TITLE

[0001 ] METHODS OF USING ACTiVTN RECEPTOR ΤΐΒ-BASED PROTEINS

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62/175,789, filed June 15, 2015, U.S. Provisional Application No. 62/268,345, filed December 16, 2015, and U.S. Provisional Application No. 62/345,530, filed June 3, 2016; each of which is herein incorporated by reference, in its entirety, for all purposes.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 15, 2016, is named 33752PCT_CRF_sequencelisting.txt and is

83,907 bytes in size.

BACKGROUND

[0004] The transforming growth factor β (TGF-β) family of proteins includes the transforming growth factors-β (TGF-β), activins, bone morphogenic proteins (BMP), nerve growth factors (NGFs), brain-derived neurotrophic factor (BDNF), and growth/

differentiation factors (GDFs). These family members are involved in the regulation of a wide range of biological processes including cell proliferation, differentiation, and other functions.

[0005] Growth/differentiation factor 8 (GDF-8), also referred to as myostatin, is a TGF-β family member expressed for the most part in the cells of developing and adult skeletal muscle tissue. Myostatin appears to play an essential role in negatively controlling skeletal muscle growth (McPherron et al.. Nature (London) 387, 83-90 (1997), Zimmers et al., Science 296: 1486-1488 (2002)). Antagonizing myostatin has been shown to increase lean muscle mass in animals.

[0006] Another member of the TGF-β family of proteins is a related growth/differentiation factor, growth/differentiation factor 11 (GDF-11). GDF-11 has approximately 90 % sequence identity to the ammo acid sequence of myostatin. GDF-11 has a role in the axial patterning in developing animals (Oh et al., Genes Dev 11 : 1812-26 (1997)), and also appears to play a role in skeletal muscle development and growth.

[0007] Activins A, B and AB are the homodimers and heterdimer respectively of two polypeptide chains, βΑ and βΒ (Vale et al., Nature 321, 776-779 (1986), Ling et al., Nature 321 , 779-782 (1986)). Activins were originally discovered as gonadal peptides involved in the regulation of follicle stimulating hormone synthesis, and are now believed to be involved in the regulation of a number of biological activities, Activin A is a predominant fonn of activin.

[0008] Activin, myostatin, GDF-1 1 , BMP9 and other members of the TGF-β superfamily bind and signal through a combination of activin type II and activin type ΪΪΒ receptors, both of which are transmembrane serine/threonine kinases (Harrison et al ,, J. Biol. Chem. 279, 28036-28044 (2,004)). Cross-linking studies have determined that myostatin is capable of binding the activin type II receptors ActRJIA and ActRIIB in vitro (Lee et al, PNAS USA 98:9306-11 (2.001)). There is also evidence that GDF-11 binds to both ActRIIA and ActRIIB (Oh et al., Genes Dev 16:2749-54 (2.002)).

[0009] TGF-β protein expression is known to be associated with a variety of diseases and disorders. Therefore, therapeutic molecules capable of antagonizing several TGF-β proteins simultaneously may be particularly effective for treating these diseases and disorders.

[0010] Related applications include: U.S. Serial No. 12/626,375, filed November 25, 2009, U.S. Serial No. 12/074,877, filed March 5, 2008, U.S. Serial No. 13/329,897, filed December 19, 2011, U.S. Serial No. 11/590,962, filed October 31, 2006, PCT7US2014/014490, filed February 3, 2014, PCT7US2015/011396, filed January 14, 2015, and PCT/US2015/035818, filed June 15, 2015; each of which is hereby incorporated by reference, in its entirety, for all purposes.

SUMMARY

[0011] Disclosed herein is a method treating obesity, treating a disease associated with obesity, increasing muscle mass, maintaining muscle mass, incresaing muscle function, maintaining muscle function, or decreasing fat mass in a subject, comprising: administering to the subject an effective dose of a protein that inhibits at least one of activin, myostatin, BMP9, an endogenous ligand of ActRIIB, and GDF-1 1, optionally wherein the disease associated with obesity is at least one of a genetic obesity syndrome, Prader willi syndrome, a hypothalamic disorder, familial hypercholesterolemia, Bardet-Biedl syndrome, Prader-Willi syndrome, a syndrome resulting from a loss of imprinted genes on 15ql 1-13, Alstrom syndrome, Cohen syndrome, Albright's hereditary osteodystrophy

(pseudohypoparathyroidism), Carpenter syndrome, MOMO syndrome, Rubinstein-Taybi syndrome, a syndrome resulting from deletions of at least one of 6ql6, lp36, 2q37, and 9q34, maternal uniparental disomy of chromosome 14, fragile X syndrome, atherosclerosis, non- alcoholic steatohepatitis, a disease where visceral fat deposition results in one or more deleterious outcomes, cerebrovascular disease, fatty liver, and Borjeson-Forssman-Lehman syndrome, and optionally wherein the polypeptide is selected from the group consisting of: (a) a polypeptide consisting of the amino acid sequence set forth in the group consisting of SEQ ID NO: 4, 6, 12, and 14; (b) a polypeptide having at least 90% sequence identity to (a), and the polypeptide has a W or a Y at the position corresponding to position 28 of the sequence set forth in SEQ ID NO:2 and a T at the position corresponding to position 44 of the sequence set forth in SEQ ID NO: 2, (c) a polypeptide having at least 95% sequence identity to (a), wherein the polypeptide has a W or a Y at the position corresponding to position 28 of the sequence set forth in SEQ ID NO: 2 and a T at the position corresponding to position 44 of the sequence set forth in SEQ ID NO:2, and (d) a polypeptide having a sequence with at least 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % identity to the sequence set forth in ammo acids 19-25, 19, 20, 21 , 22, 23, 24, or 25 through 130-134, 125, 126, 127, 128, 129, 130, 131 , 132, 133, or 134 of SEQ ID NO: 2.

[0012] Also disclosed herein is a method of reversing or preventing cachexia in a subject in need thereof, compri sing: administering to the subject an effective dose of a protein that inhibits at least one of activin, myostatin, bone morphogenetic protein 9 (BMP9), an endogenous ligand of ActRIIB, and growth differentiation factor 11 (GDF-1 1).

[0013] Also disclosed herein is a method of inhibiting solid tumor growth in a human subject, or treating a solid tumor in a human subject, comprising administering a plurality of doses of a formulation to the human subject, wherein the formulation comprises a homodimer of two polypeptides each consisting of the sequence set forth in SEQ ID NO: 10 at a concentration of 70 mg/mL, 8.8% (w/v) sucrose, 10 mM potassium phosphate buffer, 0.006% (w/v) polysorbate 20, and a pH of 6.7, wherein each dose is at least 0.5 mg/kg, and wherein each dose is administered to the subject every two weeks or every four weeks.

[0014] Also disclosed herein is a method of increasing at least one of muscle mass and muscle function in a subject, comprising administering a protein to the subject once every two weeks or once every month at a dose of at least 0.5 mg kg per administration, wherein the protein binds at least one of activin, myostatin, BMP9, and GDF-11 and comprises a polypeptide comprising the sequence set forth in SEQ ID NO:6.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0015] These and other features, aspects, and advantages will become better understood with regard to the following description, and accompanying drawings, where: [0016] Figure 1 shows ST 1587 Pancreatic Adenocarcinoma Body Weights and Tumor

Volume Plots,

[0017] Figure 2 shows an End-of- Study Comparison of Total and Host-Specifi c Body Weights for ST1587.

