 98:93069311; Yeo and Whitman, 2001, Moi. Cell 7: 949-957; Oh et at, 2002, Genes Dev. 16:27490 54). ALK4 is the primary type 1 receptor for aetivins, particularly for activin A, and ALK.-7 may serve as a receptor for aetivins as well, particularly for activin B.

As demonstrated herein, a soluble ActRHa polypeptide (sAetRlla), which shows substantial preference in binding to aetivin A as opposed to other TGF-beta family members, such as GDFS or GDP.11, is effective to promote hone growth and increase hone density in vivo. While not wishing to be bound to any particular mechanism, it is expected that the effect of sActRl la is caused primarily by an aetivin antagonist effect, given the very strong activin binding (picomolar dissociation constant) exhibited by the particular sAetRlla construct used in these studies. Regardless of mechanism, it is apparent from the data presented herein that ActRlla-aetlvin antagonists do Increase hone density in normal mice, in mouse models for osteoporosis and in a mouse model of multiple myeloma. It should be noted that bone is a dynamic tissue, with growth or shrinkage and. increased or decreased

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2016202691 27 Apr 2016 density depending on a balance of factors that produce bone and stimulate mineralization (primarily osteoblasts) and factors that destroy and demineralize hone (primarily osteoclasts). Bone growth and mineralization may be increased by increasing the productive factors, by decreasing the destructive factors, or both. The terms “promote bone growth” and “increase 5 hone mineralization” refer to the observable physical changes in bone and are intended to be neutral as to the mechanism by which changes in bone occur,

The mouse models for osteoporosis and bone growth/density that were used in the studies described herein are considered to be highly predictive of efficacy in humans, and therefore, this disclosure provides methods for using AetRIia polypeptides and other aetivin0 ActROa antagonists to promote bone growth and increase bone density in humans, AetivinAetRIia antagonists include, for example, aefivln-binding soluble ActROa polypeptides, antibodies that bind to aetivin (particularly the aetivin A or B subunits, also referred to as βΑ or βΒ) and disrupt AetRIia binding, antibodies that bind io AetRIia and disrupt aetivin binding, non-antibody proteins selected for aetivin or AetRIia binding (see e.g.,

WO/2O02/O88 i71, WO/2O06/055689,. and WO/2002/032925 for examples of such proteins and methods for design and selection of such non-antibody affini ty binding reagents), and randomized peptides selected for aetivin or AetRIia binding, often affixed to an Fe domain. Two different proteins (or other moieties) with aetivin or AetRIia binding activity, especially aetivin binders that block the type I (e.g,, a soluble type I aetivin receptor) and type II (e.g., a 0 soluble type II aetivin receptor) binding sites, respectively, may be linked together to create a bifunctionai binding .molecule.· Nucleic acid aptamers, small molecules and other agents that inhibit the activin-ActRO.a signaling axis. Various proteins have aetivin-AetRIla antagonist activity, including inhibits (he,, inhibin alpha subunit), although, inhibin does not universally antagonize aetivin in all tissues, follistabn (e.g,, foihstafin-288 and follistatin-315), FSRP, aetivin C, aipha(2)~maorogiobuiin, and an Ml OSA (methionine to alanine change at position 108) mutant aeti vin A. Generally, .alternative forms of aetivin, particularly those with alterations in the type I receptor binding domain can bind to type H receptors and fad to form an active ternary complex, thus acting as antagonists. Additionally, nucleie acids, such as antisense molecules,. siRNAs or ribozymes that Inhibit aetivin A, B, C or £, or, particularly, 30 ActRHa expression, can be used as ael^sn-AeilUla antagonists. Preferably, the aetivinAetRIla antagonist to be used will exhibit selectivity for inhibiting aetivln-mediated signaling versus other members of the TGF-beta family, and particularly with respect to GDF8 and - 13 2016202691 27 Apr 2016

GDFH, Soluble AetRllb proteins do hind to activin, however, the wild type protein does not exhibit significant selectivity in binding to activiu versus GDFS/11. and preliminary experiments suggest that this protein does not provide the desired effects on bone, while also causing substantial muscle growth. However, altered forms of AetRllb with different binding 5 properties have been identified (see, e.g,, WO 2006/012627, pp. 55-59, incorporated herein by reference) and these proteins may achieve the desired effects on bone. Native or altered ActRfib may he given added specificity for acts vie by coupling with a second, activinsefective binding agent.

The terms used in this specification generally have their ordinary meanings in 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 specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them. The scope or meaning of any use of a term will be apparent (fern the specific context in which the term is used, “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a gi ven value or range of values.

Alternatively, and particularly in biological systems, the terms about” and .0 approximately” may mean values that ate within an order of magnitude, preferably within 5fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about⁵' or ‘approximately⁵' can be inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences to each other, Including wild-type sequence to one or more mutants .(sequence variants). Such comparisons typically comprise alignments of polymer sequences, e.g.» using sequence alignment programs and/or algorithms that are well known in the art (tor example, BLAST, FASTA and MEG ALIGN, to name a few). The skilled artisan can readily appreciate that, in such alignments, where a mutation contains a residue insertion or deletion, the sequence alignment will introduce a gap” (typically represented by a dash, or “A”) in the polymer sequence not containing the inserted or deleted residue.

-142016202691 27 Apr 2016 “Homologous,”'in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evolutionary origin,” including proteins from superfamdies in the same species of organism, as wel l as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have 5 sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.

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 usage and m the instant application, the term “homologous?⁵ when modified with an adverb such as “highly?’ may refer to sequence similarity and may or may not relate to a common evolutionary origin.

2. ActRlla Polypeptides

In certain aspects, the present invention relates to ActRlla polypeptides. As used herein, the term “ActRlla” refers to a family of activin receptor type Ila (ActRlla) proteins from any species and variants derived from such ActRHa proteins by mutagenesis or other modification . Reference to ActR l la herein is understood to be a reference to any one of t he currently identified forms. Members of the ActRlla family are generally transmembrane proteins, composed of a hgand-hindiug .extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serifte/threomne kinase activity.

The »t*nn “ActRlla polypeptide includes polypeptides comprising an\ naturally occurring polypeptide of an ActRlla family member as well as any variants thereof (including mutants, fragments, fusions, and peptidotnimetic forms) that retain a useful activity. For example, ActRlla polypeptides include polypeptides derived from the sequence: of any known ActRHa. having a sequence at least about 80% identical to the sequence of an ActRlla polypeptide, and preferably at least 85%. 90%, 95%, 97%, 99% or greater identity. For example, an ActRlla polypeptide of the invention may hind to and inhibit the function of an. ActRHa protein and/or activin. Preferably, an ActRlla polypeptide promotes bone growth

152016202691 27 Apr 2016 and. bone mineralization. Examples of AetR.Ua polypeptides include human AetRlla precursor polypeptide (SEQ ID NO: I) and soluble human AetRlla polypeptides (e.g,, SEQ ID NOs: 2, 3, 7 and 12),

The human AciRHa precursor protein sequence is as follows;

⁵ NGAAARLAFAyFLXGGGEGAXLGRSEYQECLrgKANWEKDRTNQTGVEF

CYGDNDKHRHCFATWKKX SGS IB! VK.QGCWWD1 NCXDRTDCV32KKDSP EVX FCCCEGNMCNBKFSYFPEaffiVTQFTSNFVTPKFFYYNIu g v GRVpL

ML I AG IV XCAFWVYRHHKMAY P PVLVPTQDPG RP PPS PL.LGLK RLQLLE VKARGEFGCVW'KAQLI.EEYVAVKXFP.XQDK'QSbIQRSYE\.^?YSLPGMKHER

l.LQFIGAEKRGTSVDVDLWLITAEHEKGSLSDFLKANWS^NELCHIAE

TRARGLAYLHEDXPGLKDGHRPAXSHRDXKSKEVLlKhNLTAClADFGL ZiLKFEAGKSAGDTHGQVGTRRYMAPEY’LEGAlRFQRDAFLRXDMYAMGL VLWELASRCTAA'DGRyDBYMLRPEEElGQH'RSLEDMQEVVVHKKK'RFVL RDYWQRHAGMAMLCETXEECWDHDAEARLEAGCVGERXTQMQRLTNIIT

TEDIVTWTMVTNVDFRPKESSL (SEQ ID NO; .1)

The signal peptide is single underlined; the extracellular domain is in bold and the potential N-linked glycosylafion sues ,ue double underlined.

The human AetRlla soluble (extracellular.}, processed polypeptide sequence is as follows;

XLGRSETQECLFFNAEWEKDRTEQTGVEFCyGDKDRRRKCX’ATViFN 1SG SXEIYKQGCWWDXNCYQRTDCVBKEDSPEVYPCCCEGERCNEKESYEP ^f'^SEQ ^{U) NO;} 2)

The C-iemiinal “tail” of the extracellular domain is underlined. The sequence with, the “tail” deleted (a Δ15 sequence) is as fellows:

ILGRSETQECLFEFAENEKDRTEGTGVEFCYGDKDKERHCPATWKRXEG SIEIVRQGCWEDDIECYDRTDCVEKKDSPEVYFCGCEGNMCEEKESYFP

EM (SEQ1D NOG)

The nucleic acid sequence encoding human AetRlla precursor protein is as feliows(nncleotides 164* 1705 of Genbank entry NMDX) 1616);

ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATCTCCTGTTCTYCAGGYGC

TATACTTGGTAGATCAGAAACTCAGGAtSTGTCTTTTCTTTAATGCTAATTGGGAWtAG

ACAGAACCAA'rCAAACTGGTG'rTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCAT

TGTTTTCCTACCTGGAAGAATATTTCTGGTYCCATYGAAATAGTGAAACAAGGTTGTTG * 16 2016202691 27 Apr 2016

GC'rGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTG AAGTATATTTTTGTT«3CTGT<2AGGGCAATATG?GTAATG.AAAAG?TT^rfCTTATTTTCCA v ·, 4 „ vj -. % t 'i · ·, K \ V ’ c j \ , ν'. V < Λ

CATCCTGCTC’rATTCCTTGGTGCCACTTATG'TTAATTGCSGGGATTGTCATTTGTGCAT 5 TTTGGGTGTACAGGCATCACAAGATGSCCTACCCTCCTGTACTTGTTCCA&C’rCA&d&C

CCAGGACCACCCCCACCTfCTCCATl'AG'PAGGGTTGAAACCACTGCAGTTATTAGAAGT GAAAGCAAGGGGAAGAT’TTGGTTGTGTCTGG.AAAGCCCAGTTGCTTAACGAATATGTGG CTGTCAAAAY&TTTCGAAT&CAGGACAAAC&G’FCATGGCAAAATGAATACGAAGTC'rAC agtttgcctggaatgaagcatgagaacatattacagttcattggtgcagaaaaacgagg 0 CACCAGTGTTGATGTGGATCTTTGGCTGAGCAGAGCArTTCArGAAAAGGGTTGAGTAT

CAGACTTTCTTAAGGCTAATGTGGTCTCTTGGAATGAACTGTGTCA'rATTGCAGAAACC ATGGCTAGAGGATTGGCATA'rTTACATGAGGATA'fACCTGGCCTAAAAGATGGCCACAA ACCTGCCATATCTCACAGGGACATGAAAAGTAAAAATGTGCTGTTGAAAAACAACCTGA CAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATTTGAGGCTGGCAAGTCTGCAGGC 5 GATACCCA'i'GGACAGGTTGGTACCCGGAGGTACATGGGTCCAGAGGTATTAGAGGGTGC

TATAAACTTCCAAAGGGATGGATTTTTGAGGATAGA’rATGTATGCCATGGGAGTAGTCC TATGGGAACTGGC-TTCTCGCTGTACTGGGGCAGATGGACCTGTAGATGAATAGAGGTTG CCATTTGAGGAGGAAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTG? GCATAAAAAAAAGASGCCTGTTTTAAGAGATTATTGGGAGAAACATGCTGGAATGGCAA.

TGCTCTGTGAAAGCA'rTGAAGAATGG'rGGGATGACGACGCAGAAGCCAGGTTATCAGCT

GGATGTGTAGGTGAAAGAATTACCCAGATGCAGAGACTAACAAATATTATTACCACAGA GGAGATGGTAACAGTGGGGACAATGGTGACAAATGTTGAGT'rGCCTCCCAAAGAATCTA T \/i AT ί·. J A.. (SEQ ID NO: 4)

The nucleic acid sequence encoding a human ActRIla soluble (extracellular) S polypeptide is as follows:

atagttggtagatcagaaactcaggagtgtcttttcttgaatgctaattgggaaaaaga

CAGAACCAATCAAACTGGTGTTGAACCGTGTTATCGTGACAAAGATAAACGGCGGQATT ' G’TTTTGCTACCTGGAAGAATATTTCTGGS’TCCATTGAAATAGtGAAACAAGGTTGTTGG

CTGGATGATATCAACGGC'rATGACAGGACTGAT'rGTGTAGAAAAAAAAGACAGCCCTGA

AGTATATTTTTGGTGCTGGGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAG

AGATGGAAGTCACACAGCGCACTTCAAATCCAGTTACACCTAAGCCACCC (SEQ ID NO. S)

In a specific embodiment, the invention relates to soluble ActRIla polypeptides, As described herein, the term “soluble ActRIla polypeptide⁵' generally refers to polypeptides comprising an extraeel tolar domain of an ActRIla protei n. The term 'soluble ActRIla polypeptide, as used herein, includes any naturally occurring extracellular domain of an

-172016202691 27 Apr 2016

Actllila protein as well as any variants thereof (including mutants, fragments and peptidomimetic forms), Aft aetivin-hinding AetRlla polypeptide is one that .retains the ability to bind to acdvin, particularly activin A A, AB orBB. Preferably, aa aotivin-bniding AetRlla polypeptide will hind to activin AA with a dissociation constant of I nM or less. Amino acid 5 sequences of human AetRlla precursor protein is provided below.. The extracellular domain of an AetRlla protein binds to activin and is generally soluble, and thus can he termed a soluble, aetivin-brnding AetRlla polypeptide. Examples of soluble, aetivin-bmding ActR.Oa pclvpeptnles un-iudc dw smnh'.e pnlvpepnde diustr Fed ® SI Q IP N< A. 2, T T 12 «md 1 SEQ ID NO.? is referred to as ActRUa-hPe. and is described further In the Examples. Other 0 examples of soluble, acdvm-bi.nding AetRlla polypeptides comprise a signal sequence in addition to the extracellular domain of an AetRlla protein, for example, the honey bee rnehitin leader sequence (SEQ ID NO: 8), the tissue plarnmogen activator {TPA) leader (SEQ ID NO: 9) or the native .AetRlla leader (SEQ ID NO: 10}.. The A efRIIadiFc polypeptide illustrated, in SEQ ID NO: 13 uses a TEA leader.

Functionally active fragments of ActRI la polypeptides can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an AetRlla polypeptide. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moe or t~Boo chemistry. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function as antagonists (inhibitors) of AetRlla protein or signaling mediated by activin.

Functionally active variants of AetRlla polypeptides can be obtained by screening libraries of modified polypeptides recombinantly produced from the corresponding mulagenixed nucleic acids encoding an AetRlla polypeptide. The variants can be produced and tested to identify those that can frmetion as antagonists (inhibitors) of AetRlla protein or signaling mediated by activin. In certain embodiments, a functional variant of the AetRlla polypeptides comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3. 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 front SEQ ID NOs: 2 or 3.