[0018] Figure 3 shows ST1584 Pancreatic Adenocarcinoma Body Weights and Tumor Volume Plots.

[0019] Figure 4 shows an End-of- Study Comparison of Total and Host-Specific Body- Weights for ST 1584.

[0020] Figure 5 shows ST1 185 Pancreatic Adenocarcinoma Body Weights and Tumor Volume Plots.

[0021 ] Figure 6 shows an End-of- Study Comparison of Total and Host-Specific Body- Weights for ST1 185.

[0022] Figure 7 shows ST1 145 Squamous Head & Neck Body Weights and Tumor Volume Plots.

[0023] Figure 8 shows an End-of-Study Comparison of Total and Host-Specific Body Weights for ST 1145.

[0024] Figure 9 shows that average ECK values were determined as being 0.36 nM with 95% Confidence Intervals from 0.24 nM to 0.56 nM for STM 434, and as 0.45 nM, with 95% Confidence Intervals from 0.29 nM to 0.72 nM for STM 217 binding with huBMP9.

[0025] Figure 10 shows a kinetic analysis of STM 434 binding with human BMP9. Hie kinetic parameters were determined as ka-1.4e7 1 Ms, kd-1.7e-4 1/s, and Kd— 12 pM for STM 434 binding with huBMP9.

[0026] Figure 11 shows the impact of STM 434 on FSH, lean body mass, and 6 minute walk- distance. 6 minute walk distance is measured in meters (Y axis).

[0027] Figure 12 shows best overall response by tumor type and time on trial for subjects with granulosa tumors. Estimated months on trial for granulosa is shown for each subject.

DETAILED DESCRIPTION

Compositions

[0028] Described herein is an isolated protein comprising a stabilized human activin IIB receptor (svActRIIB) polypeptide and related formulations. Proteins and polypeptides can be characterized by their ability to bind to at least one of four TGF-β proteins, myostatm (GDF- 8), activin A, BMP9 or GDF-1 1, to inhibit the activities of at least one of these proteins, and. optionally, to have improved manufacturability properties compared with other ActRIIB soluble receptors. The stabilized human activin IIB receptor polypeptide can be

characterized by amino acid substitutions at both positions E28 and S44 with reference to the extracellular domain of ActRIIB, as set forth in SEQ ID NO: 2. In one embodiment, a stabilized human activin IIB receptor polypeptide can have a further substitution of alanine at position 64 with respect to SEQ ID NO: 2. In some aspects, a protein is an antibody, an ActRIIB-based protein, and/or an Fc -Fusion protein. In some aspects, an Fc-Fusion protein is an ActRIIB Fc-Fusion protein. In some aspects, an ActRIIB protein comprises a sequence with at least 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % identity to the sequence set forth in amino acids 19-25, 19, 20, 21, 22, 23, 24, or 25 through 130-134, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO: 2, optionally wherein the Fc is IgG, optionally wherein IgG is human IgG. In some aspects, an ActRIIB protein comprises or consists of the sequence set forth in SEQ ID NO:6 or SEQ ID NO: 10. In some aspects, an ActRIIB protein comprises a sequence with at least 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % identity to the sequence set forth in SEQ ID NO: 6. In some aspects, an ActRIIB protein comprises a sequence the differs by less than 1 -2, 1, 2, 3, 4, or 5 amino acids from the sequence set forth in SEQ ID NO:6. In some aspects, a protein inhibits at least two or three of activin, myostatin, BMP9, and GDF-1 1. In some aspects, a protein inhibits activin, myostatin, and GDF-11 , In some aspects, a protein inhibits activin .

[0029] As used herein the term. "TGF-β family members" or 'TGF-β proteins" refers to the structurally related growth factors of the transforming growth factor family including activins, and growth and differentiation factor (GDF) proteins (Kingsley et al. Genes Dev. 8: 133-146 (1994), McPherron et al., Growth factors and cytokines in health and disease, Vol. IB, D. LeRoith and C. Bondy. ed., JAI Press Inc., Greenwich, Conn, USA: pp 357-393).

[0030] GDF-8, also referred to as myostatin, is a negative regulator of skeletal muscle tissue (McPherron et al. PNAS USA 94: 12457-12461 (1997)). Myostatin is synthesized as an inactive protein approximately 375 ammo acids in length, having GenBank Accession No: AAB86694 for human. The precursor protein is activated by proteolytic cleavage at a tetrabasic processing site to produce an N-terminal inactive prodomain and an approximately 1 9 amino acid C-terminal protein which dimerizes to form a homodimer of about 25 kDa. This homodimer is the mature, biologically active protein (Zimmers et al.. Science 296, 1486 (2002)). [0031] As used herein, the term "prodomain" or "propeptide ^" refers to the inactive N- terminal protein which is cleaved off to release the active C-terminal protein. As used herein the term "myostatin" or "mature myostatin" refers to the mature, biologically active C~ terminal polypeptide, in monomer, dimer or other form, as well as biologically active fragments or related polypeptides including allelic variants, splice variants, and fusion peptides and polypeptides. The mature myostatin has been reported to have 100% sequence identity among many species including human, mouse, chicken, porcine, turkey, and rat (Lee et al., PNAS 98, 9306 (2001)).

[0032] As used herein GDF-1 1 refers to the BMP (bone morphogenic protein) having Swissprot accession num ber 095390, as well as variants and species homologs of that protein. GDF-1 1 is involved in the regulation of anterior/posterior patterning of the axial skeleton (McPherron et al. Nature Genet. 22 (93): 260-264 (1999): Gamer et al, Dev. Biol. 208 (1), 222-232 (1999)) but postnatal functions are unknown.

[0033] Activin A is the homodimer of the polypeptide chains βΑ. As used herein the term "activm A" refers to the activin protein having GenBank Accession No: NM 002192. Activms A, B, and AB are the homodimers and heterodimer respectively of two polypeptide chains, βΑ and βΒ. As used herein, "activin" refers to activin A, B, and AB, as well as variants and species homologs of that protein.

[0034] Receptor Polypeptides

[0035] As used herein, the term activin type II B receptors (ActRIIB) refers to human activin receptors having accession number NP_001097 or variants thereof, such as those having arginine at position 64 substituted with alanine. The term soluble ActRIIB (wild type) refers to the extracellular domain of ActRIIB, e.g., amino acids 1 to 134 (with signal sequence), or amino acids 19 through 134 of SEQ ID NO: 2 (without signal sequence), or amino acids 20, 21, 22, 23, 24, or 25 through 134 of SEQ ID NO: 2 (without signal sequence).