- 182016202691 27 Apr 2016

Functional variants may be generated by modifying tbe structure of an ActRIla polypeptide for such purposes as enhancing therapeutic efficacy, or stability - e.g., ex vivo shelf life and resistance to proteolytic degradation In vivo). Such modified ActRIla polypeptides when selected'to .retain aefivin binding, are considered -functional equivalents of 5 the naforally-occumng ActRIla polypeptides. Modified ActRIla polypeptides can also be produced, for instance, by amino acid substitution, deletion, or addition. For instance, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with, a glutamate, a threonine with a serine, or a similar replacement of an. amino acid with a structurally related amino acid (e.g,, conservative mutations) will not have a 0 major effect on the biological activity of the resulting molecule·. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether a change In the amino acid sequence of an ActRHa polypeptide results in. a functional homblog can be readily determined by assessing the ability of the variant ActRIla polypeptide to produce a response in cells in a fashion similar to the wild-type·. ActRIla 5 polypeptide.

in certain embodiments, the present invention contemplates specific mutations of the ActRIla polypeptides so ns to alter the glyeosylation. of the polypeptide. Such .mutations may be selected so as to introduce or eliminate one or more glyeosylation sites, such as O-ltnked or N-linked glyeosylation sites. Asparagine-linked glyeosylation recognition sites generally comprise a tripeptide sequence, asparaglne-X-ihreonine (or asparagines-X-serine) (where “X” Is any amino acid) which is 'specifically recognized by appropriate cellular glyeosylation enzymes. The alteration may also bo made by the addition ofi or substitution by,, one or more serine or threonine residues to the sequence of the wild-type ActRIla polypeptide (for O linked glyeosylation sites), A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a gfycosylafion recognition site i and/or amino acid deletion at the second position) results In non-glycosylation at the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties co an ActRIla polypeptide is by chemical or enzymatic coupling of glycosides to the ActRIla polypeptide, Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (e) free: sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine,-threonine, or hydroxy pro line; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of

- 192016202691 27 Apr 2016 glutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston (1981) CRC Crit. Rev., Biochenn, pp, 259-306. incorporated by reference herein. Removal of one or more carbohydrate moieties present on an .ActRlla polypeptide may be accomplished chemically and/or enzymatically. Chemical degiyeosyfotfon may 5 involve, for example, exposure of the ActRHa polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or ah sugars except the linking sugar (N-aeetylglueosamine orNacetylgalactosamine), while leaving the amino acid sequence intact. Chemical deglycosylatfon is -further described by Hafcimuddin et al. (1987) Arch. Rioehem. Biophys,

259:52 and by Edge et al. (1981) Anal, Rioehem, 118:131, Enzymatic cleavage of carbohydrate moieties on ActRlla polypeptides can be achieved by the use of a variety of endo- and exo-glyeosidases as described by Thotakura et ah (1987) Meth, Errxytnoi. 138:350. The sequence of an ActRlla polypeptide may be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect and plant cells may all Introduce 5 differing giycosylation patterns that can be affected by the amino acid sequence of the peptide. In general, ActRlla proteins for use in humans will be expressed in a mammalian ceil tine that provides proper giycosylation, such as ΗΈΚ293 or CHO cell lines, although other mammalian expression cell lines, yeast cell lines with engineered giycosylation enzymes and Insect cells are expected to be useful as well.

This disclosure further contemplates a method of generating mutants, particularly sets .d „omh.nafooal mamma fo .u ActRlla pfevpepmk, as we I <w truncation mutants; pools of combinatorial mutants are especially useful for identifying functional variant sequences. The purpose of screening such combinatorial libraries may he to generate, for example, ActRlla polypeptide variants which can act as either agonists or antagonist, or alternatively, which possess novel activities all together, A variety of screening assays are provided 'below, and such assays may be used to evaluate variants. For example, an ActRlla polypeptide variant may be screened for ability to bind to an ActRlla ligand, to prevent binding of an ActRlla ligand to an ActRiia polypeptide or to interfere with signaling caused by an ActRlla ligand.

The activity of an ActRlla polypeptide nr its variants may also be tested in a cell30 based or in vivo assay. For example, the effect of an ActRiia polypeptide variant on the expression of genes involved in bone production or bone destruction may be assessed. This

- 20 2016202691 27 Apr 2016 may, as needed, he performed Ift the presence of one or more recombinant AetRIIa ligand proteins (e.g,, activin), and cells rnay be transfected so as to produce an AetRIIa polypeptide and/or variants thereof,, and optionally, an AetRIIa ligand. Likewise, an AetRIIa polypeptide may be administered to a mouse or other animat and one or more bone properties, such as 5 density or volume may be assessed. The healing rate for bone fractures may also be evaluated. Dual-energy x-ray absorptiometry (DEXA) is a. wen-established, non-invasive, quantitative technique for assessing bone density in an animal. In humans central DEXA systems may be used to evaluate bone density In the spine and pelvis. These are the best predictors of o verall bone density. Peripheral DEXA systems may be used to evaluate hone 0 density in peripheral bones, including, for example, the bones of the hand, wrist, ankle and foot. Traditional x-ray imaging systems, including CAT scans,, may be used to evaluate hone growth and fracture healing. The mechanical strength of bone may also be evaluated,

Combinatorially-derived variants can be generated which have a. selective or generally increased potency relative to a naturally occurring AetRIIa polypeptide. Likewise, mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding a wild-type AetRIIa polypeptide. For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction of, or otherwise inactivation of a native AetRlia polypeptide. Such variants, and the genes vdach encode them, can be utilized to alter

AetRIIa polypeptide levels by modulating the half-life of the AetRIIa polypeptides. For instance, a short bait-life can give rise to more transient biological effects and can allow tighter control of recombinant AetRIIa polypeptide levels within the patient. In an Fc fusion protein, mutations may be made in the linker (If any) and/or the Fc portion to alter the halflife oft.be protein.

A combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion, of potential AetRIIa polypeptide sequences. For Instance, a mixture of synthetic oiigonuci.eot.ides can be enzymatically ligated into gene sequences such that the degenerate set of potential AetRIIa polypeptide nucleotide sequences are expressible as Individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g,, for phage display).

2016202691 27 Apr 2016

There are many ways by which the library of potential homologs ears be generated from, a degmerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then he ligated into an appropriate vector for expression, The synthesis of degenerate 5 oligonucleotides is well-known in the art. (see for example, Narang, SA. (1983) Tetrahedron bt: Itakura et al., (19811 Recombinant DNA, Proc. 3rd Cleveland Sympos.

Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp2?3~28.9; Itakura et at, (1984) Anno, Rev. Biochem. 53:323; Itakura et at, (1984) Science I98: 1056; Ike et ah, (1983) Nucleic Acid Res, 11:477). Such techniques have been employed in the directed evolution of 0 other proteins (see, for example, Scott et al,, (1990) Science 249:386-390; Roberts et at, (1992) FNAS USA 89:2429-2433; Devlin et al, (1990) Science 249: 404-406; Cwirla et at, (1990) PNAS USA 87: 6378-6382; as well as UR, Patent Nos: 5,223,409, 5,198,346, and $,096,815),.

Alternatively, other tonus ofnuuagenesis ears be utilized to generate a combinatorial 5 library. For example, ActRlla polypeptide variants can be generated and Isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et at, (1994) Biochemistry 33:4565-1572; Wanget al., ¢1994) j. Biol. Cheat 269:3095-3099; Baliatet al.. i1993) Gene 137:109-118: Gmdhcrg et ah. (1993) Fur. J. Biochem. 218:597601; Nagashima et. at, (1993) J. Biol. Chem, 268:2888-2892; Lowman et al,, (199.1)

Biochemistry 30:10832-40838: and Cunningham et al, (1989) Science 244:4081-1085), by linker scanning mutagenesis (Gustin et ah, (1993) Virology 193:653-660; Brown et ah, (1992} Mot Cell Biol. 12:2644-2652; MeKnight et al._s (1982) Science 257 U6); by saturation mutagenesis (Meyers et ah, ¢1986) Science 232:613); by PCR mutagenesis (Leung et si., (1989) Method Coll Mol Biol 1:11-19); or by random mutagenesis, including chemical mutagenesis, etc, (Miller et ah, (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et at, ( .1994} Strategies in Mol. Biol 7:32-34). Linker scanning mutagenesis, particularly in a combinatorial setting, Is an attractive method for identifying truncated (bioactive) forma of AetRBa polypeptides.

A wide range of techniques are known in the art for screening gene products of .30 combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will

-222016202691 27 Apr 2016 be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of AetRila polypeptides. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and 5 expressing the combinatorial genes under conditions in which detection of a desired acti vity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferred assays include activin binding assays and aetivrn-niediated cell signaling assays.

in certain embodiments, the ActR.Ha polypeptides of the invention may further comprise post-translational modifications in addition to any that are naturally present in the AetRila polypeptides. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipldation, and acylation. As a result, the modified AetRila polypeptides may contain non-amino acid elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. Effects of such non-amino acid elements on the functionality of a ActRITa polypeptide may be tested as described herein for other ActR Ila polypeptide variants. When an AetRila polypeptide is produced in cells by cleaving a nascent form of the AetRila polypeptide, post-translafionai processing may also be important for correct folding and/or function ofthe protein. Different cells (such as CH'O, ffeta, MDCK, 293, WI3S, NIH-3T3 or HEK293) have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to. ensure the correct modification and processing of the AetRila polypeptides.

In certain aspects, functional variants or modified forms of the AetRila polypeptides include fusion proteins having at least a portion of the ActR Ida polypeptides and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, polyhisfidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein <3, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin, A fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt- 'conjugated resins are used. Many of such matrices are available In “kit” form, such as the Pharmacia

-232016202691 27 Apr 2016

GST purification system and the QIAexpress^rM system (Qiagen) useful with (H1SQ fusion partners, As another example, a. fusion domain may he selected so as to facilitate detection of the AciRHa polypeptides. Examples of such detection domains include the various fluorescent proteins (e,g., OFF) as well as “epitope tags,” which are usually short peptide 5 sequences for which a specific antibody is available. Well known epitope tags for which specific monoclonal antibodies are readily available include FLAG, Influenza virus hacmaggiutmm tHA) and «.-roye tat> hi some ews the fusmn domains have .1 psek-ase cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the .recombinant proteins therefrom.

The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation, in certain preferred embodiments, an AetR I la polypeptide is fused with a domain that stabilizes the ActRIIa polypeptide in vivo (a “stabilizer” domain).

By “stabilizing¹ is meant anything that increases serum half life, regardless of whether this ts because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fe portion of an iumumoglobuhn 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 r-i fusion domains that may he selected include mnltlmerizing (e.g.., dimerizing, tetramerizing) domains and functional domains (that conifer an additional biological function, such as further stimulation of hone gro wth or muscle growth, as des ired).

As a specific example, the present invention provides a fusion protein comprising a soluble extracellular domain of AeiRlla fused to an Fe domain (e.g., SEQ ID NO: 6),

THTCF?CPAPELLGGPSVF1,FPPKPKDTLMISRTFSVTCWVD{A) VSHEDPEVRFWYVDG VSVdBAKTKPREEQypsiwpvVSVLTVLHQDWLMGKEYKCKiAiVSNKALPVPIEKTISKAK

EQPREPQVYTLPPSREEMTKrtQVSLTCLVKGPYPSDiAVEWESBGQPEBNYKTTPPVLOSDG PFELYSKLTVDKSPWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

Optionally, the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domain having one or more of these mutations (e.g., Asp-265 mutation) has reduced aluhty of binding to the Fey receptor relative to a wildtype Fc domain, in other cases, the mutant Fc domain ha ving one or more of these

2016202691 27 Apr 2016 mutations (e.g., .Asn -434 mutation) has increased ability of binding to the MHC class F related Fc-receptor (FcRN) relative to a wildlype Fc domain.

It is understood that different elements of the fusion proteins may be arranged, in any manner that is consistent with the desired functionality. For-example,.an ActRHa polypeptide 3 may be placed C-terminal to a heterologous domain, or, alternatively, a heterologous domain may he placed C-terminal to an ActRHa polypeptide. The ActRIla polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C~ or N-terminal to either domain or between the domains,

Q In certain embodiments, the ActRHa polypeptides of the present invention contain one or more modifications that are capable of stabilizing the ActRIla polypeptides. For example, such modifications enhance the in vitro half life of tbe ActRIla polypeptides, enhance circulatory' half life of the ActRHa polypeptides or reduce proteolytic degradation of the ActRIla polypeptides. Such stabilizing modifications Include, but are not. limited to, fusion proteins (including, for example, fusion proteins comprising an ActRIla polypeptide and a stabilizer domain), modifications of a giycosylation site (including, for example, addition of a glycosyiaiion site to an ActRIla polypeptide), and modifications of carbohydrate moiety (including, for example, removal of carbohydrate moieties from an ActRIla polypeptide). In the case of felon proteins, an ActRHa polypeptide is fused to a. stabilizer domain such as an

IgG'molecule (e.g., an Fc domain). As used herein, the term “stabilizer domain’’ not only refers to a fusion domain (e.g., Fc) as in. the case of fusion proteins,, but also includes nonprofetnaceous modifications such as a carbohydrate moiety, or nonproteinaceous polymer, such as polyethylene glycol.

In certain embodiments, the present invention makes available Isolated and/or purified forms of the ActRHa polypeptides, which are isolated from, or otherwise substantially free of, other proteins. ActRIla polypeptides will generally be produced by expression from reeombi n an t n ncl ei c acid s.

2016202691 27 Apr 2016

3. Nucmw Acids Encoding, ActRIht Polypeptides

In certain aspects, the invention provides isolated and/or recombinant nucleic acids encoding any of the ActRlla polypeptides (e.g,, soluble ActRlla polypeptides), including fragments, functional variants and fusion proteins disclosed herein. For example, SEQ IB 5 NO; 4 encodes the naturally occurring human ActRlla precursor polypeptide, while SEQ IO NO; 5 encodes the processed extracellular domain of ActRlla. The subject nucleic acids may be single-stranded or double stranded. Such nucleic acids may be DN A or RNA molecules. These nucleic acids may be used, for example, in methods for making ActRlla polypeptides or as direct therapeutic agents (e.g., in a gene therapy approach).

In certain aspects, the subject nucleic acids encoding ActRHa polypeptides are further understood to include- nucleic acids that are variants of SEQ ID NO: 4 or 5. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants.

In certain embodiments, the Invention provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 9?%,- 98%, 99% or 100% identical to SEQ ID NO; 4 or 5. One of ordinary skill in the art will appreciate that-nucleic acid sequences complementary to SEQ ID NO: 4 or 5, and variants of SEQ ID NO: 4 or 5 are also within the scope of this invention. In further embodiments, the nucleic acid sequences of the invention can he isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a

DNA library.

In other embodiments, n ucleic acids of the invention also Include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NO: 4 or 5, complement sequence of SEQ ID NO: 4 or 5, or fragments thereof. As discussed above, one of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied. One of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can he varied. For example, one could perform the hybridization at 6.0 x sodium ehloride/sodlum citrate (SSC) at about 45 °C, followed by a wash of 2.0 x SSC- at SO °C. For example, the salt concentration In the wash step can he selected from a low stringency of about 2.0 x SSC at SO °C to a high stringency of about 0,2 x

SSC at 50 ^eC. in addition, the temperature in the wash step can he Increased from low • 26 2016202691 27 Apr 2016 stringency conditions at room temperature, about 22 °C, to high stringency conditions at about 65 °C, Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed, In one-embodiment, the invention provides nucleic acids which hybridize under lew stringency conditions of 6 x SSC at room S temperature followed by a wash at. 2 x SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ ID NOs; 4 or 5 due to -degeneracy in the genetic code are also within the scope of the invention. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, C AU and CAO are synonyms for 0 histidine) may result in feilenf ’ mutations which do not affect the amino acid sequence of the protein,- However, it is expected that DNA sequence polymorphisms that do lead to changes m the amino acid sequences ofthe subject proteins will exist among mammalian ceils. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding a particular protein may exist among 5 individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this 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. Regulatory nucleotide sequences will generally be appropriate to the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, said one or more regulatory nucleotide sequences may include, hut are not limited to, promoter sequences, leader or signal, sequences, ribosomal, binding sites, transcriptional start and termination sequences, translational start and termination. sequences, and enhancer or activator sequences,

Constitutive or inducible promoters as known in the art. are contemplated by the invention. 'Die promoters may he cither naturally occurring promoters, or hvnod promoters that combine elements of more than one promoter. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may he inserted in a chromosome, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. -'Selectable marker genes are well known in the art and will vary with the host cell used.

-272016202691 27 Apr 2016 la certain aspects of the invention, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding an AetRIla polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of the AetRIla polypeptide. Accordingly, the term 5 regulatory sequence includes. promoters, enhancers, and other expression control elements. Exemplary regulatory sequences arc described in Goeddel; Gene Exprexfow? Ibchnofogy; Afo/kods /«Academic Press, San Diego, CA (19.90), For instance, any of a wide variety of expression control sequences that, control the expression of a DN A sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding an 0 AetRIla polypeptide. Such useful expression control sequences, include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T? RNA. polymerase, the major operator and promoter regions of phage lambda , the control regions for fd coat, protein, the promoter for 5 3-phospboglyceraie kinase or other glycolytic enzymes, the promoters of acid phosphatase, eg.. PhoS, the promoters of the yeast ά-mating factors, the polyhedron promoter of the hacufovlrus system and other sequences kno wn to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof It should be understood that the design of the expression vector may depend on such factors as the 0 choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such: as antibiotic markers, should also he considered,

A recombinant nucleic acid of the in vention can be produced by ligating the cloned 25 gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian},. or both. Expression vehicles for production of a recombinant AetRIla polypeptide include plasmids and other vectors. For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBLderived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derlved plasmids lor expiession in prokaryotic cells, such as £'. co.//.