[0036] Stabilized receptor pol ^y peptides

[0037] Also provided herein is an isolated protein comprising a stabilized Aetl!B receptor polypeptide (referred to herein as "sv ActRIIB polypeptide"). As used herein the term "sv ActRIIB protein" refers to a protein comprising a stabilized ActRIIB polypeptide. These polypeptides and proteins are characterized as having the ability to bind and inhibit the activity of any one of activin A, myostatin, BMP9 or GDF-11, in addition to having improved manufacturability characteristics. [0038] The stabilized ActRIIB polypeptide can be characterized by having an amino acid substitution at both position 28 and 44 with respect to SEQ ID NO: 2, For consistency, the amino acid positions on the stabilized ActRIIB polypeptides and proteins are referred to with respect to the positions in SEQ ID NO: 2, regardless of whether the polypeptide is mature or truncated , As used herein, the tenn "mature" refers to a polypeptide or peptide without its signal sequence. As used herein, the term "truncated" refers to polypeptides having N terminal amino acids or C terminal amino acids removed,

[0039] In one embodiment, the isolated stabilized activin ΠΒ receptor polypeptide

(svActRIIB) has the polypeptide sequence set forth in SEQ ID NO: 2. In another embodiment, the polypeptide has the sequence set forth in amino acids 19 through 134 of SEQ ID NO: 2. In another embodiment, the polypeptide has the sequence set forth in amino acids 23 through 134 of SEQ ID NO: 2. In another embodiment, the polypeptide has the sequence set forth in ammo acids 25 through 134 of SEQ ID NO: 2. In another embodiment, the polypeptide has an ammo acid sequence with at least 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 % or 99 % identity to any one of the polypeptides above. In one embodiment, the polypeptide is capable of binding myostatin, activin A, BMP9 or GDF-11. In one embodiment, the isolated stabilized activin IIB receptor polypeptide (sv ActRIIB) comprises a sequence with at least 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 % or 99 % identity to the sequence set forth in ammo acids 19-24, 19, 20, 21, 22, 23, 24, or 25 through 130-134, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO: 2.

[0040] In one embodiment, the isolated stabilized activin IIB receptor polypeptide

(sv ActRIIB) has the polypeptide sequence set forth in SEQ ID NO: 2, except for an amino acid substitution at position 28, and an amino acid substitution at position 44, wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T. In another embodiment, the polypeptide has the sequence set forth in amino acids 19 through 134 of SEQ ID NO: 2, except for an amino acid substitution at position 28, and an amino acid substitution at position 44, wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T. In another embodiment, the polypeptide has the sequence set forth in amino acids 23 through 134 of SEQ ID NO: 2, except for an amino acid substitution at position 28, and an amino acid substitution at position 44, wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T. In another embodiment, the polypeptide has the sequence set forth in amino acids 25 through 134 of SEQ ID NO: 2, except for an amino acid substitution at position 28, and an amino acid substitution at position 44, wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T. In another embodiment, the polypeptide has an amino acid sequence with at least 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 % or 99 % identity to any one of the polypeptides above, wherein the polypeptide has an amino acid substitution at position 28, and an amino acid substitution at position 44, optionally wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T, and wherein the polypeptide is capable of binding myostatin, activm A, BMP9 or GDF-i 1. In one embodiment, the substitution of the above polypeptides at position 28 is W, and the substitution at position 44 is T, wherein the polypeptide is capable of binding myostatin, activin A, BMP 9 or GDF-11.

[0041] In one embodiment, svActRIIB polypeptide includes a signal sequence, for example, the sequences shown in SEQ ID NO: 4, 8, 12, and 16. However, various signal peptides can be used in the preparation of the polypeptides of the instant application. The signal peptides can have the sequence set forth in amino acids 1 to 19 of SEQ ID NO: 4, for example. Any other signal peptides useful for expressing svActRIIB polypeptides can be used. In other embodiments, the signal sequence is removed, leaving the mature peptide. Examples of svActRIIB polypeptides lacking a signal sequence includes, for example, the sequences shown in SEQ ID NO: 6, 10, 14 and 18.

[0042] In one embodiment, the protein comprises a stabilized activm IIB receptor polypeptide, wherein the polypeptide is selected from the group consisting of polypeptides having the sequence set forth in the group consisting of SEQ ID NO: 4, 6, 12 and 14. These polypeptides represent amino acids 25 to 134 of SEQ ID NO: 2, wherein the polypeptide has an ammo acid substitution at position 28, and an amino acid substitution at position 44, wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T, and wherein the polypeptide is capable of binding myostatin, activin A, BMP 9 or GDF-I 1, with and without a signal sequence different from that shown in SEQ ID NO: 2. In another embodiment the protein comprises a polypeptide having at least 80-100%, 90-100%, 85-95%, 90-95%, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity to SEQ ID NO: 4, 6, 12 or 14, optionally wherein the polypeptide has a W or Y at position 28 and a T at position 44, and wherein the polypeptide is capable of binding myostatin, activin A, BMP 9 or GDF- 11. In one embodiment, the substitution at position 28 is W and the substitution at position 44 is T, wherein the polypeptide is capable of binding myostatin, activin A or GDF-11. [0043] In a further embodiment svActRIIB protein further comprises a heterologous protein. In one embodiment, the heterologous protein is an Fc domain. In a further embodiment, the Fc domain is a human IgG Fc domain. In one embodiment, the protein comprises a polypeptide having the sequence set forth in the group consisting of SEQ ID NO: 8, 10, 16 and 18. In another embodiment, the protein comprises a polypeptide having at least 80- 100%, 90-100%, 85-95%, 90-95%, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity to the sequence shown in SEQ ID NO: 8, 10, 16 or 18, optionally wherein the polypeptide has a W or Y at position 28 and a T at position 44, and wherein the polypeptide is capable of binding myostatin, activin A, BMP 9 or GDF-11. In one embodiment, the substitution at position 28 is W and the substitution at position 44 is T, wherein the polypeptide is capable of binding myostatin, activin A or GDF-11. In certain aspects, the protein comprises or consists of the sequence shown in SEQ ID NO: 10. In certain aspects, the sequence is glycosylated.

10044] In a further embodiment svActRIIB protein is STM 434, described in the Examples.

[0045] In a further embodiment, the protein comprises the any one of the polypeptides described above, wherein the amino acid residue at position 64 is alanine.

[0046] In another embodiment, the term svActRIIB polypeptide and protein encompasses proteins comprising fragments of SEQ ID NO: 2, 4, 6, 12 and 14, including N and C terminal truncations, wherein position 28 is W or Y, and position 44 is T, and wherein the polypeptide is capable of binding myostatin, activin A or GDF-11.

[0047] As used herein the term "derivative ^" of a svActRIIB polypeptide refers to the attachment of at least one additional chemical moiety, or at least one additional polypeptide to form covalent or aggregate conjugates such as glycosyl groups, lipids, acetyl groups, or C- terminal or N-terminal fusion polypeptides, conjugation to PEG molecules, and other modifications which are described more fully below. Stabilized ActRIIB receptor polypeptides can also include additional modifications and derivatives, including modifications to the C and N termini which arise from processing due to expression in various cell types such as mammalian cells, E. coli, yeasts and other recombinant host cells.

[0048] svActRIIB proteins can further comprise heterologous polypeptides attached to svActRIIB polypeptide either directly or through a linker to form a fusion protein. As used herein the term "fusion protein" refers to a protein having a heterologous polypeptide attached to another polypeptide such as an svActRIIB. Heterologous polypeptides include but are not limited to Fc polypeptides, his tags, and leucine zipper domains to promote oHgomerization and further stabilization of the stabilized ActRIIB polypeptides as described in, for example, WO 00/29581, which is herein incorporated by reference. In one embodiment, the heterologous polypeptide is an Fc polypeptide or domain , in one embodiment, the Fc domain is selected from a human IgGl Fc (SEQ ID NO: 23), a modified IgGl Fc, IgG2 Fc (SEQ ID NO: 22), and IgG4 Fc (SEQ ID NO: 24) domain, Sv ActRIIB protein can further comprise all or a portion of the hinge sequence of the TgGl, IgG2, or IgG4. Exemplary sv ActRIIB polypeptides are selected from polypeptides consisting of the sequences as set forth in SEQ ID NO: 8, 10, 16 and 18, as well as those polypeptides having substantial similarity to these sequences, wherein the substitutions at positions 28 and 44 are retained. As used herein, "substantial similarity" refers to sequences that are at least 80- 100%, 90-100%, 85-95%, 90-95%, 80 % identical, 85 % identical, 90 % identical, 95 % identical, 96 % identical, 97 % identical, 98 % identical, 99 % identical to any of SEQ ID NO: 8, 10, 16, and 18, wherein the polypeptides retain W or Y at position 28 and T at position 44, and wherein the polypeptide is capable of binding myostatin, activm A or GDF- 1 1 . In one embodiment, the substitution at position 28 is W and the substitution at position 44 is T, wherein the polypeptide is capable of binding myostatin, activm A or GDF-11.