-282016202691 27 Apr 2016

Some mammalian expression vectors'contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that, are expressed in eukaryotic cells. The peDNAE'anip, pcDNAI/neo, pRe/CMV, pSV2gpt, pSVlneo, pSV2-dh.fr, pTk2, pRBVneo, pMSG, pSVT7, pko-neo and pMyg derived vectors > are examples of mammalian expression vectors suitable for transfection of euka*volte cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pSR322, to facilitate replication and drug resistance selection In both prokaryotic and eukaryotic ceils. Alternatively, derivatives of viruses such as the bovine papilloma virus (BRV-1), or EpsteinBarr virus (pHEBo, pREP-derived and p205) can he used for transient expression of proteins 3 in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in. the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Afofocafor Cfonmg .,4

Lah&ratary Manual, 3rd Ed., ed. by Sambrook, Fritsch and Mania!is (Cold Spring Harbor Laboratory Press, 2001). in some instances, it may be desirable to express the recombinant polypeptides by the use of a baeulovirus expression system. Examples of such baeulovirus expression systems include pVL~de.rived vectors (such as pVL'1392, pVL!393 and pVL94l), pAcUW-derived vectors (such as pAeUWi), and pBlueBac-derived vectors (such as the E-gal containing pBlueBac III).

in a preferred embodiment, a vector will be designed for production of the subject ActRila polypeptides in CHO ceils, such as a Pcrov-Seript vector (Straiagene, La folia,

Calif), peDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCl-neo vectors (Promega, Madison, Wise,). As will be apparent, the subject gene constructs can he used to cause expression ofthe subject ActRila polypeptides in ceils propagated in culture, e.g», to produce proteins, including fusion proteins or variant proteins, for purification.

This disclosure also pertains to a host cell transfected with a recombinant gene including a coding sequence (e.g., SEQ ID NO: 4 or 5} for one or more of the subject ActRila polypeptides. The host cell may be any prokaryotic or eukaryotic cell. 'For example, an

ActR.Ha polypeptide of the invention may be expressed in bacterial cells such as S, eo/i,

2016202691 27 Apr 2016 insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

Accordingly, the present invention further pertains to methods of producing the subject ActRHa polypeptides,. For'example, a host cell transfected with an expression vector 5 encoding an ActRHa polypeptide can be cultured under appropriate conditions to allow expression of the ActRHa polypeptide to occur. The ActRHa polypeptide may be secreted and isolated from a mixture of cells and medium containing the ActRlla polypeptide. Alternatively, the ActRlla polypeptide may be retained eytoplasmieally or in a membrane fiaeOon and thecelfe hai'ested, lysed nod the pro-tens isekned. A exil eulturo includes best 0 ceils, media and other byproducts. Suitable media for cell -culture are well known in the art. The subject. ActRHa polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, iromunoaffinity purification with antibodies specific for particular epitopes of the ActRHa 5 polypeptides and affinity purification with an agent, that binds to a domain fused to the

ActRHa polypeptide ie.g., a protein A column may be used to purify an ActRlla-Fc fusion). In a preferred embodiment, the ActRl la polypeptide is a fusion protein containing a domain which facilitates its purification. In a preferred embodiment, purification is achieved by a series of column chromatography steps, including, tor example, 'three or more of the 0 following, in any order: protein A chromatography, Q ^ephroo'-e chromatography, phenyl sepharose chromatography, size exclusion chromatography, and cation, exchange chromatography. The purification could be completed with viral filtration and buffer exchange. As demonstrated herein, AetR.Ha-h.Fc protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by Sl>$ PAGE. This 25 level of purity was sufficient to achieve desirable effects on hone in mice and an acceptable safety profile in mice, rats and non-human primates.

In another embodiment, a fusion gene coding tor a purification leader sequence, such as a po)y«(His/cnierokinase clea vage site sequence at the N-terminus of the desired portion of the recombinant ActRlla polypeptide, cars allow purification of the expressed fusion protein by affinity chromatography using a NR* metal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide tbe

- 30 2016202691 27 Apr 2016 purified: ActRIia polypeptide:(e,g„, see Hochuli et ah, (IPS?) J. Cfowmumgrnpijy 411:177; and Janknecbt et at, PZfofo' £ZS4 88:8972).

Techniques for snak ing fusion genes are well known, Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in 5 accordance with conventional techniques, employing biunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation, in another embodiment, the fusion gene can he synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR 0 amplification of gene ‘fragments can be carded out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate-a chimeric gens sequence (sec, for example, Cmrcnt Frozocofr m Mo/eca/er Sio/ogy, eds. Ausubel et ah, John Wiley & Sons: 1992),

A AfenafiycActmnmid^ActRM

The data presented herein demonstrates that antagonists of activin-ActRHa signaling can be used to promote bone growth and bone mineralization. Although soluble ActRIia polypeptides, and particularly Actrlla-Fc, are preferred antagonists, and although such antagonists may affect bone through a mechanism other than activin antagonism (e.g,, activin inhibition may be an indicator of the tendency of an agent to inhibit the activities of a spectrum of molecules, including, perhaps, other members of the TOF-beta soper.fimnly, and such collective inhibition may lead to. the desired effect on hone), other types of activinAetRIIa antagonists are expected to be useful, including anti-activin (e„g., A, B, C or E) antibodies, anti-'ActRIla antibodies, antisense, RNAi or ribozyme nucleic acids that inhibit the production of ActR.Ha and other inhibitors of activin or ActRIia, particularly those that disrupt activin-ActRIia. binding.

An antibody that is specifically reactive with an ActRIia polypeptide (e,g„ a soluble ActRIia polypeptide) and which either binds competitively to ligand with the-ActRIia polypeptide or otherwise inhibits AcfRUa-mediated signaling may be used as an antagonist of

AetRIIa polypeptide activities. Likewise, an antibody that is specifically reactive with an activin A polypeptide and which disrupts ActRIia binding may he used as an antagonist.

-31 2016202691 27 Apr 2016

By using immunogens derived from an ActROa polypeptide or an aetivin polypeptide, anti-protein/anti-peptide antisera, or'monoclonal antibodies can be made by standard protocols (see, for example, Antibodies; A Laboratory Manual ed, by Harlow and Lane (Cold Spring Harbor Press; I988)), A mammal, such as a mouse, a hamster or rabbit can be immunized 5 with an immunogenic form of the AetRIia polypeptide, an antigenic fragment which is capable⁴ of el iciting an. antibody response, or a fusion protein. Techniques for conferring immunogens city on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of an ActRl la. or aetivin polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by 0 detection of antibody titers in plasma o? serum. Man-hoi ELISA or other immunoassays can be used, with the immunogen as antigen to assess the. levels of antibodies.

following immunization of an animal with an antigenic preparation, of an AciRUa polypeptide, antisera can be obtained and, if desired, polyclonal antibodies can he isolated front the serum, To produce nwoct m n antibodies, antibody-producing cells (lymphocytes) S can be harvested from an immunized animal, and fused by standard somatic ceil fusion procedures with immortalizing cells such as myeloma ceils to yield hybridoma ceils. Such techniques are well known in the art, and include, for example, the hybridoma technique (origtftally developed by Kohler and Milstein, (197$) Nature, 256:495-497), the human B cell hybridoma technique (foozbar et al, (1^83) Immunology Today, 4: 72), and the BBV0 hybridoma technique to produce human, monoclonal antibodies (Cole et ah, (1985)

Monoclonal Antibodies and Cancer Therapy, -Alan R. Liss, Inc, pp, 77-96),, Hybridoma cells can be screened rmmunochemieaily for production of antibodies specifically reactive with an ActRHa polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.

The term “antibody⁵ as used herein is intended to include fragments thereof which are also specifically reactive with a subject polypeptide. Antibodies can he fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ah)y fragments can he generated by treating antibody with pepsin. The resulting F(abjy fragment can be treated to reduce disulfide bridges to produce f ab fragments. The antibody of the present invention is further intended to include bispecific, single-chain, chimeric, humanized and fully human molecules

-52 2016202691 27 Apr 2016 having affinity for an.ActRHa or activin polypeptide conferred by at least one CDR, region of the antibody. An antibody may further comprise a label attached thereto and able to be detected (e.g,, the label can be a radioisotope, fluorescent compound, enzyme or enzyme cofactor).

In certain embodiments, the antibody is a recombinant antibody, which term encompasses any antibody generated in part by techniques of molecular biology, including CDR-grafted or chimeric antibodies, human or other antibodies assembled from libraryselected antibody domains, single chain antibodies and single domain antibodies (e.g._s human Vh proteins or camelid V_Hu proteins). In certain embodiments, an antibody of the invention is a monoclonal antibody, and in certain embodiments, the invention makes available' methods for generating novel antibodies. For example, a method for generating a monoclonal antibody that binds specifically to an ActRlla polypeptide or activin polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the. antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the antigen. Once obtained, a hybridoma can be propagated in a cell culture, optionally m culture conditions where the hybrldoma-derived cells produce the monoclonal antibody that binds specifically to the antigen. The monoclonal antibody may be purified from the cell culture.

The adjective “specifically reactive with” as used in reference to an antibody Is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of Interest (e.g., an ActRlla polypeptide) and other antigens that are not of interest that the antibody is useful for, at minimum, detecting the presence of the antigen of i nterest in a particul ar type of biological sample. In certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity in binding may be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and cross30 reacting polypeptides. One characteristic that influences the specificity of an antihodymnrigen interaction is ibe affinity of the antibody for the antigen. Although the

2016202691 27 Apr 2016 desired specificity may be reached with a range of different affinities, generally preferred antibodies will have an affinity (a dissociation constant) of about 1 0'°, 10*'» 10'\ I0”’ or less. Given the extraordinarily tight binding bet ween activin and ActRHa, it is expected that a neutralizing anii-activin or anti-ActRlla antibody would generally have a dissociation constant of 10'^!° or less.

In addition, the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, if an antibody is to he used for binding an antigen in solution, it may be desirable to test solution binding, A variety of different techniques are available for testing interaction between antibodies and 0 antigens to identify particularly desirable antibodies. Such techniques include ELIS As,

V-x »ΐ surface plasrnon resonance binding assays (e.g., the Biaeore”'' binding assay, Biaeore AB, Uppsala, Sweden), sandwich assays (e.g,, the paramagnetic bead system of iOBN International, inc., Gaithersburg, Maryland), western blots, immunopreeipitahon assays, and immunohistochemistry.

Examples of categories of nucleic acid compounds that are activin or ActRlla antagonists include antisense nucleic acids, RNAi constructs and catalytic nucleic acid constructs. A nucleic acid compound may be single or double stranded, A double stranded compound may also include regions of overhang or non-complementarity, where one or the other of the strands is single stranded. A single stranded compound may include regions of self-complementarity, meaning that the compound forms a so-called “hairpin” or “stem-loop'⁵ structure, with a region of double helical structure, A nudetc acid compound may comprise a nucleotide sequence that is complementary to a region consisting of no more than 1000,. no more than 500, no more than 250, no more than 100 or no more than 50, 35. 30, 25,22, 20 or 18 nucleotides of the full-length ActRlla nucleic acid sequence, or activin βΑ or activin βΒ nucleic acid sequence. The region of complementarity will preferably be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, and optionally beiween 15 and 25 nucleotides. A region of complementarity may fall within an intron, a coding sequence or a noncoding sequence of the target transcript, such as the coding sequence portion. Generally, a nucleic acid compound will have a length of about 8 to about 500 nucleotides or base pairs in length, and optionally the length will be about 14 to about 50 nucleotides. A nucleic acid maybe a ONA (particularly for use as an antisense), RNA or

- 34 2016202691 27 Apr 2016

RNA:DNA. hybrid. Anyone strand may include' a mixture of DMA and RNA, as well as modified forms: that cannot readily be classified as either DNA or RNA. Likewise, a double stranded compound may he DN A :DNA, DMAtRMA or RMAtRMA, and any one strand may also include a mixture Of DNA and RNA, as well as modified .forms that cannot readily he 5 classified as either DMA or RN A, A nucleic acid compound may include any of a variety of modifications, including one or modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including internucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid). An antisense nucleic acid compound will preferably have a length of about 15 to about 30 nucleotides and will often contain one ot 0 more modifications to improve eharaeterisfies such as stability in the serum, in a cell or in a: place-where the compound is likely to be delivered, such as the stomach in the case of orally delivered compounds and the lung for inhaled compounds. In the case of an RNAi construct, the strand complementary to the target transcript will generally be RNA or modifications thereof. The other strand may he RNA, DMA or any other variation, 3'hc duplex portion of 5 double stranded or single stranded “hairpin” RN Ai construct wi ll preferably have a length of 18 to 40 nucleotides in length and optionally about 21 to 23 nucleotides in length, so long as It serves as a Dicer substrate, -Catalytic or enzymatic nucleic acids may be rihoxymes or DNA enzymes and may also contain modified forms. Nucleic acid compounds may inhibit expression of the target by about 50%, 75%, 90% or more when contacted with cells under 0 physiological conditions and at a concentration where a nonsense or sense control has little or no effect. Preferred concentrations for testing the effect of nucleic acid compounds arc I , 5 and 10 micromolar, Nucleic acid compounds may also be tested for effects on, for example, bone growth and mineralization.

In certain aspects, the present invention relates to the use of AetRIIa polypeptides (e.g.. soluble AetRIIa polypeptides) and activin polypeptides to identify compounds (agents) which are agonist or antagonists of the activin-AetRIIa signaling pathway. Compounds identified through this screening can be tested to assess their ability to modulate bone growth or mineralization in vitro, Optionally, these compounds can further he tested in animal models to assess their ability to modulate tissue growth in vivo.

- 35 ~

2016202691 27 Apr 2016

There are numerous approaches to screening for therapeutic agents: for modulating tissue growth by targeting activin and ActRHa polypeptides. In certain embodiments, highthroughput screening of compounds can be carried out to identify agents that perturb aciivin or ActRlla-medlated effects on bone. In certain embodiments, the assay is carried out to 5 screen and identify compounds that specifically inhibit or reduce binding of an ActRHa polypeptide to aedvin. Alternatively, the assay can be used to identify compounds that enhance binding of an ActRHa polypeptide to activia. In a further embodiment, the compounds can be identified by their ability to interact with an activin or ActRHa polypeptide.

A variety of assay formats will suffice and, In light of the present disclosure, those not expressly described herein will nevertheless be comprehended by one of ordinary skill in the art. As described herein, the test compounds (agents) of the invention may be created by any combinatorial chemical method. Alternatively, the subject compounds may he naturally occurring biomolecules synthesized in vivo or in vitro. Compounds (agents) to be tested tor

S their ability to act as modulators of tissue growth can be produced, for example, by bacteria, yeast, plants or other organisms (e,g., natural products), produced chemically (e.g,, small molecules, including peptidomimeiics}, or produced recombinantly, Tost compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomirnetics, sugars, hormones, and nucleic acid molecules. In a specific embodiment, the test agent is a small organic molecule having a molecular weight ofless than about 2,000 dal tons.

The test compounds of the invention can he provided as single, discrete entities, or provided in libraries of greater complexity, such as made by combinatorial chemistry. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and ether classes of organic compounds. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps. Optionally, the compounds may be optionally derivatizod with other compounds and have derivaiizmg groups that facilitate isolation of the compounds. Nonlimiting examples of derivahzing groups include biotin, fluorescein, digoxygenin, green fluorescent protein, isotopes, polyhisiidine, magnetic beads, glutathione S transferase (GST), phofoaetivatihle erossbnkers or any combinations thereof

- 362016202691 27 Apr 2016

In many drag screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may he derived with purified or semi-purified proteins, are often preferred as “primary” screens in that they can he generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity or hioavaiiahility ofthe test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect ofthe drug on the molecular target as may be manifest in an alteration of binding 0 affinity between an AetRila polypeptide and activin.