[0049] sv ActRIIB polypeptides can optionally further comprise a "linker". Linkers serve primarily as a spacer between a polypeptide and a second heterologous polypeptide or other type of fusion or between two or more stabilized ActRIIB polypeptides. In one embodiment, a linker is made up of amino acids linked together by peptide bonds, preferably from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. One or more of these amino acids can be glycosylated, as is understood by those of skill in the art. In one embodiment, the 1 to 20 amino acids can be selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In one embodiment, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Exemplary linkers are polyglycines, particularly (G!y)j (SEQ ID NO: 51), (Gly)g (SEQ ID NO: 52), poly(Gly-Ala), and polyaianines. One exemplary suitable linker is (Gly^Ser (SEQ ID NO: 25). In a further embodiment, sv ActRIIB can comprise a "hinge linker", that is a linker sequence provided adjacent to a hinge region or a partial hinge region of an IgG, as exemplified in SEQ ID NO: 27. Hinge sequences include IgG2Fc, IgGlFc, and IgG4Fc.

[0050] Hinge linker sequences can also be designed to improve manufacturability and stability of svActRIIB-Fc proteins. In one embodiment, the hinge linkers of SEQ ID NO: 27, 38, 40, 42, 44, 45, and 46 are designed to improve manufacturability with the IgG2 Fc (SEQ ID NO: 22) when attached to svActRIIB polypeptides. In one embodiment, the hinge linker sequences is designed to improve manufacturability when attaching svActRIIB polypeptides to a human IgGI Fc (SEQ ID NO: 23) or a modified human IgG l Fc.

[0051] Linkers can also be non-peptide Sinkers. For example, aSkyi linkers such as -NH- (CH ')s-C(O)-. wherein s = 2-20 can be used. These alky! linkers may further be substituted by any non-sterically hindering group such as lower aiky! (e.g., Ci-Cr,) lower acyl, halogen (e.g., CI, Br), CN, NH ₂ , phenyl, etc.

[0052] svActRIIB polypeptides disclosed herein can also be attached to a non-polypeptide molecule for the purpose of conferring desired properties such as reducing degradation and/or increasing half-life, reducing toxicity, reducing immunogenicity, and/or increasing the biological acti vity of svActRIIB polypeptides. Exemplary molecules include but are not limited to linear polymers such as polyethylene glycol (PEG), polylysine, a dextran; a lipid; a cholesterol group (such as a steroid); a carbohydrate, or an oligosaccharide molecule.

[0053] svActRIIB proteins and polypeptides can have improved manufacturability properties when compared to other ActRlIB soluble polypeptides. As used herein, the term

"manufacturability" refers to the stability of a particular protein during recombinant expression and purification of that protein. Manufacturability is believed to be due to the intrinsic properties of the molecule under conditions of expression and purification.

Examples of improved manufacturability characteristics include uniform giycosyiation of a protein, increased cell titer, growth and protein expression during recombinant production of the protein, improved purification properties, and improved stability at low pH. svActRIIB proteins and polypeptides demonstrate the improved manufacturability, along with retention of in vitro and in vivo activity, compared with other soluble ActRIIB polypeptides. Further, additional hinge linker sequences can confer additional manufacturability benefits.

[0054] As used herein, the term a "svActRIIB polypeptide activity" or "a biological activity of a soluble ActRIIB polypeptide" refers to one or more in vitro or in vivo activities of svActRIIB polypeptides. Activities of svActRIIB polypeptides include, but are not limited to, the ability to bind to myostatin or activin A or GDF-11 , and the ability to inhibit or neutralize an activ ity of myostatin or activin A or GDF-11. As used herein, the term

"capable of binding" to myostatin, activin A, BMP 9 or GDF-11 refers to binding measured by methods known in the art, such as the KinExA™ method. In vitro inhibition of myostatin, activin A, BMP 9 or GDF-1 1 can be measured using, for example, the pMARE C2CI 2 cell- based assay. In vivo activity, is demonstrated by increased lean muscle mass in mouse models. In vivo activities of svActRIIB polypeptides and proteins include but are not limited to increasing body weight, increasing lean muscle mass, and increasing the ratio of lean muscle to fat mass. Therapeutic activities further include reducing or preventing cachexia caused by certain types of tumors, preventing the growth of certain types of tumors, and increasing survival of certain animal models. Further discussion of svActRIIB protein and polypeptide activities is provided below.

[0055] In another aspect, an isolated nucleic acid molecule comprising a polynucleotide encoding an svActRIIB polypeptide is provided.

[0056] In one embodiment, the pol nucleotide encodes a polypeptide having the sequence set forth in SEQ ID NO: 2, except for an amino acid substitution at position 28, and an amino acid substitution at position 44, wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T. In another embodiment, the polynucleotide encodes a polypeptide having the sequence set forth in amino acids 19 through 134 of SEQ ID NO: 2, except for an ammo acid substitution at position 28, and an amino acid substitution at position 44, wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T. In another embodiment, the polynucleotide encodes a polypeptide having the sequence set forth in amino acids 23 through 134 of SEQ ID NO: 2, except for an amino acid substitution at position 28, and an ammo acid substitution at position 44, wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T. In another embodiment, the polynucleotide encodes a polypeptide having the sequence set forth in amino acids 25 through 134 of SEQ ID NO: 2, except for an amino acid substitution at position 28, and an ammo acid substitution at position 44, wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T. In another embodiment, the polynucleotide encodes the a polypeptide having an amino acid sequence at least 80-100%, 90-100%, 85-95%>, 90-95%>, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % identity to any one of the polypeptides above, wherein the polypeptide has single amino acid substitution at position 28, and an amino acid substitution at position 44, optionally wherein the substitution at position 28 is selected from W or Y, and the substitution at position 44 is T, and wherein the polypeptide is capable of binding myostatin, activin A, BMP 9 or GDF-11. In one embodiment, the polynucleotide of the above embodiments encodes a polypeptide wherein the substitution at position 28 is W and the substitution at position 44 is T. [0057] In one embodiment, the isolated nucleic acid molecule comprises a polynucleotide encoding a polypeptide having the sequence set forth in the group consisting of SEQ ID NO: 4, 6, 12, and 14. In another embodiment, the nucleic acid comprises a polynucleotide encoding a polypeptide having at least 80-100%, 90-100%, 85-95%, 90-95%, 80 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity- to SEQ ID NO: 4, 6, 12 or 14, optionally w herein the polypeptide has a W or Y at position 28 and a T at position 44, and wherein the polypeptide is capable of binding activin A, GDF-11, or myostatin. In one embodiment, the polynucleotide of the above embodiments encodes a polypeptide wherein the substitution at position 2,8 is W and the substitution at position 44 is T, and wherein the polypeptide is capable of binding activin A, GDF-11 or myostatin,

[0058] In another embodiment, an isolated nucleic acid molecule further comprises a polynucleotide encoding at least one heterologous protein. In one embodiment, the heterologous protein is an Fc domain, in a further embodiment, the Fc domain is a human IgG Fc domain. In another embodiment, the nucleic acid molecule further comprises polynucleotides encoding linkers and hinge linkers set forth in, e.g., SEQ ID NO: 25, 27.