Merely to illustrate, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified ActRHa polypeptide which is ordinarily capable of binding to activin. To the mixture ofthe compound and AetRila polypeptide is then added a composition containing an ActRHa ligand. Detection and 5 quantification of ActRIIa/aefi vin -complexes provides a means for determining the compound's efficacy at inhibiting (or potentiating) complex formation between the .ActRHa polypeptide and activin. The efficacy ofthe compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. For 0 example, in a control assay, isolated and purified activin is added to a composition containing the ActRHa polypeptide, and the formation of ActRHa/aetlvin complex is quantitated in the absence of the test compound, It will be understood that, in general, the order in which the reactants may be admixed can fee varied, and can be admixed sinmlianeously. Moreover, in place of purified proteins, cellular extracts and lysates may fee used to render a suitable cell25 free assay system.

Complex formation between the ActRHa polypeptide and activin may be defected fey a variety of techniques. For instance, modulation ofthe formation of complexes can be quantitated using, for example, detectafely labeled proteins such as radiolabeled (e.g,, ^j2P, ‘C or -fl), fiooreseently labeled {e.g., FITC), or enzymatically labeled AetRila polypeptide or activin, by Immunoassay, or by chromatographic detection.

2016202691 27 Apr 2016

In certain embodiments, the present invention contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays in measuring, either directly or indirectly, the degree of interaction between an.ActRHa polypeptide and its binding protein. Further, other modes of detection, such as those based on optical 5 waveguides (PCT Publication W(> 96/26432 and U.S. Pat. No. 5,677J96), surface olasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodiments of the invention.

Moreover, the present invention, contemplates the use of an interaction trap assay, also known as the “two hybrid assay/’ for identi lying agents that disrupt or potentiate interaction 0 between an ActRIla polypeptide and its binding protein. See for example, U.S, Pat, No. 5,283,317; Zervos et al, (19931C til 72:223-232; Madura et at (1993) J Biel Chem 268:12046-12.054; Bartel et a! 11 1) Biotedimqoes 14:920-924; and Iwabuchi et al. (1.99.3)

Ondogene 8:1693-1.696), In a specific embodiment, the present invention contemplates the use of reverse two hybrid systems to identify compounds (e.g.,, small molecules or peptides) that dissociate interactions between an ActRIla polypeptide and its binding protein. See tor example, Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Bioteohnoi 17:374-81; and U.S, Pat, Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain, embodiments, the subject compounds are identified by their ability to interact with an ActRIla. or activin polypeptide of the invention. The interaction between the compound and the AetRila or activin polypeptide may be covalent or non-eovalent. For example, sm.h uuwaohon can he idenirifrd U the proktn krel uxmg sn woo hioeketuwai methods, including photo-crosslinking, radiolabeled ligand binding, and affinity chromatography (Jakoby WB et ah, 1974, Methods in Enzymotegy 46; 1). In certain eases, the compounds may be screened in a mechanism based assay, such as an assay to detect compounds which bind to an activin or ActRIla polypeptide. This may include a solid phase or fluid phase binding event, Alternatively, the gene encoding an activin or ActRIla polypeptide can be transfected with a reporter system (e.g,, β-galaetosidase, luclferase, or green fluorescent protein) into a cell and screened against the library preferably by a high throughput screening or with individual members of the library. Other mechanism based binding assays may be used, for example, binding assays which detect changes in free energy. Binding assays can be performed with the target fixed to a well, bead or chip or ~ 38 2016202691 27 Apr 2016 captured by an immobilized antibody or resolved by capillary eieeirophoresis. The bound compounds may he detected usually using colorimetric or fluorescence or surface plasmon resonance. .

In certain aspects, the present invention provides methods and agents tor modulating 5 '(stimulatingor inhibiting) bone formation and increasing bone mass. Therefore, any compound identified can be tested in whole cells or tissues, in vitro or in vivo, to confirm their ability to modulate bone growth, or mineralization. Various methods known in the art can he utilized for this purpose.

For example, the effect, of the ActRlla or activin polypeptides or test compounds on 0 bone or cartilage growth can be determined by measuring induction of Msx2 or differentiation of osteoprogenitor cells into osteoblasts in cell based assays (see, e.g,, .Dalutski et at, Hat Cenet. 2001, 27(1):84-8:, Hino et al., Front Bioseh 2004, 9:1520-9). Another example of cell-based assays Includes analyzing the osteogenic activity of the subject ActRlla or activin polypeptides and test compounds in mesenchymal progenitor and 5 osteoblastic cells. To illustrate,, recombinant adenoviruses expressing an activin or ActRlla polypeptide can be constructed to infect pluripotent mesenchymal progenitor C3Hl QT1/2 cells, preosteoblastic C2C12 cells, and Osteoblastic TE-85 ceils. Osteogenic activity is then determined by measuring the induction of alkaline phosphatase, osteocalcin, and matrix mineralization (see, e.g., Cheng et ah, J bone Joint Surg Am, 2003, 85-A(8):l 544-52).

The present invention also contemplates in vivo assays to measure bone or cartilage growth. For example, Namkung-Mattbai et al., Bone, 28;80-8o <2001) discloses a rat osteoporotic model in which hone repair during the early period after fracture is studied.

Kubo et aL, Steroid Biochemistry & Molecular Biology, 68:197-202 (1999) also discloses a rat. osteoporotic model In which hone repair during the late period after fracture is studied,

Andersson et al,_s 1 Endocrinol. 170:529-537 describe a .mouse osteoporosis model in which mice are ovariectomixed, which causes the mice to lose substantial bone mineral content ami bone mineral density, with the trabecular bone losing roughly 50% of bone mineral density. Bone density could be increased in the ovarieetomized mice by administration of factors such as parathyroid hormone, In certain aspects, the present In vend on makes use of fracture healing assays that are known in the art. These assays include fracture technique, histological analysis, and biomechanical analysis, which are described in, for example, l.I.S. Pat. No.

- 39 2016202691 27 Apr 2016

6,521,750, which is incorporated by reference in its entirety for its disclosure of experimental protocols for causing as well as measuring the extent of fractures, and the repair process,

6- tembiyLlheweylfollses

Jn certain embodiments, acb vin~ActR.il a antagonists (e.g,, ActRHa -polypeptides) of the present invention can he used for treating or preventing a disease or condition that is associated with bone damage, whether, e.g., through breakage, loss or demineralization. As demonstrated herein, aetivin-AeiRila antagonists, and particularly ActRHa-Fc constructs, are effective in treating or preventing cancer-related bone loss. In certain embodiments, the present invention provides methods of treating or preventing bone damage in an individual in need thereof through administering to the individual a therapeutically effective amount of an activln-ActRiia antagonist, particularly an ActRHa polypeptide. In certain embodiments, the present invention provides methods of promoting bone growth or mineralization in an individual in need thereof through administering to the individual a therapeutically effective amount of an acti vin-A ctRIIa antagonist, particularly an ActRHa polypeptide, These methods are preferably aimed at therapeutic and prophylactic treatments of animals, and more preferably, humans. In certain embodiments, the disclosure provides for the use of aetivin-A ctRIIa antagonists (particularly soluble ActRHa polypeptides and neutralizing antibodies targeted to aetivin or ActRHa) for the treatment of disorders associated with low bone density or decreased bone strength.

As used herein, a therapeutic that ’'prevents” a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence ofthe disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms ofthe- disorder or eondufon relative to the untreated control 'sample. The term 'Treating” as used herein includes prophylaxis of the named condition or amelioration or elimination ofthe condition once it has been established. In either case, prevention or treatment may be discerned in the diagnosis provided by a physician and the intended result of administration ofthe therapeutic agent.

The disclosure provides methods of inducing bone and/or cartilage formation, preventing bone loss, increasing bone mineralization or preventing the demineralization of bone. For example, the subject aetivin-AeiRHa antagonists have application in treating ~ 40 2016202691 27 Apr 2016 osteoporosis and the hearing of bone fractures and-cartilage defects in humans and other animals. ActRlla or aetivin polypeptides may be useful in patients that are diagnosed with subclinieal low bone density, as a protective measure against the development of osteoporosis.

In one specific embodiment, methods and compositions oftbe present invention may find medical utility in the healing of bone fractures and cartilage defects in humans and other animals. Ihe subject methods and compositions may also have prophylactic use m closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma-induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery. In certain cases, the subject activin-AetRlie antagonists may provide an environment to attract bone-·fanning cells, .stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. Acfivm-ActRlla antagonists ol the invention may also be useful in the treatment, of osteoporosis.

Methods and compositions oftbe invention can be applied to conditions characterized by or causing bone loss, such as osteoporosis (including secondary osteoporosis), hyperparathyroidism, Cushing’s disease, Paget’s disease, thyrotoxicosis, chronic diarrheal state or malabsorption, renal tubular acidosis, or anorexia nervosa.

Osteopcm.sis may be caused by, or associated with, various factors. Being female..

particularly a post-menopausal female, having a low body weight, and leading a sedentary lifestyle are all risk factors for osteoporosis (loss of hone mineral density, leading to fracture risk). Persons having any oftbe .following profiles may be candidates tor treat·cent with an ActRlla antagonist: a post-menopausal woman and not taking estrogen or other hormone replacement therapy; a person with a personal or .maternal history of hip- fracture- or smoking;

a post-menopausal woman who is tall (over 5 feet 7 inches) or thin (less than 125 pounds): a man with clinical conditions associated with bone loss; a person using medications that are: known to cause hone loss, including corticosteroids such as Prednisone^{1 M}, various antiseizure medications such as Dilantin^rM and certain barbiturates, or high-dose thyroid replacement drugs: a person having type 1 diabetes, liver disease, kidney disease or a family history of osteoporosis; a person having high bone turnover (e.g,, excessive collagen in urine samples); a person with a thyroid condition, such as hyperthyroidism; a person who has „41 .

2016202691 27 Apr 2016 experienced a fracture after only mild trauma; a person who has had x-ray evidence of vertebral fracture or other signs of osteoporosis.

As noted above, osteoporosis can also result as a condition associated with another disorder or from the use of certain medications. Osteoporosis resulting from drugs or another 5 medical condition is 'known as secondary osteoporosis, tn a condition known, as Cushing’s disease, the excess amount of cortisol produced by the body results in. osteoporosis and fractures. The most common medications associated with secondary osteoporosis are the eorticosieroids, a class of drugs that act like cortisol, a hormone produced naturally by tbe adrenal glands. Although adequate levels of thyroid hormones (which are produced by the Q thyroid gland) are needed for the developmen t of the skeleton, excess thyroid hormone can decrease bone mass over time. Antacids that contain aluminum can lead to bone loss when taken in high doses by people with kidney problems, particularl y those undergoing -dialysis. Other medications that can cause secondary osteoporosis include phenytom (Dilantin) and barbiturates that are used to prevent seizures; methotrexate (Rheumatrex, tmrnunex, Folex 5 PFS), a drug for some forms of arthritis, cancer, and immune disorders; cyclosporine (Sandimmune, Neorai), a drug used to treat some autoimmune diseases and to suppress the immune system in organ transplant patients; luteinizing hormone-releasing hormone agonists (Lupron, Zoladexy used io treat prostate cancer and endometriosis; heparin (Caleiparine, Liqnaemin), an antidotting medication; and cholestyramine (Questran) and colestipol 0 (Colestid), used to treat high ehoiesieroL Bone toss resulting from cancer therapy is widely recognized and termed cancer therapy induced hone loss (CTiSL). Bone metastases can. create cavities in the bone that may be corrected by treatment with aetiviu-ActRIIa antagonists.

In a preferred embodiment, activln-ActRUa antagonists, particularly a soluble 25 ActRIla, disclosed herein may be used in cancer patients. Patients having certain tumors (e.g, prostate, breast, multiple myeloma or any turnor causing hyperparathyroidism) are at high risk for hone loss due to tumor-indneed bone toss as welt as hone metastases and therapeutic agents. Such patients may be treated with act!vin-ActRIla antagonists even in the absence of evidence of bone loss or hone metastases. Patients may also be moni tored for evidence of bone loss or bone metastases, and may be treated with activin-ActRiia antagonists in the event that indicators suggest an increased risk. Generally, DEXA scans are employed to

- 42 2016202691 27 Apr 2016 assess changes in bone density, while indicators of bone remodeling may be used io assess the likelihood of bone metastases. Serum markers may be monitored. Bone specific alkaline phosphatase (BSAP) is an enzyme that is present in osteoblasts. Blood levels of BSAP are increased in patients with hone metastasis and other conditions that result in increased bone 5 remodeling. Osteocalcin and procollagen peptides are also associated with bone ton-nation and bone metastases. Increases in BSAP have been detected in patients with hone metastasis caused by prostate cancer, and to a lesser degree, in bone metastases from breast cancer.

Bone Morphogenetic Protein-7 (BMP-?) levels are high in prostate esneer that has metastasized to bone, but not in bone metastases due to bladder, skin, fiver, or lung cancer,

Type I Carboxy-terminal fefonvpnde (ICTP) is a crosslink found in collagen that is formed during to the resorption of none. Since hone is constantly being broken down and reformed, ICTP will be found throughout the body, However, at the site of bone metastasis, the level will be significantly higher than in an area of normal bone, ICTP has been found in high levels in hone metastasis due to prostate, lung,.and breast cancer. Another collagen crosslink, 5 Type I N-terminal telopeptide (NTx), is produced along 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. Also, the levels of NTx increase with the progression of the bone metastasis. Therefore, this marker can he used to both detect metastasis as well as measure the extent of the disease. Other markers of resorption include 0 pyridinoline and deoxypyridmoline. Any increase in resorption markers or markers of bone metastases indicate the need for activin-AetRlla antagonist therapy in a patient,

Activin-ActRlla antagonists may be conjointly administered with other pharmaceutical agents. Conjoint administration may be accompli shed by administration of a single co-formulation, by simultaneous, administration or by administration at separate times.

Activin-ActRIla antagonists may be particularly advantageous if administered with other bone-active agents. A patient may benefit from conjointly receiving aetivin-ActR I la antagonist and taking calcium supplements, vitamin D, appropriate exercise and/or, in some eases, other medication. Examples of other medications ineode, bisphosphonates (alendronate., ibandronate and risedronate), calcitonin, estrogens, parathyroid hormone and raloxifene. The 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 ... 43 .·

2016202691 27 Apr 2016 formation, Anti-resorptive medications slow or stop the bone-resorbing portion'of the boneremodeling cycle but do not slow the bone-forming portion of the cycle. As a result, new formation continues at a greater rate than bone resorption, and bone density may increase over time. Teripsratide, a form of parathyroid hormone, increases the rate of bone formation 5 in the bone remodeling cycle. Alendronate is approved for both the prevention (5 mg per day or 35 mg once a week) and treatment (10 mg per day or 70 mg once a week) of postmenopausal osteoporosis. .Alendronate reduces bone loss, increases bone density and reduces the risk of spine, wrist and hip fractures. Alendronate also is approved for treatment of glucocorticoid-induced osteoporosis m men and women as a result of long-term use of 0 these medications' (i.e, . prednisone and cortisone) and for the treatment of osteoporosis in men. 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. Ihandronate: is approved .for the prevention and treatment of postmenopausal osteoporosis. Taken as a once-a-monlh pill ¢150 mg), ibandronate should be taken on the same day each month. Ibandronate reduces bone loss, increases bone density and reduces the risk of spine fractures, Risedronate is approved for the prevention and treatment of postmenopausal osteoporosis; Taken daily (5 mg dose) or weekly (35 mg dose or 35. mg dose with calcium),, risedronate slows bone loss, increases bone density and reduces the risk of spine and non-spine fractures. Risedronate also is 0 approved for use by men and women fo prevent and/or treat glucocorticoid-induced osteoporosis that results from long-term use of these medications (he., prednisone or cortisone). Calcitonin is a naturally occurring hormone involved in calcium regulation and bone metabolism, in women who are more than 5 years beyond menopause, calcitonin slows bone loss, increases spinal bone density, and may relieve the pain associated with bone 25 fractures. Calcitonin reduces the risk of spinal fractures. Calcitonin is available as an injection (50-100 IO daily) or nasal spray (200 1U daily). Estrogen therapy (ET)ZHormone therapy (HT) is approved for the prevention of osteoporosis. EThas been shown to reduce bone loss, increase bone density in both the spine and hip, and reduce the risk of hip and spinal fractures in postmenopausal women. ET is administered most commonly in the form of 30 a pill or skin patch that delivers a low dose of approximately 0,3 mg daily or a standard dose of approximately 0,625 mg daily and is effective even when started after age 70. When estrogen is taken alone, it can increase a woman’s risk of developing cancer of the uterine . 44 .