[0059] In one embodiment, the nucleic acid molecule comprises a polynucleotide encoding a polypeptide consisting of the sequence set forth in the group consisting of SEQ ID NO: 8, 10, 16 and 18. In another embodiment, the nucleic acid comprises a polynucleotide encoding a polypeptide having at least 80-100%, 90-100%, 85-95%, 90-95%, 80 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 % sequence identity to the group consisting of SEQ ID NO: 8, 10, 16 and 18, optionally wherein the polypeptide has a W or Y at position 28 and a T at position 44, and wherein the polypeptide is capable of binding activin A, GDF-1 1, or myostatin. In one embodiment, the polynucleotide of the above embodiments encodes a polypeptide wherein the substitution at position 28 is W and the substitution at position 44 is T, and wherein the polypeptide is capable of binding myostatin, activin A or GDF-1 1.

[0060] In one embodiment, the isolated nucleic acid molecule comprises a polynucleotide having the sequence selected from the group consisting of SEQ ID NO: 3, 5, 11 or 13, or its complement. In another embodiment, the isolated nucleic acid molecule comprises a polynucleotide having the sequence selected from the group consisting of the sequence SEQ ID NO: 7, 9, 15 and 17, or its complement. In a further embodiment the isolated nucleic acid molecule hybridizes under stringent or moderate conditions with SEQ ID NO: 3, 5, 7, 9, 1 1, 13, 15 or 17 wherein the encoded polypeptide is substantially similar to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18, optionally wherein the polypeptide comprises an amino acid sequence having W or Y at position 28, and T at position 44, and wherein the encoded polypeptide is capable of binding or inhibiting activin A, myostatin or GDF-11.

[0061] Nucleic acid molecules include DNA in both single-stranded and double-stranded form, as well as the RNA complement thereof, DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA may be isolated by conventional techniques, such as by using the DNA of SEQ ID NO: 3, 5, 11 or 13, or a suitable fragment thereof, as a probe. Genomic DN A encoding ActRIIB polypeptides is obtained from genomic libraries which are available for a number of species. Synthetic DNA is available from chemical synthesis of overlapping oligonucleotide fragments followed by assembly of the fragments to reconstitute part or all of the coding regions and flanking sequences. RNA may be obtained from procaryotic expression vectors which direct high-level synthesis of mRNA, such as vectors using T ^' 7 promoters and RNA polymerase. cDNA is obtained from libraries prepared from mRNA isolated from various tissues that express ActRIIB. The DNA molecules include full length genes as well as polynucleotides and fragments thereof. The full length gene may also include sequences encoding the N-terminal signal sequence.

[0062] Also provided are the nucleic acid molecule describe above, wherein the

polynucleotide is operably linked to a transcriptional or translational regulatory sequence.

[0063] In another aspect expression vectors containing the nucleic acid molecules and polynucleotides are also provided, and host cells transfoiTned with such vectors, and methods of producing svActRIIB polypeptides are also provided. The term "expression vector" refers to a plasmid, phage, virus or vector for expressing a polypeptide from a polynucleotide sequence. Vectors for the expression of sv ActRIIB polypeptides contain at a minimum sequences required for vector propagation and for expression of the cloned insert. An expression vector comprises a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a sequence that encodes sv ActRIIB polypeptides and proteins to be transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. These sequences may further include a selection marker. Vectors suitable for expression in host cells are readily available and the nucleic acid molecules are inserted into the vectors using standard recombinant DNA techniques. Such vectors can include promoters which function in specific tissues, and viral vectors for the expression of sv ActRIIB polypeptides in targeted human or animal cells. An exemplary expression vector suitable for expression of svActRITB is the pDSRa, (descnbed in WO 90/14363, herein incorporated by reference) and its derivatives, containing svActRIIB polynucleotides, as well as any additional suitable vectors known in the art or described below.

Polypeptides

[0064] In some embodiments, compositions disclosed herein include a polypeptide that is less than 100% identical to an amino acid sequence disclosed herein. In some embodiments, the polypeptide is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or between 99 and 100% identical to a sequence disclosed herein.

[0065] The term "percent identical" in the context of two or more amino acid or nucleic acid sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent identity can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the TWO sequences to be compared.

[0066] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0067] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appi. Math. 2:482 (1981), by the homology alignment algorithm of Needlernan & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0068] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403-410 ( 1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information

(www ,ncbi .nl m .nih .gov/) .

Variants

[0069] The compositions described herein also encompass variants of the polypeptides described herein. As used herein, the term "variants ^" ' refers to polypeptides having one or more amino acid residues inserted, deleted or substituted into the original amino acid sequence and which retain at least a portion of the function of the polypeptide described herein. As used herein, fragments of the polypeptides are included within the definition of "variants". It is understood that any given peptide or peptibody may contain one or two or all three types of variants. Insertional and substitutional variants may contain natural amino acids, as well as non-naturaiiy occurring amino acids or both. Variants can include, e.g., polypeptides that include a leader or signal sequence: polypeptides with additional ammo terminal residues, e.g., Metl or Lys 2; polypeptides with expression tags, e.g., histidine tags; and polypeptides expressed as fusion proteins.

[0070] Variants of the polypeptides described herein can include amino acid

substitutions. Stereoisomers (e.g. , D-amino acids) of the twenty conventional (naturally occurring) ammo acids, non-naturally occurring ammo acids such as -, α-disubstituted amino acids, N-aikyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of non-naturally occurring amino acids include, for example: aminoadipic acid, beta-alanine, beta- aminopropionic acid, aminobutyric acid, piperidmic acid, ammocaprioic acid,

aminoheptanoic acid, aminoisobutyric acid, aminopimelic acid, di aminobutyric acid, desmosme, diammopimelic acid, diammopropionic acid, N-ethylglycme, N-ethylaspargine, hyroxylysine, allO-hydroxylysine, hydroxyproline, isodesmosine, allo-isoleucine, N- methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine, 4-hydroxyproline, γ-carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-Ν-methylarginine, and other similar amino acids and ammo acids (e.g. , 4-hydroxyproline).

[0071] Naturally occurring residues may be divided into (ove lapping) classes based on common side chain properties:

1) neutral hydrophobic: Met, Ala, Val, Leu, He, Pro, Tip, Met, Phe;

2) neutral polar: Cys, Ser, Thr, Asn, Gin, Tyr, Gly; 3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

[0072] Substitution with naturally occurring amino acids can be conservative or non- conservative. Conservative amino acid substitutions involve exchanging a member of one of the above classes for another member of the same class. Conservative changes may encompass unconventional amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include

peptidomimetics and other reversed or inverted forms of amino acid moieties.