2016202691 27 Apr 2016 lining (endometrial cancer). To eliminate this risk, healthcare providers prescribe the hormone progestin in combination with estrogen (hormone replacement therapy or HT) for those women who have an intact uterus. BT/HT relieves menopause symptoms and has been shown to have a beneficial effect on bone health. Side effects may include vaginal bleeding, breast, tenderness, mood disturbances and gallbladder disease. Raloxifene, 60 nag a day, is approved for the prevention, and treatment of postmenopausal osteoporosis. It is from a class of drugs called Selective Estrogen Receptor Modulators (SER.Ms) that have been developed to provide the beneficial effects of estrogens without their potential disadvantages.

Raloxifene increases bone mass and reduces tbe risk of spine fractures. Data are not yet 0 a vailable to demonstrate that raloxifene can reduce the risk of hip and other non-spine fractures, Teriparatide, a form, of parathyroid hormone, is approved for the treatment of osteoporosis in postmenopausal women and men. who are at high risk for a fracture. This medication stimulates new bone formation and significantly increases bone mineral density.

In postmenopausal women, fracture reduction was noted in the spine, hip, foot, ribs and wrist, 5 In men, fracture reduction was noted in the spine, but there were insufficient data to evaluate fracture reduction at other sites. Teriparatide is self-administered as a daily injection for up to 24 months,

7. Phana^emical^ai^SiiM

In certain embodiments, activin-AotRlia antagonists (e.g,, ActRlla polypeptides) of the present invention are formulated with a -pharmaceutically acceptable earner. For example, an ActRHa polypeptide can he administered alone or as a component of a pharmaceutical formulation (therapeutic composition). The subject compounds rosy be formulated for administration in any convenient way for use in human or veterinary medicine.

In certain embodiments, the therapeutic method of the invention includes administering the composition systemieaiiy, or locally as an Implant or device. When administered, the therapeutic composition for use in this invention is in a pyrogen-free, physiologically acceptable form. Therapeutically useful agents other than the ActRlla antagonists which may also optionally be included in the composition as described above, ^Λ 2016202691 27 Apr 2016 may be administered simultaneously or sequentially with the subject compounds {e.g., ActRIIa. polypeptides) in the methods of the invention.

Typically, AeiROa antagonists will be administered parentally, and particularly Intravenously or subcutaneously. Pharmaceutical compositions suitable for parenteral 5 administration .may comprise one or more .ActRIIa polypeptides in combination with one or more pharmaceutically acceptable sterile Isotonic aqueous or nonaqueous solutions, dispuOeuv suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation iso tonic with the blood of the 0 intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may he 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 vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper thndity can he maintained, for 5 example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Further, the composition may be encapsulated or injected in a form for delivery to a target tissue site (e.g., bone). In certain embodiments, compositions of the present Invention may Include a matrix capable of delivering one or more therapeutic compounds (e.g., AotROa polypeptides) to a target tissue site (e.g., bone), providing a structure for the developing tissue and optimally capable of being resorbed into the body. For example, the matrix may provide slow release of the ActRIIa polypeptides. Such matrices may be formed of materials presently in use for other implanted medical applications.

The choice of matrix material is based on hiocornpatlbdity, biodegfadabihty, mechanical properties, cosmetic appearance and interface properties, The particular application of the subject compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, triealciumphosphate, hydroxyapatite, poiyiaetic acid and polyanhydrides.. Other potential materials are biodegradable and biologically well defined, such as bene or dermal collagen.

further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are norobiodegradabie and chemically defined, such as sintered

.. 46 ..

2016202691 27 Apr 2016 hydroxyapatite, bieglass, aluminates, or other ceramics, Matrices may be comprised of combinations of any of the abo ve m entioned types of material, such as polylactio acid and hydroxyapatite or collagen and trieaieiomphosphal.e. The biocersnues may be altered in composition, such as in calcium-aluminate-phosphate and processmgto alter pore size, particle size, particle shape, and biodegradabi I ity.

in certain embodiments, methods of the invention can be administered tor orally, e.g., in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragaeanth), powders, grannies, er as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an 0 elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia} and/or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient. An agent may also be administered as a bolus, electuary or paste.

in solid dosage forms for oral administration (capsules, tablets, pills, dragees, $ powders, granules, and the 11 key one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: :(1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, earboxymethyleeilulose, alginates, gelatin, polyvinyl pyrrol!done, sucrose, and/or acacta, (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; /6) absorption accelerators., such as quaternary ammonium compounds; (?) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9). lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and. mixtures thereof; and (10) coloring agents. In the ease of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

2016202691 27 Apr 2016

Liquid dosage forms for oral administration include· pharmaceutically acceptable emulsions, microerouisions, solutions, suspensions, syrups, and elixirs, in addition fo the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the • art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, 5 isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol· benzyl benzoate, propylene glycol, 1,3-btnyiene glycol, oils (in particular, cottonseed, groundnut, oom, germ, olive, castor, and sesame oils). glycerol, teunhydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragaeanth, and mixtures thereof

The compositions ofthe invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol· phenol sorbic acid, and the like, if may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the .0 compositions,· in addition, prolonged absorption of the injectable pharm aceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

It is understood that the dosage regimen will be determined by the attending physician considering various factors which modify the action of the subject compounds of the invention (e.g., ActRila polypeptides). The various factors include, hot are not limited to, amount of bone weight desired to be formed, the degree of bone density loss, the site of bone damage, the condition ofthe damaged bone, the patient’s age. sex, and diet, the severity of any disease that may be contributing to bone loss, time of administration, and other clinical factors. Optionally, the dosage may vary with the 'type of matrix used in the reconstitorion and the types of compounds in the composition The addition of other known growth factors io .the final composition, may also affect the dosage, Progress can be monitored by periodic

- 48 2016202691 27 Apr 2016 assessment of bone growth and/or repair, for example, X-rays (including D.RXA), histornoiphometric determinations, and tetracycline labeling.

Experiments with primates and humans have demonstrated that effects of ActRffa-Fc on hone are detectable when the compound is dosed at intervals and amounts sufficient to 5 achieve serum concentrations of about 200 ng/ml, with half-maximal effects on anabolic hone bio-markets occurring at a dosage of 0.3 rog/kg or the equivalent in terms of area-under•curve, in humans, serum levels of 200 ng/ml may he achieved with a single dose of 0.1 ,mg/kg or greater and serum levels of 1000 ng/ml may be achieved with a single dose of 0 J rng/kg or greater. The observed serum, half-life of the molecule is between about 2.5 and 35 0 days, substantially longer than most Fc fusion proteins, and thus a .sustained effecti ve serum level may he achieved, for example, by dosing with about 0.05 to 0.5 mg/kg on a weekly or biweekly basis, or higher doses may be used with longer intervals between dosings. For example, doses of 0,1, 0.3,0.5, 0.7, I, 2 or 3 rog/kg, or values in between, might he used, on a monthly or bimonthly basis, and the effect on. hone may be sufficiently durable that dosing is 5 necessary only once every 3, 4,5, h, 9, 12 or more months. Longer intervals between doses are further supported by the duration of the pharmacodynamic effect, which is longer than the duration of drug i n the serum, PD effects are observed for at least 120 days in human patients.

In certain, embodiments,, the present, invention also provides gene therapy for the in 0 vivo production of ActRIla polypeptides. Such therapy would achieve its therapeutic effect by introduction of the AeiR-Ha polynucleotide sequences into cells or tissues having the disorders as listed above. Delivery of ActRIla polynucleotide sequences can be achieved using a recombinant expression vector such, as a chimeric virus ora colloidal dispersion system. Preferred for therapeutic delivery of ActRIla polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can he utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an R.NA virus such as a retrovirus,. Preferably,-the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, hut are not limited to; Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (RaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus <RSV). A number of

.. 40 2016202691 27 Apr 2016 additional retroviral vectors can incorporate ondhple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. Retroviral vectors can be made target-specific by attaching, for example, a sugar, a giycohpid. or a protein. Preferred targeting is accomplished bv using an antibody. Those $ of skill in the art will recognize that specific polynucleotide sequences can he inserted into the retroviral genome or attached to a viral envelope to allow target specific deli very of the retroviral vector containing the AetRIia polynucleotide. in a preferred embodiment, the vector is targeted to bone or cartilage.

Alternatively, tissue culture cells can be directly transfected with plasmids encoding ;) the retroviral structural genes gag, pot and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for AetRIia polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, nncrospheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles v filch are-useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and he delivered to cells in a biologically active .form (see e,g,. Fraley, et ah, Trends ΰ Bioehem, Sci., 6.:77, 1981). Methods for efficient gene transfer using a liposome vehicle, are known in the art, see e.g., Mannlno, et ah, Bioleehniqoes. 6:682., 1988, The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids useful In liposome production include phosphatidyl compounds., such as phosphatidylglyceroi, phosphatidylcholine, phosphaiidylserme, phosphatidylethanolamine, sphingoiipids, cerehrosides, and gangliosides. Illustrative phospholipids include egg phosphatidylcholine, dipalnritoylphosphatidyleholine, and distearoylpbosphatidylcholirte. The targeting of hposomes is also possible based on, for example;, organ-specificity, cell-specificity, and organelfe-speeifieuy and is known in the art.

- 50 2016202691 27 Apr 2016

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of 5 certain embodiments and embodiments of the present invention, and are not intended to limit the invention.

J^«IiLlLAetBM

Applicants constructed a soluble AetRU'a fusion protein that has the extracellular domain of human ActRlla fused to a human or mouse Fc domain with a minimal linker in 0 between, The constructs are referred to as ActRHa-hFe and ActRIla-m'Fe, respectively.

AetRlIa-hFc is shown below as purified from CHO cell lines (SEQ ID NO: ?).:

I LORS ETQBCLFFNAN W EKDRTNQTGVEPCYODK DK R RHCFATWK.N1SGSIEI VKQG CWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMi'NEK.FSYFPEMEVTQFrS'NFVTPK

NWYYDG5:.EyH.NA.KIKPRE.EQ>|NSTY.RyySVLiy.EBQQWi,NGKEYKCKySNKA.LF yPlEKTISKAKGQPREPOVYTtPFSKEEMTKNOVSLTCLVKGPYPSDIAyEWHSNGQP

SPGK

The ActROa-hFc and ActRHa-mFc. proteins were expressed in CHO cell lines. Three 0 -different leader sequences were considered:

(i) Honey bee mellhin (HBML): MRFLVNVAEVPMVVY'iSYIYA (SEQ ID NO: 8) (ii) Tissue Plasminogen Activator (TPA): MDAMK.RGLCCVLLLCGAVFVSP (SEQ ID NO: 9) rid} Native: MGAAAK.LAFAVFL1SCSSGA (SEQ ID NO: 10).

The selected form employs the TEA leader and has the following unprocessed amino acid sequence:

MDAMKRGtCCVLLLCGAVFVSPGAAltGRSETQECLFFNANWEKDRTNQTGVEPCY

GDfoDKRRHCFA'rWRNiSGSIEIVKQGCWLDDlNCYDRTDCVeKKDSPEVYFCCCEG

NMCNBRFSYFPEMEVTQPTSNPVTPOPTGGGTHTCPPCPAPELLGGPSVFLFPPKPK -51 2016202691 27 Apr 2016

VL'TVL

QVSLTCLVKGFYFout?\ v cv* cams

QGNVESCSVMHEALBNBYTQKSL8LSPGK (SEQ ID NO;13)_:

This polypeptide is encoded by the following nucleic acid sequence:

ATGGATGCAATGAAGAGAGGGCfCTGCTGTGTGCTGCIGCTGTGIGGAGGAGTGT

TCGTTTCGCCCGGCGCGGCTATAGTTGGTAGATCAGAAACTGAGGAGTGTC’rrrr

TTTAATGCITAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTT

ATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATA’rrraOG ttccattgaatagtgaaacaaggttgttggctggatgatatca.actggtatgaca

GGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATA'nTCrGTTGCTGTGA

GGG€AATATGTGTAATGAAAAGTFTTCTTA'nTfCCGGAGATGGAAGTC.ACAC-AG

CCCACTTCAAATCCAGTrACACCTAAGCCACCCACCGGTGGTGGAACTC’ACACAT

GCCCACCGTGGCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTrCCTCTrCCC

CCCAAAACCCAAGGACACCCTCATGATCTGCCGGACCCCTGAGGTCACATGCGTG

GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT{?AACTGGTACGTGGAC

GGGGTfiGAGGTGCATAATGCCAAGACAAAGCCGGGGGAGGAGCAGTACAACAG

CACGTAC C GT GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGC

AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGTCCCCATCGAGAAA

ACCATCTCGAAAGCCAAAGGGCAGCCCCGAGAACeACAGGTGTACACCCTGCCC

CCATCCCGGGAGGAGATGACGAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA

GGCTrCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG

AACAACTACAAGACCACGCCTCCCGTGGTGGACTCCGACGGCTCCITCTTCCTCT

ATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT

GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT

GTCTCCGGGTAAATGAGAATTC (SEQ ID NO: 14)

Both AetRll'a-hFc and ActRlla-mFc were remarkably amenable to recombinant expression. As shown in figure 1, the protein was purified as a single, well-defined peak of protein. N-terminai sequencing revealed a single sequence of -ILGRSTQE (SEQ I D NO:

11), Purification could .he achieved by a series of column chromatography steps, including, lor example, three or more of the following, in any order: protein A chromatography, Q

2016202691 27 Apr 2016 sepharose chromatography,, plmnylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography. The purification coaid 'be completed with viral filtration and butter exchange. The AetRlla-hFc protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS 5 PAGE.

AetRlla-hFc and ActRlIa-mPc showed a high-affinity for ligands, particularly activin A. GDF-11 or Activin A (AetA”) were immobilized on a Biacore CM5 chip using standard amine coupling procedure. AetRlla-hFc and Aet.RIIa-ro.Fc proteins were loaded onto the system, and binding was measured, AetRlla-hFc bound to activin with a dissociation 0 constant (Ko) of 5xI O'⁵², and the protein bound to GDPI1 with a K_D of 9.96x 1 θΛ See figure

2. ActRlla-mFc behaved similarly.

An A-204 Reporter Gene Assay was used to evaluate the effects of .ActRHa-hFc proteins on signaling by GDF-1 1. and Activin A. Cell, line: Human Rhabdomyosarcoma (derived from muscle). Reporter vector: pGE3{GAGA)12 (Described in Dennler et al, 1.998, 5 EM BO 17: 3091-3100.) See Figure 3. The CAGAI2 motif is present in TGF-Beta responsive genes ( PAU gene) , so this vector is of general use for factors signaling through Smad2 and 3.

Day 1: Split A-204 cells into 48-well plate,

Day 2: A-204 cells transfected with 10 pgpGL3(CAGA)I2 or pGL3(CAGA).12 (10 0 pg)> pRLCMV (1 gg) and Fugene.

Bay 3:.Add factors (diluted into fnediumA 0.1 % BSA). Inhibitors need to be preincuhated with Factors for I hr before adding io cells, 6 hrs later, cells rinsed with PBS, and lyse cells.

This is followed by a Lueiferase assay. Typically in this assay, in the absence of any 25 inhibitors, Activin A shows roughly 10 fold stimulation of reporter gene expression and an

BD50 - 2 ng/mt GDF-11: 16 fold stimulation, ED50: - 1.5 ng/mt GDF-8 shows an effect similar to GDP-11.

As shown in figure 4, ActRHa-hFc and ActRlla-mFc inhibit GDF-8 mediated signaling at pfoomolar concentrations. As shown in figure 5, three different preparations of

ActRIIa-hFc inhibited GDF-11 signaling with an 1C50 of approximately 200 pM.