Methods of Making

[0073] Also provided are methods of making svActRJIB polypeptides. A variety of other expression/host systems may be utilized . These systems include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, piasmid or cosmid DN A expression vectors; yeast transformed with yeast expression vectors: insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, T ^' MV) or transformed with bacterial expression vectors (e.g., T ^' i or pBR322 piasmid); or animal cell systems. Mammalian cells useful in recombinant protein production include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (0 10) cell lines, or their derivatives such as Veggie CHO and related cell lines which grow in serum- free media (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient in DHFR (see Urlaub et al., 1980, Proc, Natl Acad. Sci. USA 77:4216-20) COS cells such as the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al, 1981 , Cell 23: 175), W 138, BHK, HepG2, 3T3 (ATCC CCL 163), RJN, MIX K . A549, PC12, K562, L cells, C 127 cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see

McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains deri ved from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Mammalian expression allows for the production of secreted or soluble polypeptides which may be recovered from the growth medium. [0074] Using an appropriate host-vector system, svActRIIB polypeptides are produced recombinantly by culturing a host cell transformed with an expression vector containing the nucleic acid molecules under conditions allowing for production. Transformed cells can be used for long-term, high-yield polypeptide production. Once such cells are transformed with vectors that contain selectable markers as well as the desired expression cassette, the cells can be allowed to grow in an enriched media before they are switched to selective media, for example. The selectable marker is designed to allow growth and recovery of cells that successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell line employed. An overview of expression of recombinant proteins is found in Methods of Enzymology, v. 185, Goeddell, D.V., ed., Academic Press (1990).

[0075] In some cases, such as in expression using procaryotic systems, the expressed polypeptides may need to be "refolded" and oxidized into a proper tertiary structure and disulfide linkages generated in order to be biologically active. Refolding can be

accomplished using a number of procedures well known in the art. Such methods include, for example, exposing the solubilized polypeptide to a pH usually above 7 in the presence of a chaotropic agent. The selection of chaotrope is similar to the choices used for inclusion body solubilization, however a chaotrope is typically used at a lower concentration.

Exemplary chaotropic agents are guanidine and urea. In most cases, the refolding/oxidation solution will also contain a reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential which allows for disulfide shuffling to occur for the formation of cysteine bridges. Some commonly used redox couples include

cysteine/cystamine, glutathione/dithiobisGSH, cupric chloride, dithiothreitol DTT/dithiane DTT, and 2-mercaptoethanol (bME)/dithio-bME. In many instances, a co-solvent may be used to increase the efficiency of the refolding. Commonly used cosolvents include glycerol, polyethylene gly col of various molecular weights, and arginme.

[0076] In addition, the polypeptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d.Ed., Pierce Chemical Co. (1984); Tarn et al., J Am Chem Soc, 105:6442, (1983): Mernfield, Science 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284; Barany et al., Int J Pep Protein Res, 30:705-739 ( 1987). [0077] The polypeptides and proteins can be purified according to protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the proteinaceous and non-proteinaceous fractions. Having separated the peptide polypeptides from, other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). The term "isolated polypeptide" or "purified polypeptide" as used herein, is intended to refer to a composition, isolatable from, other components, wherein the polypeptide is purified to any degree relative to its naturally-obtainable state. A purified polypeptide therefore also refers to a polypeptide that is free from the environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms the major component of the composition, such as constituting about 50 %, about 60 %, about 70 %, about 80 %, about 85 %, or about 90 % or more of the proteins in the composition. The term isolated can include a synthesized component such as a polypeptide.

[0078] Various techniques suitable for use in purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies (immunoprecipitation) and the like or by heat denaturation, followed by centrifugation; chromatography such as affinity chromatography (Protein-A columns), ion exchange, gel filtration, reverse phase, hydroxylapatite, hydrophobic interaction

chromatography, isoelectric focusing, gel electrophoresis, and combinations of these techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified polypeptide.

Exemplar - purification steps are provided in the Examples below.

[0079] Various methods for quantifying the degree of purification of polypeptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific binding activity of an active fraction, or assessing the amount of peptide or polypeptide within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a polypeptide fraction is to calculate the binding activity of the fraction, to compare it to the binding activity of the initial extract, and to thus calculate the degree of purification, herein assessed by a "-fold purification number," The actual units used to represent the amount of binding activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the polypeptide or peptide exhibits a detectable binding activity.

Methods of Use

[0080] Also provided are methods, proteins, and compositions for reducing or neutralizing the amount or activity of at least one of myostatin, activm A, BMP 9 or GDF-1 1 in vivo and in vitro, svActRIIB polypeptides have a high binding affinity for myostatm, activin A, and GDF-1 1 , and are capable of reducing and inhibiting the biological activities of at least one of myostatm, activin A and GDF-11 , In some aspects, a protein is an antibody or an Fc-Fusion protein. In some aspects, an Fc-Fusion protein is an ActRIIB Fc-Fusion protein. In some aspects, an ActRIIB Fc-Fusion protein comprises a sequence with at least 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % identity to the sequence set forth in amino acids 19-25, 19, 20, 21 , 22, 23, 24, or 25 through 130-134, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO: 2, optionally wherein the Fc is IgG, optionally wherein IgG is human IgG , In some aspects, an ActRIIB Fc-Fusion protein comprises or consists of the sequence set forth in SEQ ID NO:6 or SEQ ID NO: 10. In some aspects, a protein inhibits at least two of activm, myostatin, and GDF-11. In some aspects, a protein inhibits activin, myostatin, and GDF-1 1. In some aspects, a protein inhibits activin.

[0081] In one aspect, methods and reagents are provided for treating myostatin-related and/or activin A related disorders in a subject in need of such a treatment by administering an effective dosage of an sv ActRIIB composition to the subject. As used herein the term

"subject" refers to any animal, such as mammals including humans.

[0082] The compositions are useful for increasing lean muscle mass in a subject. The compositions may also be useful to increase lean muscle mass in proportion to fat mass, and thus decrease fat mass as percentage of body weight in a subject.

[0083] The disorders that can be treated by an svActRIIB include but are not limited to various forms of muscle wasting, as well as metabolic disorders such as diabetes and related disorders, and bone degenerative diseases such as osteoporosis.

[0084] Muscle wasting disorders also include dystrophies such as Duchenne's muscular dystrophy, progressive muscular dystrophy, Becker's type muscular dystrophy, Dejerine- Landouzy muscular dystrophy, Erb's muscular dystrophy, and infantile neuroaxonal muscular dystrophy. Additional muscle wasting disorders arise from chronic diseases or disorders such as amyotrophic lateral sclerosis, congestive obstructive pulmonary disease, cancer, AIDS, renal failure, organ atrophy, androgen deprivation, and rheumatoid arthritis, [0085] Over-expression of mvostatin and/or activin may contribute to cachexia, a severe muscle wasting syndrome. Cachexia results from cancers, and also arises due to rheumatoid arthritis, diabetic nephropathy, renal failure, chemotherapy, injury due to bums, as well as other causes. In another example, serum and intramuscular concentrations of myostatin- immunoreactive protein was found to be increased in men exhibiting AIDS-related muscle wasting and was inversely related to fat-free mass (Gonzalez-Cadavid et al ,, PNAS USA 95: 14938-14943 (1998)). Mvostatin levels have also been shown to increase in response to bums injuries, resulting in a catabolic muscle effect (Lang et al, FASEB J 15, 1807-1809 (2001)). Additional conditions resulting in muscle wasting may arise from, inactivity due to disability such as confinement in a wheelchair, prolonged bed rest due to stroke, illness, spinal chord injury, bone fracture or trauma, and muscular atrophy in a microgravity environment (space flight). For example, plasma myostatin immunoreactive protein was found to increase after prolonged bed rest (Zachwieja et al. J Gravit Physiol . 6(2): 11(1999). It was al so found that the mu scles of rats exposed to a microgravity environment during a space shuttle flight expressed an increased amount of myostatin compared with the muscles of rats which were not exposed (Lalani et al ., J.Endocrm 167 (3):417-28 (2000)).