- 53 2016202691 27 Apr 2016

The ActROa~hFe was very stable in pharmaeokinefic studies. Rats were dosed with I mg/kg, 3 mg/kg or 10 mg/kg of AetRlla-hFc protein sod plasma levels ofthe protein were measured at 24» 48, 72, 144 and 168 hours, in a separate study, rats were dosed at 1 rngfeg, 10 mg/kg or 30 mg/kg. in rats, AetRHa-hFc had an 11-14 day serum half life and circulating levels of the drag were quite high after two weeks (11 gg/ml, 110 pg/ml or 304 μ-g/ml for initial administrations of t nig'^fkg_s 10 mg/kgor 30 mg/kg, respectively,) In eynornolgus monkeys» the plasma half life was substantially greater than 14 days and circulating levels of the drug were 25 pg/mk 304 gg/mi or 1440 pg/ηύ for initial administrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively. Preliminary results in humans suggests that the serum half life is between about 20 and 30 days.

Eiasfole2yActfefemFfo

Normal female mice (BALB/c) were dosed with AciRHa-mFc at a level of 1 mg/kg/dose, 3 mg/kg/dose or Π) mg/kg/dose, with doses given twice weekly. Bone mineral density and bone mineral content were determined by DEXA, see figure 6.

In BALB/c female mice, DEXA scans showed a significant increase (>20%) in bone mineral density and content as a result, of AciRria-mFe treatment. See figures 7 and 8,

Thus, antagonism of AetRila caused increased bone density and content m normal female mice. As a next step, the effect of AetRlla-niPc on hone in a mouse model for osteoporosis was tested,

Anderssori et al, (2001), established that ovarieetomized mice suffered substantial bone loss (rougly 50% loss of trabecular bone six weeks post-operation), and that bone loss in these mice could be corrected with candidate therapeutic agents, such as parathyroid hormone.

Applicants used C57BL6 female mice that were ovarieetomized (OVX) or sham operated at 4-5 weeks of age. Eight weeks after surgery, treatment wi th AetRfta~roFc (10 mg/kg, twice weekly) or control (PBS) was initiated. Bone density was measured by CT scanner.

As shown in figure 9, untreated, ovarleetomized nnce shewed substantial loss of 30 trabecular bone density relative to the sham controls after six weeks. ActRlla-mFc treatment ~ 54 2016202691 27 Apr 2016 restored bone density to the level of the sham operated mice. At 6 and 12 weeks of the treatment, ActRIla-mFe caused substantial increase in trabecular bone of OVX mice. See figure 10, After 6 weeks of treatment, bone density increased by 24% relative to PBS controls. After 12 weeks, the increase was 27%.

In the sham operated mice, AetRIia-mFc also caused a substantial increase in trabecular bone, See figure 11. After 6 and 12 weeks, the treatment produced a 35% increase relative to controls..

In an additional set of experiments, ovariectomized (OVX) or sham operated mice as described above were treated with ActRIla-mFc (10 rngfeg, twice weekly) or control (PBS) over twelve weeks. Similar to the results described above tor ActRlla-anFe, OVX mice ieccumg ‘\clRlh-mFe evhibncd .m increase m trabecular bone deusdy of 153,, gs as _ca{s_s four weeks and 25% after 12 weeks of treatment (Figure 12). Sham operated mice receiving AetRIia-mFc similarly showed an increase in trabecular hone density of 22% by as early as four weeks and of 32% after 12 weeks of treatment ( Figure 13),

After twelve weeks of treatment with ActRIla-mFc, whole body and ex vivo femur

DEXA analysis showed that treatment induces an increase in bone density in both ovariectomized and sham operated mice (Figures 14A and I4B, respectively). These results are also supported by ex vivo pQCT analysis of the femoral midshaft which demonstrated a significant increase in both xual and cortical bone Jensdy after twelve weeks of treatment with ActRIla-mFc. Vehicle-treated control ovariectomized mice exhibited bone densities that were comparable to vehicle-treated, control sham operated mice (Figure 15). in addition to hone density, bone content Increased following ActRlIa-mFC treatment, fit Wvo pQCT analysis of the femoral midshaft demonstrated a significant Increase in both total and cortical bene content after twelve weeks of treatment with AetRila-mFc while both ovariectomized and sham operated vehicle control-treated mice exhibited comparable bone content (Figure 16). & vivo pQCT analysis of the femoral midshaft also showed that ActRIla-mFc treated mwe did not show a change in periosteal circumference; however ActRIla-mFc treatment resulted in a decrease in endosteal circumference indicating an increase in cortical thickness due to growth on the inner surface of the femur ( Figure 17),

Mechanical testing of femurs determined. that ActRIla-mFc was able to increase the extrinsic characteristics of the bone (maximal load, stiffness and energy to break) which

-552016202691 27 Apr 2016 contributed to a significant increase in the intrinsic properties, {ultimate strength) of the bones. Ovariectomixed mice treated, with ActRHa-mFc exhibited increased hone strength to levels beyond sham operated, vehicle treated controls, indicating a complete reversal ot'the osteoporotic phene-type (Figure 18).

These data demonstrate that an activin-ActRHs antagonist can increase bone density in normal female mice and, furthermore, correct defects in bone density,, hone content, and ultimately bone strength, in a mouse model of osteoporosis.

In a Rather set of experiments, mice were ovariectomized or sham operated at 4 weeks, and beginning at 12 weeks received either placebo or ActRHa-mFc (2 times/week, lOmgdig) (also referred, to as RAF-11 in Figures 19-24), for a further period of 12 weeks. A variety of bone parameters were evaluated. As shown in Figure 19, ActRHa-mFc increased vertebral trabecular hone volume to total volume ratios (BY/TV) in both the OVX and SHAM operated mice. ActRHa-mFc also improved the trabecular architecture (Figure 20), Increased cortical thickness (Figure 21) and improved hone strength (Figure 22), As shown 5 in Figure 23, ActRHa-mFc produced desirable effects at a range of doses from 1 mg/kg to 10 mg/kg.

Bone histomorphometry was conducted at a 2 week time point in sham operated mice. These data, presented in f igure 24, demonstrate that ActRHa-mFc has a dual effect, both inhibiting bone resorption and promoting bone growth. Thus ActRHa-mFc stimulates hone growth (anabolic effect) and inhibits bone resorption (anti-catabohc effect). BV ~ Bone volume; TV “total tissue volume. BV/TV Is a measure of the percentage of bone volume that is mineralized, BS ~ Eroded, surface; BS -- Bone surface, ES/BS is a measure of hone erosion, and the decrease caused by RAP-011 demonstrates an anti-resorptive or anticatabohe effect, Ms/Bs is the mineralizing surface/hone surface ratio, which is an indicator ofbone growth, or anabolic effect. Similarly, mineral apposition, rate (MAR) and bone formation rate per hone surface per day .(BFR/BSd) indicate bone growth. Measures of osteoblasts (Nob/BPm) and osteoclasts (Noc/BFm) are taken in order to probe the mechanism of action,

A second bone histomorphometry experiment was conducted in female C578L/6 mice, beginning at an age of twelve weeks. Mice were dosed mtraperitoneally twice per week with 10 mg/kg ActRlla-mfc for two weeks, four weeks, eight weeks or twelve weeks.

- 56 2016202691 27 Apr 2016

Each group was sacrificed five days after the last dose and bones taken for analysis. Mice were ealeein labeled nine days and two days prior to euthanasia. As shown in Figure 25, the metrics show that ActROa-mFc promotes bone growth and mineralization and has both anabolic and anti-catabolic effects. See for example foe BV/TV ratio, the ESZBS ratio and 5 the MS/BS ratio. The anabolic effects appear to persist throughout foe dosing regimen, while the anti-resorptive effects appear to he shorter lived in the mice.

EMffiEteSfAeBliamfofoamehorates^^

WSted

Multiple myeloma patients exhibit a hone loss disorder characterized by increased osteoclast activity and decreased bone formation by osteoblasts. The 5T2MM model of myeloma in mice is based on the use of tumor cells (5T2M.M cells) from a type of spontaneous tumor that develops in aged mice and causes effects in mice that are similar to those seen in human multiple myeloma patients. See, e.g,, Vanderkerken et al., Methods Moi

Med, 2005; 113;191 -205, AetRIia-mPe was tested tor effects in this model.

5T2MM cells injected Into €5?Bi/KatwRij mice promoted an Increase in osteoclast surface, the formation of osteolytic lesions and caused a decrease in bone area. Bone disease was associated with a decrease in osteoblast number, osteoblast surface and a reduction in mineralization.

i) Mice bearing 5T2MM cells were treated with ActRlla-mFc (RAP-01Ii lOmg/kg, i.p, twice weekly), ora vehicle, from the time of 5T2MM injection, for a total of 12 weeks. MicroCT analysis of the proximal tibia and lumbar vertebrae demonstrated a 39% and 2153 reduction in cancellous bone volume (p<0.001 and p<0,0I) and a 37% and 15% reduction in trabecular number (p«0.O I and p<0.05) in 5T2MM-bearing mice compared to naive mice.

RAP-011 completely prevented 5T2MM~induced decreases in trabecular volume and number in both tibia (p<0.00l and p<0.05) and vertebrae (p<0.Ol and. p<0.05) when compared to vehicle treated mice. Bone volume was 19% higher in the tibia (ρ^ 168) and 12%. higher in vertebrae (p<0.05) of RAP-011 treated mice when compared to naive mice. RAP-011 prevented the development, of osteolytic bone lesions (p<0,05). This effect is illustrated in

Figure 26. While a preliminary assessment ofthe data faded to Identify significant effects on serum, paraprotein (a biomarker of multiple myeloma tumor cells) or myeloma burden in this - 57 2016202691 27 Apr 2016 study, a further analysis indicated that serum paraprotein was substantially decreased in ail hut one of the treated ani mals, and further that the vol ume of healthy bone marrow was substantially increased, indicating a decrease in the myeloma tumor cell burden.

Therefore., ActlNla-roPe may he used to decrease the effects of hone disease resulting 5 from multiple myeloma and to treat the tumor cells themselves,

ActRlla-hfe fusion protein was expressed in stably transfected CHO-DUK.X B i 1 cells from a pAID4 vector (SV40 orl/enhaneer, CMV promoter), using a tissue plasminogen 0 leader sequence of S EQ ID NO:9, The protein, purified as described above in Example 1, bad a sequence of SEQ ID NO:?. Tbe Fc portion is a human IgGl Fc sequence, as shown in SEQ ID NO:7. Sialic acid analysis showed that die protein contained, on average, between about 1,5 and 2,,5 moles of sialic acid per molecule of ActROa-hFc fiision protein.

This purified protein showed a remarkably long serum half-life in all animals tested, including a half-life of 25-32 days in human patients (see Example 5, below). Additionally, the CHO cell expressed material has a higher affinity for activin B ligand than that reported for an ActRBa-hFc fusion protein expressed m human to) eelfr tdel Re <rt ah, J ifrol f’hera.

2()09 Dee 17;279(51 ):53126-35,) Additionally, the use of the tPa leader sequence provided greater production than other leader sequences and, unlike ActRlla-Fc expressed with a native leader, provided a .highly pure N-tormmal. sequence. Use of the native leader sequence resulted in two major species of ActROa-Fc, each having a different N-terminal sequence.

fcA2;M)if.52.HumanT;hnw?_LLTnal

The protein described in Example 4 was administered to human patients in a 25 randomized, double-bund, placebo-controlled study that was conducted to evaluate, primarily, the safety of the protein in healthy, postmenopausal women. Forty-eight subjects were randomized in cohorts of 6 to receive either a single dose of ActRIIa-hPe or placebo (5 active;! placebo). Dose levels ranged from 0.01 to .3,0 tng/kg intravenously (IV) and 0.03 to 0,1 mg/kg subcutaneously (SC), All subjects were followed for 120 days. Subjects were excluded from study participation if they took medications affecting bone metabolism within - 5S 2016202691 27 Apr 2016 months of study entry. Safety evaluations were conducted following each cohort to determine dose -escalation. In addition to pharmacokinetic (PK) analyses, the biologic activity of AetRIIa-hFe Was also assessed by measurement of biochemical markers of bone formation and resorption, and FSH levels.

No serious adverse events were reported in this study. Adverse events t Ahsj were generally mild and transient. Preliminary analysis of AEs included headache, elevated laboratory values, cold symptoms, emesis or vomiting, intravenous Infiltration, and hematoma at Ifocefion site,

PR analysis of ActRlIa-h.Fc displayed a linear profile with dose, and a mean half-life

O of approximately .25-32 days. The area-under-curve (AUC) for AetRIIa-hFe was linearly related to dose, and the absorption after SC dosing was essentially complete (see Figures 27 and 28), These data indicate that SC is a desirable approach to dosing because it provides equi valent hioavailahillty and serum-half life for the drug while avoiding the spike in serum concentrations of drug associated with the first few days of IV dosing (see Figure 28),

AetRIIa-hFe caused a rapid, sustained dose-dependent increase in serum levels of bonespecific alkaline phosphatase (BAP), which Is a marker for anabolic hone growth, and a dosedependent decrease in C-terminal type I collagen, ielopeptide and tartrate-resistant acid phosphatase 5b levels, which are. markers for bone resorption. Other markers, such as P1NP showed inconclusive results, BAP levels showed near saturating effects at the highest dosage of drug, todicatiag that half-maximal effects on this anabolic bone biomarker could be achieved at a dosage of 0.3 mg/kg, with increases ranging up to 3 rng/kg. Calculated as a relationship of pharmacodynamic effect to AUC for drug, the Γ C50 Is 51,465 (day*ng/ml). See Figure 29. These hone biomarker changes were sustained for approximately 120 days at the highest dose levels tested. There was also a dose-dependent decrease in serum FSH 25 levels consistent with inhibition of activin.

A single dose of ActRila-hFe given to healthy postmenopausal women was safe and well-folemted for the range of dose levels tested. 'The prolonged PR and pharmacodynamic effects suggest that intermittent dosing would be appropriate for future studies. For example, dosing on the basis of serum hall'-hfe could be performed on a monthly basis, or on the order of once every two, three, four, five or six weeks. Additionally, because the phanriaeodynamie effect extends for beyond the serum residence of the drug, dosing could be

- 59 performed on the basts oftbe pharmacodynamic effect, meaning that, dosing every three months or every two. three, four, five, six or even twelve months may be effective to produce the desired effect in patients. This clinical trial demonstrates that, in humans, ActRHa-hFc is an osteoanabohe agent with biological evidence of both an increase in hone formation and a 5 decrease in hone resorption.

2016202691 27 Apr 2016 feymle6:Xz>admhd^^^^

Bisphosphonates are a class of drugs that are widely used to treat disorders associated with low bon© mineral density, including osteoporosis and cancer-related bone loss.

Bisphosphonates have a potent anti-resorptive activity, inhibiting osteoclasts. Perhaps because osteoclasts are required both for bone breakdown and bone growth, bisphosphonates appear to diminish the effects of parathyroid hormone (PTH), one of the only known anabolic bone growth agents (Black et ah, N Engl J Med. 2003 Sep 25:349(13): 1.207-15; Samadfam et al., Endocrinology. 2007 Jurgl48(61:2778-87.)

To test the utility of ActRlla-Fc treatment in patients that had previously or were concomitantly receiving bisphosphonate or other anti-resorptive therapy, mice were tested with combined ActRlia-niFe and zoledronate, a bisphosphonate compound. 12 week old C57B176N mice were treated as follows:

Group 1 0 Group 2

Group 3 Group 4

Group 5 week

PBS

Acthlla-mEc (RAP-011) (10 uuykg) twice per week (with Group 3 and 4) Zofedronic Acid (ZOL) singe dose (20 mg/kg)

ZOL (1 dose), 3 days later ActRUa-mPe (RAP-011)(1 mg/'kg) twice per week •ZOL (1 dose), 3 days later ActRlla-mFc (RAP-011) (10 mg/kg) twice per

Total BMD was determined by DEXA scan (PiXI) prior to dosing and at 3 and 8 weeks of treatment.

As shown in Figure 30, total BMD increased markedly in all treatment groups, with the combination of ZOL and ActRlla-mFc producing the greatest effects. These results

- 60 2016202691 27 Apr 2016 .indicate that ActROa-Fc proteins can he used to increase bone density, even in patients that have received bisphosphonate therapy.

E5hmRk.2i.AotRIU-Fe.Ai»<omte^^ycms BmM^O^aiisedMtest.Caneor

Metastaseg it is estimated that 65 to 75 percent of breast cancers metastasize to the bone, causing substantial damage to the bone structure, increasing fracture risk and causing pain and other side effects. We tested the effects of AeiR.lIa~Fc in a mouse model of breast cancer that has metastasized to the hone,

A subline of the human breast cancer cell line MDA-MB-231 (done 2287} was cultured in vitro and cells harvested at a density of 5 x 106 cells/ml, MDA-MB-231 is a cell line that is highly competent for seeding into bone and causing bone damage similar to that caused by bone melasiases. 10 ml of cells were injected into the tibia of 6 week old female athymic nude mice on study day 0, On study day .10 mice received AelRfla-roFc (10 rog/fcg/ twice weekly/ subcutaneous) (n=8) or PBS vehicle (m-7). Disease progression was assessed by dual energy x-ray absorptiometry (PtXl'Mus) at weekly intervals. Mice were treated with ActRHa-tnFc for 4 weeks and then sacrificed and tibae (both tumor injected and untumored) were collected from each animal. Tibiae were then processed and prepared for mieroCT and histololgieal analysis.

intratibial injection of MDA-M.B-231 cells into athymic nude mice promoted the development of osteolytic bone lesions in the injected tibia compared to the contralateral leg MieroCT analysis of the proximal tibia, demonstrated a 6254, redaction in cancellous honevolume in the MDA-MB-231 bearing tibiae compared to the untumored tibia in PBS vehicle treated mice, ActRlla-mFc treatment led to an Increase of 70% or 14784 in the naive or tumor bearing tibia respectively compared to vehicle (P«0,01 for both). The tumor bearing tibiae of ActRlla-mFc treated mice had a similar cancellous bone density as the naive tibiae of the VEH treated mice (p-OJRh

Thus, ActRlla-mFc is able to eliminate the bone damage associated with the presence of breast tumor cells in the bone.

-61 2016202691 27 Apr 2016

Example 8 (Alternative ActRI larEp.PrQtelus

An alternative construct may have a deletion of the C-tenninal tail (the final .15 antino acids of the extracellular domain of 'AetRlla. The-sequence for such a construct is presented below (Fc portion underlined)(SEQ ID NO: 12):

ItORSETQECLFFNANWEK.DRTNQTGVEPCYGDKDRRRHCFATWK,NlSGSlE-IVKQG CWLDDINCYDR.TDCVEK.KDSPEVYFCeCEGNMCN£RFSYFFEMTOGOTHTCPPCFA PELLGGPSVf LFFPKPKDTEPdlSRTFEVTCYVVDVSHEDPEVKbNWYVDGVEVHNAK· TK.PREFOYNSTYRVVSVLTVn-lOPWl.NGKFYKCKVSNKAtPVFlEKTiSKAKGQPRE ΡΡΥΥΊ1 PPSRPhhnRNOYSl.TGl.YKGFhTSDlAYiAV£SNGPPENNYI<TTPPYl.PSPG SFPLYSKITVPRSRWQQGNyFSCSVMHEALHNBYd'ORSLSLSPGR

INCORPOR A ΓΙΟΝ BY R EF FRENCH

AH publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and. individually indicated to be incorporated by reference.

While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their lull scope of equivalents, and the specification, along with such variations.

-622016202691 28 Jul2016

Claims (17)

1. Use of an anti-activin antibody or fragment thereof in the manufacture of a medicament for treating or preventing multiple myeloma in a human patient.

2. The use according to claim 1, wherein the antibody or fragment thereof is a chimeric antibody.

3. The use according to claim 1, wherein the antibody or fragment thereof is a humanized antibody.

4. The use according to claim 1, wherein the antibody or fragment thereof is a human antibody.

5. The use according to any one of claims 1 -4, wherein the antibody or fragment thereof is a monoclonal antibody.

6. The use according to any one of claims 1 -5, wherein the antibody or fragment thereof is an F(ab)₂ fragment.

7. The use according to any one of claims 1 -5, wherein the antibody or fragment thereof is an Fab fragment.

8. The use according to any one of claims 1 -5, wherein the antibody or fragment thereof is a single chain antibody.

9. The use according to any one of claims 1 -5, wherein the antibody or fragment thereof is a bi-specific antibody.

10. The use according to any one of claims 1 -9, wherein the antibody or fragment thereof further comprises one or more agents selected from: a radioisotope, a fluorescent compound, and an enzyme or enzyme co-factor.

11. The use according to any one of claims 1 -10, wherein the antibody or fragment thereof binds to activin A.

12. The use according to any one of claims 1 -10, wherein the antibody or fragment thereof binds to activin B.

13. The use according to any one of claims 1 -10, wherein the antibody or fragment thereof binds to activin A and B.

2016202691 28 Jul2016

-6414. The use according to any one of claims 1-13, wherein the antibody or fragment thereof inhibits activin from binding to ActRlla.

15. The use according to any one of claims 1 -14, wherein the antibody or fragment thereof reduces tumor burden in the patient.

16. The use according to any one of claims 1 -15, wherein the antibody or fragment thereof treats or prevents bone loss in the patient.

17. The use according to any one of claims 1 -16, wherein the antibody or fragment thereof is for administration to the patient in combination with one or more bone-active agents selected from: a calcium supplement, vitamin D, a bisphosphonate, calcitonin, estrogen, parathyroid hormone, and raloxifene.

18. A method for treating or preventing multiple myeloma in a human patient, the method comprising administering to the patient an effective amount of an anti-activin antibody or fragment thereof.

19. The method according to claim 18, wherein the antibody or fragment thereof is a chimeric antibody.

20. The method according to claim 18, wherein the antibody or fragment thereof is a humanized antibody.

21. The method according to claim 18, wherein the antibody or fragment thereof is a human antibody.

22. The method according to any one of claims 18-21, wherein the antibody or fragment thereof is a monoclonal antibody.

23. The method according to any one of claims 18-22, wherein the antibody or fragment thereof is an F(ab)₂ fragment.

24. The method according to any one of claims 18-22, wherein the antibody or fragment thereof is an Fab fragment.

25. The method according to any one of any one of claims 18-22, wherein the antibody or fragment thereof is a single chain antibody.

26. The method according to any one of any one claims 18-22, wherein the antibody or fragment thereof is a bi-specific antibody.

2016202691 28 Jul2016

-6527. The method according to any one of any one of claims 18-26, wherein the antibody or fragment thereof further comprises one or more agents selected from: a radioisotope, a fluorescent compound, and an enzyme or enzyme co-factor.

28. The method according to any one of any one of claims 18-27, wherein the antibody or fragment thereof binds to activin A.

29. The method according to any one of any one of claims 18-27, wherein the antibody or fragment thereof binds to activin B.

30. The method according to any one of any one of claims 18-27, wherein the antibody or fragment thereof binds to activin A and B.

31. The method according to any one of any one of claims 18-30, wherein the antibody or fragment thereof inhibits activin from binding to AetRIla.

32. The method according to any one of any one of claims 18-31, wherein the antibody or fragment thereof reduces tumor burden in the patient.

33. The method according to any one of any one of claims 18-32, wherein the antibody or fragment thereof treats or prevents bone loss in the patient.

34. The method according to any one of any one of claims 18-33, wherein the antibody or fragment thereof is administered to the patient in combination with one or more bone-active agents selected from: a calcium supplement, vitamin D, a bisphosphonate, calcitonin, estrogen, parathyroid hormone, and raloxifene.

1/30

2016202691 27 Apr 2016

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2016202691 27 Apr 2016

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2016202691 27 Apr 2016

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2016202691 27 Apr 2016 •PHPH~0'25-WQ'l„subst'i tute_Seq'uence_Li sting (2) . txt SEQUENCE LISTING <110> KNOPF, JOHN

KUMAR, RAVINDRA SEEHRA, JASBIR <120> ACTIVIN-ACTRIIA ANTAGONISTS AND USES FOR PROMOTING BONE GROWTH IN CANCER. PATIENTS <130> PHPH-025-WO1 <140> PCT/US20Q8/Q01354 <141> 2008-02--01 <150> 66/900,580 <153> 2007-02-09 <150> 60/932,762 <151> 2007-05-31 <15 0> 60/937,365 <151> 2007-06-26 <150> 61/000,528 <151> 2007-10-25 <16O> 18 <170> Patentlrt ver, 3.3 <210> 1 <211> 513 <212> PRT <213> Homo sapiens <400> I

Met :i Gl y Ala Ala Al a 5 Lys Leu Ala Phe Al a 10 Val Phe Leu Τ' Mi . _ Ii e Ser 15 Cys Ser Ser Gly Ala II e Leu Gl y Arg ser Glu Thr Gin G 1 a Cys Leu Phe 20 25 30 Phe Asn Al a Asn Trp Glu Lys Asp Arg Thr Asn Girt Thr Gly Val Gl u 35 40 45 Pro CVS Tyr G 1 y Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp 50 55 SO Lys 65 Asn il e Ser Gly Ser lie Glu lie Val Lys Gin Gly cys Trp Leu 70 75 SO Asp Asp 11 e Asn Cys Tyr Asp Arg Thr Asp cys Val Glu Lys Lys Asp 85 90 95 Ser Pro Glu Vsl Tyr Phe cys Cys CVS Gl u Gly Asn Met cys Asn Gl U 100 To 5 110 Lys phe Ser Tyr Phe Pro Glu Met Gl u Val Thr Gin pro Thr Ser Asn 115 120 125 Pro val Th r Pro Lys ! ί M pro Tyr Tyr Asn lie Leu Leu Ty r Ser Leu 130 135 140 Val pro Leu Met Leu Il e Al & Gly lie Val Il e Cys Ala Phe Trp val 145 ISO 155 160 Tyr Arg Hi s Hi s Lys Met Al a Tyr Pro Pro val Leu val Pro Thr Gin 165 170 175

Page 1

2016202691 27 Apr 2016

PHPH-025-WOl_.,Substi tute„Sequence_Li sting (2) . txt

Asp pro Gly Pro 180 Pro Pro pro Ser Pro 185 Leu Leu Gly Leu Lys 190 Pro Leu Gin Leu Leu 195 Glu val Lys Ala Arg 200 Gly Arg Phe Gly cys 205 Val Trp Lys A1 a Gin 210 Leu Leu Asn Glu Tyr 215 Val Ala Val Lys Il e 220 Phe Pro lie Gin Asp 225 Lys Gin Ser Trp Gin 230 Asn Glu Tyr Glu val 235 Tyr ser Leu Pro Gly 240 Met Lys His Glu Asn 245 lie Leu Gin Phe lie 250 Gly Ala Gl u Lys Arg 255 sly Thr ser val ASP 260 Val Asp Leu Trp Leu 265 Il e Thr Ala Phe Hi s 77Ω Glu Lys Gly Ser Leu 275 Ser Asp Phe Leu Lys 280 Ala Asn val val ser 285 Trp Asn Gl u Leu cys 290 Hi s lie Ala G1 J Thr 295 Met Ala Arg Gly Leu 300 Ala Tyr Leu Hi 5 Gl u 305 ASp lie Pro Gly Leu 310 Lys Asp Gly Hi 5 Lvs 315 Pro Ala He Ser Hi £ 320 Arg Asp lie Lys Ser 32 5 Lys Asn Val Leu Leu 330 Lys Asn Ash Leu Thr 335 Ala Cys lie Ala ASP 340 Phe Gly Leu Al a Leu 345 Lys Phe Glu Ala Gly 350 Lys Ser Ala Gly Asp 3 55 Thr Hi s Gly Gin Val 360 Gly Thr Arg Arg Tyr 365 Met Ala pro Glu Val 370 Leu Glu Gly Ala lie 375 Asn Phe Gin Arg Asp 380 Al a Phe Leu Arg lie 385 Asp Met Tyr Al a Met 390 Gly Leu Val Leu Trp 395 Glu Leu Al a Ser Arg 400 Cvs Thr Ala A . 3 ASP 405 Gly Pro val Asp Glu 410 Tyr Met Leu Pro Phe 415 Glu Glu Glu lie Gly 420 Gin Hi s Pro Ser Leu 425 Glu Asp Met Gin Glu 430 Val Val Va i Hi s Lys 435 Lys Lys Arg P ro Val 440 Leu Arg Asp Tyr Trp 445 Gin Lys His Ala Gly 450 Met Ala Met Leu Cys 455 Glu Th r He Gl u Glu 460 cys Trp Asp His Asp 465 Ala Glu Ala Arg Leu 470 Ser Ala Gly Cys Val 475 Gly Glu Arg He Thr 480 Gin Met G1 n Arg Leu 485 Thr Asn lie: lie Thr 490 Thr Glu Asp Il e Val 495 Thr va Leu val Thr Met 500 Val Thr Asn val Asp 505 Phe Pro Pro tys Glu 510 Ser Ser

<210> 2 <211> 11,5

Page 2

2016202691 27 Apr 2016 phph-025-woi_Sub.sti'tute„Secfuence_Listi ng (2) . txt <212> PRT <213> Homo sapiens

<400> 2 lie 1 Leu Gly Arg Ser 5 Glu Thr Gin Glu cys 10 Leu Phe Phe Asn Ala 15 Asn Trp Glu Lys Asp 20 Arg Thr Asn Gin Thr Gly 25 Val Gl u Pro Cys 30 Tyr c i y Asp Lys Asp 35 Lys Arg Arg Hi s cys 40 Phe Ala Thr Trp Lys 45 Asn 11 e Ser Gly Ser lie 50 Glu lie val Lys 55 Gin Gly cys Trp Leu 60 Asp Asp 11 e Asn cys 65 Tyr Asp Arg Thr Asp CVS 70 val Glu Lys Lys 75 Asp Ser Pro Gl U Val 80 Tv.r phe cys cys cys 85 Glu Gly Asn Met Cys 90 Asn Glu Lys Phe Ser 95 Tyr Phe Pro Glu Met Glu Val Thr Gin Pro Thr Ser Asn Pro val Thr pro

100 105 110

Lys Pro Pro 115 <210> 3 <21I> 100 <212> PRT <213> Homo sapiens

<400> 3 GI U Cys 10 Leu Phe Phe Asn Al a 15 Asn lie 1 Leu Gly Arg Ser 5 Glu Tnr Gl n T rp Glu Lvs ASD Arg Thr Asn Gin Thr Gly Val Glu Pro cys Tyr Gly 20 25 30 Asp Lys Asp Lys Arg Arg Hi s Cys Phe Al 3. Thr Trp Lys Asn I is Ser 35 40 45 Gly Ser lie Glu II e Val Lys Gin Gly k,ys Trp Leu Asp Asp Il e Asn 50 55 60 Cys Tyr Asp Arg Thr Asp cys val Glu cys Lys Asp Ser Pro Glu Val 65 70 75 80 Tyr Phe Cys cys Cys 6IU Gly Asn Met Cys Asn Glu Lys Phe ser Tyr 85 90 95

Phe Pro Glu Met loo <21O> 4 <211> 1542 <212> DNA <23 3> Homo sapiens <400> 4 atgggagctg ctgcaaagtt ggcgtttgcc gtctttctta tctcctgttc ttcaggtgct 60 atacttggta gatcagaaac tcaggagtgt cttttcttta atgctaattg ggaaaaagae 120 agaaccaatc aaactggtgt tgaaccgtgt tatggtgaca aagataaacg gcggcattgt 180 tttgctacct ggaagaatat ttctggttcc attgaaatag tgaaacaagg ttgttggctg 240 gatgatatca actgctatga caggactgat tgtgtagaaa aaaaagacag ccctgaagta 300 tatttttgtt gctgtgaggg caatatgtgt aatgaaaagt. tttcttattt tccagagatg 360