[0086] In addition, age-related increases in fat to muscle ratios, and age-related muscular atrophy appear to be related to myostatin. For example, the average seram myostatin- immunoreactive protein increased with age in groups of young (19-35 yr. old), middle-aged (36-75 yr. old), and elderly (76-92 yr old) men and women, while the average muscle mass and fat-free mass declined with age in these groups (Yarasheski et al. J Nutr Aging 6(5): 343- 8 (2002)). In addition, myostatin has no been found to be expressed at low levels in heart muscle and expression is upregulated in cardiomyocytes after infarct (Sharma et al, J Ceil Physiol. 180 (1): 1-9 (1999)). Therefore, reducing myostatin levels in the heart muscle may improve recovery of heart muscle after infarct.

[0087] Myostatin also appears to influence metabolic disorders including type 2 diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, and obesity. For example, lack of myostatin has been shown to improve the obese and diabetic phenotypes of two mouse models (Yen et al. FASEB J. 5:479 ( 1994). svActRIIB polypeptides of the present disclosure are suitable for treating such metabolic disorders. Tlierefore, administering the compositions will improve diabetes, obesity, and hyperglycemic conditions in suitable subjects. In addition, compositions containing svActRJIB polypeptides can decrease food intake in obese individuals.

[0088] Administering stabilized ActRIIB polypeptides can improve bone strength and reduce osteoporosis and other degenerative bone diseases, it has been found, for example, that myostatin-deficient mice showed increased mineral content and density of the mouse humerus and increased mineral content of both trabecular and cortical bone at the regions where the muscles attach, as well as increased muscle mass (Hamrick et al. Calcif Tissue Int 71 (i):63-8 (2002)). In addition, svActRIIBs can be used to treat the effects of androgen deprivation in cases such as androgen deprivation therapy used for the treatment of prostate cancer, for example.

[0089] Also provide are methods and compositions for increasing muscle mass in food animals by administering an effective dosage of svActRJIB proteins to the animal. Since the mature C-terminal myostatin polypeptide is similar or identical in all species tested, svActRJIB polypeptides would be expected to be effective for increasing lean muscle mass and reducing fat in any agriculturally important species including cattle, chicken, turkeys, and pigs.

[0090] In some aspects, disclosed herein are methods of treating obesity or a disease associated with obesity, increasing muscle mass, or decreasing fat mass in a subject, comprising: administering to the subject an effective dose of a protein that inhibits at least one of activin, myostatin, and GDF-1 1.

[0091] Muscle mass can refer to lean body mass (LBM). LBM can be measured by DEXA scan or other methodologies known in the art.

[0092] Muscle function can be measured using 6 minute walk distance, stair climbing power test, or handgrip strength test. See Nightingale et al ., Systematic review of timed stair tests, JRRD, Volume 51 , Number 3 (2014) pages 335-350. See also Temel et al., Anamorelin in patients with non-small-cell lung cancer and cachexia (ROMANA 1 and ROMANA 2): results from two randomized, double-blind, phase 3 trials, Lancet Oncol 2016; 17: 519-31 , [0093] In some aspects, a disease associated with obesity is at least one of a genetic obesity syndrome, Prader willi syndrome, a hypothalam ic disorder, familial hypercholesterolemia, Bardet-Biedl syndrome, Prader-Willi syndrome, a syndrome resulting from a loss of imprinted genes on 15ql 1-13, Alstrom syndrome, Cohen syndrome, Albright's hereditary osteodystrophy (pseudohypoparathyroidism), Carpenter syndrome, MOMO syndrome, Rubinstein-Taybi syndrome, a syndrome resulting from deletions of at least one of 6q l 6, I 36, 2q37, and 9q34, maternal uniparental disomy of chromosome 14, fragile X syndrome, atherosclerosis, non-alcoholic steatohepatitis, a disease where visceral fat deposition results in one or more deleterious outcomes, cerebrovascular disease, fatty liver, and Borjeson- Forssman-Lehman syndrome.

[0094] In some aspects, a subject is a human subject in need treatment or administration. In some aspects, a subject has at least one of a genetic obesity syndrome, Prader willi syndrome, a hypothalamic disorder, familial hypercholesterolemia, Bardet-Biedl syndrome, Prader-Willi syndrome, a syndrome resulting from a loss of imprinted genes on 5ql 1-13, Alstrom syndrome, Cohen syndrome, Albright's hereditary osteodystrophy

(pseudohypoparathyroidism), Carpenter syndrome, MOMO syndrome, Rubinstein-Taybi syndrome, a syndrome resulting from deletions of at least one of 6ql6, lp36, 2q37, and 9q34, maternal uniparental disomy of chromosome 14, fragile X syndrome, atherosclerosis, nonalcoholic steatohepatitis, a disease where visceral fat deposition results in one or more deleterious outcomes, cerebrovascular disease, fatty liver, and Borjeson-Forssman-Lehman syndrome. In some aspects, a subject has at least one of insulin resistance, chronic kidney disease, cancer, and a catabolic condition.

[0095] In some aspects, a protein is an antibody or an Fc-Fusion protein. In some aspects, an Fc-Fusion protein is an ActRIIB Fc-Fusion protein. In some aspects, an ActRIIB Fc-Fusion protein comprises a sequence with at least 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % identity to the sequence set forth in amino acids 19-25, 19, 20, 21, 22, 23, 24, or 25 through 130-134, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO: 2, optionally wherein the Fc is IgG, optionally wherein IgG is human IgG. In some aspects, an ActRIIB Fc-Fusion protein comprises or consists of the sequence set forth in SEQ ID N 0:6 or SEQ ID NO: 10. In some aspects, a protein inhibits at least two of activin, myostatin, and GDF-11. In some aspects, a protein inhibits activin, myostatin, and GDF-11. In some aspects, a protein inhibits activin.

[0096] In some aspects, a method disclosed herein decreases at least one of total fat mass, subcutaneous fat mass, and visceral fat mass. In some aspects, a method disclosed herein results in a greater percent decrease in visceral fat mass relative to the percent decrease in total fat mass. In some aspects, a method disclosed herein increases at lease one of lean body- mass and appendicular lean mass. In some aspects, a method disclosed herein results in an increase in at least one of lean body mass and appendicular lean mass by at least 1-50%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In some aspects, a method disclosed herein results in a decrease in at least one of total fat mass, subcutaneous fat mass, and visceral fat mass by at least 1-99%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more,

[0097] svActRIIB polypeptides and compositions also antagonize the activity of activm A, as shown in the in vitro assays below. Activin A is known to be expressed in certain types of cancers, particularly gonadal tumors such as ovarian carcinomas, and to cause severe cachexia, (Ciprano et al . Endocrinol 141 (7):2319-27 (2000), Shou et al., Endocrinol 138 (1 !):5000-5 (1997); Coerver et al, Mol Endocnnol 10(5):534-43 (1996); Ito et al. British J Cancer 82(8): 1415-20 (2000), Lambert-Messerlian, et al, Gynecologic Oncology 74:93-7 (1999). Therefore, the compositions of the present disclosure may be used to treat conditions related to activin A overexpression, as well as myostatin expression, such as cachexia from certain cancers and the treatment of certain gonadal type tumors.