Page 3

2016202691 27 Apr 2016

PHPH-025-WOl_Substitute_.5equence„Listing (2) .txt gaagtcacac agcccacttc aaatccagtt acacctaagc cacectatta caacatcctg 420 ctctattcct tggtgccact tatgttaatt gcggggattg tcatttgtgc attttgggtg 480 tacaggcatc acaagatggc ctaccctcct gtacttgttc caactcaaga cccaggacca 540 cccccacctt ctccattact agggttgaaa ccactgcagt tattagaagt gaaagcaagg 600 ggaagatttg gttgtgtctg gaaagcccag ttgettaacg aatatgtggc tgtcaaaata 660 tttccaatac aggacaaaca gtcatggcaa aatgaatacg aagtctacag tttgcctgga 720 atgaagcatg agaacatatt acagttcatt ggtgcagaaa aacgaggcac cagtgttgat 780 gtggatcttt ggctgatcac agcatttcat gaaaagggtt cactatcaga ctttcttaag 840 gctaatgtgg tctcttggaa tgaactgtgt catattgcag aaaecatggc tagaggattg 900 gcatatttac atgaggatat acctggccta aaagatggcc acaaacctgc catatctcac 960 agggacatca aaagtaaaaa tgtgctgttg aaaaacaacc tgacagcttg cattgctgac 1020 tttgggttgg cctiaaaatt tgaggctggc aagtctgcag gcgataccca tggacaggtt 1080 ggtacccgga ggtacatggc t'ccagaggta ttagagggtg ctataaactt ccaaagggat 1140 gcatttttga ggatagatat gtatgccatg ggattagtcc tatgggaact ggcttctcgc 1200 tgtactgctg cagatggacc tgtagatgaa tacatgttgc catttgagga ggaaattggc 1260 cagcatccat ctcttgaaga catgcaggaa gttgttgtgc ataaaaaaaa gaggcctgtt 1320 ttaagagatt attggcagaa acatgctgga atggcaatgc tctgtgaaac cattgaagaa 1380 tgttgggatc acgacgcaga agccaggtta tcagctggat gtgtaggtga aagaattacc 1440 cagatgcaga gactaacaaa tattattacc acagaggaca ttgtaacagt ggtcacaatg 1500 gtgacaaatg ttgactttcc tcccaaagaa tctagtctat ga 1542 <210> 5 <211> 345 <212> DNA <213> Homo sapiens <400> 5 atacttggta gatcagaaac tcaggagtgt cttttcttta atgctaattg ggaaaaagac 60 agaaccaatc aaactggtgt tgaaccgtgt tatggtgaca aagataaacg gcggcattgt 120 tttgctacct ggaagaatat ttctggttcc attgaaatag tgaaacaagg ttgttggctg 180 gatgatatca actgctatga caggactgat tgtgtagaaa aaaaagacag ccctgaagta 240 tatttttgtt gctgtgaggg caatatgtgt aatgaaaagt tttcttattt tccagagatg 300 gaagtcacac agcccacttc aaatccagtt acacctaagc caccc ' 345 <210> 6 <211> 225 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <220>

<221> MOD_RES <222> (43) <223> Asp or Ala <220>

<221> MOD„RES <222> (100) <223> Lys or Ala <220>

<221> MQD_RE5 <222> (212) <223> Asn or Ala <400> 6

Thr 1 Hi s Thr Cys Pro Pro Cys Pro 5 Al a Pro 10 Glu Leu Leu Gly Gly 15 Pro Ser val Phe Leu Phe Pro Pro Lys Pro cys Asp Thr Leu Met lie Ser 20 25 30 Arg Thr Pro Gl U val Thr Cys Val val val Xaa Val Ser His Gl u Asp 35 40 45

Page 4

2016202691 27 Apr 2016

PHPi H-02 5-WO l_Su bsti tuts. _Seq u sn c e_Li sti n· 9 ¢2 ) . tX' Pro Gl 14 val cys Phe Asn Trp Tyr val Asp Gly val Glu Val His Asn 50 55 60 Ala Lys Th r Lys Pro Arg Glu Gl u Gl n Tyr ASP Ser Thr Tyr Arg Val 65 70 75 80 Val Ser Val Leu Thr Val Leu His Gl n Asp Trp Leu Asn Gly Lys G IU 85 90 95 Tyr Lys cys Xaa, Val Ser Asn uys Ala teu Pro Val Pro lie Glu Lys 10Q 105 110 Thr Il e Ser Lys Al a Lys Gly Gin Pro Arg Glu pro Gin val Tyr Thr 115 120 125 Leu Pro Pro ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Th ι- 130 135 140 Cys Leu val cys Gly Phs Tyr Pro Ser Asp lie Al a Val Glu Trp ΟΊ u 145 150 155 160 Ser Asn Gly Gin Pro Gl U Asn Asn Tyr Lys Thr Thr p ro Pro val Leu 165 170 175 Asp Ser Asp Gly Pro Phe Phe Leu Tyr Ser Lys Leu Thr val Asp Lys 180 185 190 Ser Arg Trp Glri Gin Gly Asn val Phe Ser cys Ser va i Met His Glu 195 200 205 Al a Leu Hi s Xaa Hi s Tyr Thr Gl n Lys Ser Leu Ser Leu Ser Pro Gly 210 215 220

Lys

225 <210> 7 <211> 344 <212> PRT <213> Artificial Sequence <22O>

<223> Description of Artificial sequence: Synthetic construct <400> 7

Il e Leu Gly Arg Ser 5 G1 u Thr Gin Glu Cys .1.0 Leu Phe Phe Asn Ala 15 Asn Trp Glu Lys Asp Arg Thr Asn Gin Thr Gly val Glu Pro Cys Tyr Gly 20 25 30 Asp Lys ASp Lys Arg Arg Hi s cys Phe Ala Th r Trp Lys Asn lie Ser 35 40 45 Gly Ser lie Glu II e Val LVS G Ih Gly cys Trp Leu Asp Asp lie Asn 50 5 5 60 Cys Tyr Asp Arg Thr Asp 70 Cys Val Glu Lys LVS Asp Ser pro Glu Val 65 75 80 Tyr Phe Cys cys cvs Glu Gly Asn Met CVS Asn Glu Lys Phe Ser Tyr 85 90 95 Phe Pro Gl u Met GlU val Thr Gin Pro Thr Ser Asn Pro Val Thr Pro 100 105 11.0

Page 5

2016202691 27 Apr 2016

PHPH-O25-WOl„Substitute_Sequence_Listing (2.) .txt

Lys Pro pro 115 Thr Gly Gly Gly Thr 120 His Thr Cys Pro Pro 125 cys Pro Al a Pro Glu Leu Leu Gly Gly Pro Ser val Phe Leu Phe Pro Pro Lys Pro 130 13 5 140 Lys Asp Thr Leu Met Il e Ser Arg Thr Pro Glu val Thr cys Val Val 145 150 15 5 160 Val Asp Val Ser Hi s Glu Asp Pro Gl u val Ly s Phe Asn Trp Tvr val 165 170 175 Asp Gly val Glu Val Hi s Asn A1 a Lys Thr Lys Pro Arg Glu Glu Gin 180 185 190 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr val LSI! Hi s Gin 195 200 205 Asp Trp Leu Asn Gly Lys Glu Tyr Lys cys Lys Val Ser ASH Lys Al a 210 215 220 Leu Pro val pro Il e G > U Lys Thr lie Ser Lys Al a Lys Gly Gin pro 225 230 235 240 Arg G ί U Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 245 250 255 Lys Asn Gin val Ser Leu Th r Cys Leu Val Lys Gly Phe Ty r Pro Ser 260 265 270 Asp Il e Ala val Glu Trp Gl u Ser Asn G i y Gin Pro Glu Asn Asn Tyr 275 280 285 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 290 295 300 Ser Lvs Leu Thr Val Asp Lys Ser Arg τ rp Gin Gl n Gly Asn Val Phe 305 310 315 320 Ser Cvs Ser val Met Hi s Glu Ala Leu Hi s Asn Hi s Tyr Thr Gl n Lys 325 330 335 Ser Leu Ser Leu Ser Pro Gly Lys

340 <210> 8 <211> 21 <212> PRT <213> Apis me1Ίifera <400> 8

Met Lys Phe Leu Val Asn val Ala Leu Val phe Met Val Val Tyr He 1 5 10 15

Ser Tyr lie Tyr Ala 20 <210> 9 <211> 22 <212> PRT <213> Unknown Organism <220>

<223> Description of unknown Organism: Tissue Plasminogen

Activator <400>

Page 6

2016202691 27 Apr 2016

PHPH-025-WOl_Substitute„Sequence^Listi ng ¢2}, txt

Met Aso Ala Met Lys Arg Gly Leu cys cys Val Leu Leu Leu cys Gly 1 5 TO 15

Ala Val Phe Val ser Pro 20 <210> 10 <21ί> 20 <2I2> PRT <213> unknown organism <22O>

<223> Description of unknown organism: Native peptide <4 0 u> 10

Met Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu lie Ser Cys

1 5 10 15

Ser Ser Gly Ala 20 <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220>

<223> Description of Artificial Sequence: synthetic peptide <400> 11 lie Leu Gly Arg Ser Glu Thr Gin Glu 1 5 <210> 12 <211> 329 <212> PRT <213> Artificial Sequence <22O>

<223> Description of Artificial Sequence: Synthetic construct <400> 12

lie 1 Leu rzlv vj : J Arg Ser 5 Glu Th r Gin Glu Cys io Leu Phe Phe Asn Ala 15 Asn Trp Gl U Lys asd 20 Arg Thr Asn Gl n Thr 25 Gly val Glu Pro cys 3.0 Tyr Gly Asp Lys Asp 35 Lys Arg Arg Hi s Cys 40 Phe Ala Thr Trp Lys 45 Asn lie Ser Gly Ser 50 lie Glu lie Val Lys 55 Gin Gly Cys Trp Leu 60 Asp Asp 11 e Asn CVS 65 Tyr Asp Arg Thr Asp 70 Cys Val Glu Lys Lys 75 Asp Ser Pro Glu Val 80 Tyr Phe Cys cys Cy s 85 Glu Gly Asn Met cys 90 Asn G i U Lys Phe Ser 95 Tyr Phe Pro Glu Met 100 Thr Gl y Gly Gly Thr 105 His Thr Cys Pro Pro 110 cys Pro A ί a Pro Glu Lsu Leu Gly Gly Pro Ser Val Phe Page 7 Leu Phe Pro Pro Lys

2016202691 27 Apr 2016

PHpH-025~WOl„SubSfitute_Sequence_Llsting <2).tx

115 120 125 Pro Lys Asp Thr Leu Met ϊΐ e Ser Arg Thr Pro Glu val Thr Cys Val 130 13 5 140 Val Val Asp val Ser His Glu Asp Pro Glu val Lys Phe Asn Trp Tyr 145 150 15 5 160 Val Asp Gly Val Glu val Hl s Asn Ala Lys Thr Lys Pro Arg Glu Gl u 165 170 175 Gl ft Tyr Asn Ser Thr Tyr Arg Val val Ser val Leu Thr Val Leu His 180 185 190 Gin Asp Trp Leu Asn dy Lys Gl U Tyr Lys Cys Lys val Ser Asn Lys 195 200 205 Ala Leu pro Val Pro Ils Glu tys Thr lie Ser Lys Al a Lys Gly Gin 210 215 220 Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg G Ϊ U Glu Met 225 230 235 240 Thr Lys Asn Gin Val Ser Leu Thr Cys Let; val Lys Gl y Phe Tyr’ Pro 245 250 25 5 Ser Asp lie. Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn 260 265 270 Tyr Lys Thr Thr Pro Pro val Leu Asp ser Asp Gly Ser Phe Phe Leu 275 280 285 Tyr Ser Lys Leu Thr val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val 290 295 300 Phe Ser CVS ser Val Met His Glu Al a Leu His Asn Hi s Tyr Thr Gin 305 310 315 320 Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 <210> 13 <211> 369 <212> PRT

<213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic construct <400> 13 Met Asp Ala Met X Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 5 10 15 Ala Val Phe Val 20 Ser Pro Glv Ala Ala lie Leu Gly Arg Ser Glu Thr 25 30 Gin Glu Cvs Leu 35 Phe Phe Asn Ala Asn Trp Glu Lys Asp Arg Thr Asn 40 45 Gin Thr Gly Val so Glu Pro cys Tyr Gly Asp Lys Asp Lys Arg Arg His 55 ' 60 Cys Phe Ala Thr 65 Trp Lys Asn lie Ser Gly Ser lie Glu lie val Lys 70 ' 75 SO Gin Gly cys Trp Leu Asp Asp lie Asn Cys Tyr Asp Arg Thr Asp Cys 85 90 95

Page 8

2016202691 27 Apr 2016

PHP H-02 5-wo 1_SU fasti tute. _5eq uenc' e„Li stin· g <2 ). txt val Glu Lys Lys Asp Ser Pro Glu val Tyr phe Cys cys cys Glu -Sly .100 IDS 110 Asn Met Cys Asn Gl u Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr 115 120 125 Gin Pro Thr Ser Asn Pro val Thr Pro Lys pro Pro Thr Gly Gl y Gly 130 135 140 Thr Hi s Thr Cys Pro Pro cys Pro Ala Pro G ϊ U Leu Leu Gly Gly Pro 145 150 155 160 Ser Val Phe Leu Phe pro pro Lys Pro Lys ASp Thr Leu Met il e Ser 165 170 1 7 C Λ.·· 7 Arg Thr Pro Glu Val Thr cys Val val val Asp Val Ser Hi s Glu Asp 180 185 190 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly val Gl u Val Hi s Asn 195 200 205 A1 a Lys Thr Lys Pro Arg Glu Glu Gl FI Tyr Asn Ser Thr Tyr Arg Val 210 215 220 val Ser Val Leu Th r Val Leu His Gl n Asp Trp Leu Asn Gly Lys Glu 225 230 235 240 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu pro val Pro lie G1 U Lys 245 250 255 Thr He Ser Lys A i a Lys sly Gl n Pro Arg Gl U Pro Gl ri val Tyr Thr 260 265 270 Leu Pro pro Ser Arg Gl ii Glu Met Th r Lys Asn Gin Val Ser Leu Thr 275 280 285 cys Leu Val Lys Gly Phe Tyr pro Ser Asp il e Ala Val Glu Trp Glu 290 295 300 ser Asn Gly Gin pro Glu Asn Asn Tyr Lys Th r Thr Pro Pro Val Leu 305 310 315 320 ASp Ser Asp Gly Ser Phe Phe Leu Tvr Ser Lys Leu Thr Val ASp Lys 325 330 335 Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser val Met Hi s Glu 340 345 350 Ala Leu Hi s Asn His Tyr Thr Gin Lys Ser Leu ser Leu Ser p ro Gly 355 360 365

Lys <210> 14 <211> 1114 <212> DNA <213> Artificial sequence <22O>

<223> Description of Artificial Sequence: synthetic construct <400> 14 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgcccggcg ccgctatact tggtagatca gaaactcagg agtgtctttt tttaatgcta 120 attgggaaaa agacagaacc aatcaaactg gtgttgaacc gtgttatggt gacaaagata 180

Page 9

2016202691 27 Apr 2016

PHPH-025-WOl_Substitute_Sequence„Listing (2).txt aacggtggta ttgttttgct acctggaaga atatttctgg ttccattgaa tagtgaaaca 240 aggttgttgg ctggatgata tcaactgcta tgacaggact gattgtgtag aaaaaaaaga 300 cagcccrgaa gtatatttct gttgctgtga gggcaatatg tgtaatgaaa agttttctta 360 ttttccggag atggaagtca cacagcccac ttcaaatcca gttacaccta agccacccac 420 cggtggtgga actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc 480 agtcttcctc ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt 540 cacatgcgtg gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt 600 ggacggcgtg gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac 660 gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta 720 caagtgcaag gtctccaaca aagccctccc agtccccatc gagaaaacca tctccaaagc 780 caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac 840 caagaaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt 900 ggagtgggag agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga 960 ctccgacggc tccttcttcc tctatagcaa gctcaccgtg gacaagagca ggtggcagca 1020 ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa 1080 gagcctctcc ctgtctccgg gtaaatgaga attc 1114 <210> 15 <211> 108 <2T2> DNA <213> Artificial Sequence <22Q>

<223> Description of Artificial sequence: Synthetic oligonucleotide <400> 15 agccagacaa gccagacaag ccagacaagc cagacaagcc agacaagcca gacaagccag 60 acaagccaga caagccagac aagccagaca agccagacaa gccagaca ' 108

<210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence; synthetic

peptide <40Q> 16

Thr Gly Gly Gly Gly

1 5 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthef

peptide <400> 17

Ser Gly Gly Gly Gly

1 5 <210> 1.8 <2ll> 6 <212> PRT <213> Artificial Sequence <22O> <223? Description of Artificial Sequence: synthetic 6xHis tag

Page 10

2016202691 27 Apr 2016 f’HPH--025-'¹Aⁱ01_mSLibst'itifte_Sec?uence~Listinq (2).txt <400> 18 ......

His Hi's His His His His

1 5

Page 11