[0098] In addition, svActRJIB polypeptides are useful for detecting and quantitating myostatin, activin A, BMP 9 or GDF-11 in any number of assays. In general, stabilized ActPJIB polypeptides are useful as capture agents to bind and immobilize myostatin, activin A, BMP 9 or GDF-11 in a variety of assays, similar to those described, for example, in Asai, ed., Methods in Cell Biology, 37, Antibodies in Cell Biology. Academic Press, Inc., New York (1993). The polypeptides may be labeled in some manner or may react with a third molecule such as an antibody which is labeled to enable myostatin to be detected and quantitated. For example, a polypeptide or a third molecule can be modified with a detectable moiety, such as biotin, which can then be bound by a fourth molecule, such as enzyme-labeled streptavidin, or other proteins. (Akerstrom, J Immunol 135:2589 (1985); Chaubert, Mod Pathol 10:585 ( 1997)).

[0099] In some aspects, a composition of the present disclosure can be used to treat cancers such as solid tumors. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, biastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary' gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophagael cancer, tumors of the biliar - tract, as well as head and neck cancer. In some aspects, a composition of the present disclosure can be used to treat cancers such as these or ovarian cancer (granulosa cell, clear cell, serous, endometrioid, germ cell tumors), head and neck cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, prostate cancer, small intestinal tumors, colon cancer, renal cell carcinoma, adenoid cystic carcinoma, gastric cancer, esophageal cancer, squamous cell carcinoma (skin), melanoma, breast cancer, bladder carcinoma, hepatocellular carcinoma, and/or uterine cancer (endometrial, cervical, leiomyoma, vulva, vaginal).

[00100] Dosages for use with the proteins and polypeptides described herein can be 0.1-

10, 0.25-5, 1-3, 0.1, 0.2, 0.25, 0,3, 0,4, 0.5, 0.6, 0.7, 0.75, 0,8, 0,9, 1, 2, 3, 4, 5, or greater than 5 mg/kg, inclusive. In some aspects each dosage can be administered every less than 1-10, 2- 9, 3-8, 4-7, 5-6, 1, 1 , 2, 3, 4, 5, 6, 7, 8, or greater than 8 weeks, inclusive. In some aspects each dosage can be administered every less tha 1-10, 2-9, 3-8, 4-7, 5-6, 1, 1 , 2, 3, 4, 5, 6, 7 days, inclusive. In some aspects each dosage is administered IV. In some aspects, the subject is administered at least 1-30, 5-20, 5-10, 10-20, 15-20, 1 -5, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses, inclusive. In some aspects, the amount of at least one of the plurality of doses is at least 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg, inclusive. In some aspects, the amount of each of the plurality- of doses is at least 0.1-30, 0,25-20, 0.25-10, 1-5, 1-3, 0, 1-1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg, inclusive. In some aspects, each dose is administered at least daily, weekly, or monthly. In some aspects, each dose is administered at least every 1-30, 5-20, 5-10, 10-20, 15-20, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, inclusive. In some aspects, treatment continues for at least 1-30, 5-20, 5-10, 10-20, 15-20, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days; at least 1-30, 5-20, 5-10, 10-20, 15-20, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks; or at least 1 -30, 5-20, 5-10, 10-20, 15-20, 1-5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 months, inclusive.

[00101] In some aspects, a subject can be selected for treatment of cancer using certain criteria such as inclusion criteria or exclusion criteria.

[00102] In some aspects the criteria are inclusion criteria. In some aspects, inclusion criteria can include: males and postmenopausal females > 18 years of age, presence of advanced solid tumors with histologic diagnosis confirming cancer, presence of recurrent metastatic or locally advanced disease after failure of at least one line of prior standard treatment (if available), measurable disease using Response Evaluation Criteria in Solid Tumors (RECIST 1.1) criteria, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, abiity to walk at least 30 meters without assistance from another person (use of assistive devices such as a cane or walking frame can be allowed), 12 months of spontaneous amenorrhea in postmenopausal women, 6 months of spontaneous amenorrhea with FSH > 40 IU/L in postmenopausal women, post-surgical bilateral oophorectomy with or without hysterectomy in postmenopausal women, prior treatment with a platinum-based chemotherapy regimen, and/or documented as unable to receive platinum-based

chemotherapy.

[00103] In some aspects the criteria are exclusion criteria. In some aspects, exclusion criteria can include: concurrent serious uncontrolled or unresolved medical condition (such as infection) limiting protocol compliance or exposing the subject to extreme risk, unresolved toxicities from prior anti-cancer therapy, such as motor or sensory neuropathy, with a CTCAE (version 4.03) Grade > 2 with the exception of alopecia, history of gastrointestinal bleeding within 6 months of starting treatment, presence of QTcF > 470 msec, history of hereditaiy prolonged QT interval, or any arrhythmia (such as bundle branch blocks) that would preclude assessment of the QT interval, myocardial infarction, unstable angina within 6 months of Cycle 1 Day 1, or congestive heart failure New York Heart Association > class II, elevated liver function tests, including total bilirubin > 1.5 x the upper limit of normal (ULN; unless subject has documented Gilbert's disease), aspartate aminotransferase (AST) or alanine aminotransferase (ALT) > 3.0 x ULN (for subjects with known liver metastasis, AST or ALT > 5 x ULN), creatinine > 1 .5 x ULN and an estimated creatinine clearance of < 60 mL/min (using the Cockcroft-Gault equation), hemoglobin < 9 g/dL; platelet < 100 x 10 ⁹ /L; absolute neutrophil count (ANC) < 1 .5 x 10 ⁹ /L (without granulocyte colony- stimulating factor support within 2 weeks of starting treatment), chemotherapy, hormonal therapy, or radiation therapy within 3 weeks of starting treatment, antibody/biologic therapy within 4 weeks of starting treatment, major surgery within 8 weeks or minor surgery within 4 weeks of starting treatment, current bowel obstruction, brain metastasis, presence of ascites or pleural effusion requiring frequent (more than 1 ^χ per week) medical intervention, presence of portal -venous shunt device or history of extensive hepatic resection (more than one segment), known human immunodeficiency virus (HIV) infection, active Hepatitis B or C infection, prior treatment with any investigational product wi thm 4 weeks of starting treatment, female of childbearing potential, or male with a female partner of childbearing potential, unwilling to use a highly effective method of contraception (i.e., one that results in pregnancy less than 1 % per year) when used consistently and correctly, such as implants, injectables, combined oral contraceptives, some intrauterine contraceptive devices, sexual abstinence, or a vasectomized partner, hypersensitivity reactions to a conventional formulation of doxorubicin HC1 or the components of liposomal doxorubicin, cumulative dose of prior doxorubin HC1 > 300 mg/'m ² , or cumulative dose of prior epirubicin > 500 mg/m ² , decreased cardiac ejection fraction less than the lower limit of normal by a MUGA scan or an echocardiogram (ECHO) within 28 days of starting treatment, and/or women who are breast-feeding.

Pharmaceutical compositions and formulations

[00104] Pharmaceutical compositions containing proteins and polypeptides disclosed herein are also provided. In some aspects, a protein is an antibody or an Fc-Fusion protein. In some aspects, an Fc -Fusion protein is an ActRIIB Fc-Fusion protein. In some aspects, an ActRIIB Fc-Fusion protein comprises a sequence with at least 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % identity to the sequence set forth in amino acids 19-25, 19, 20, 21, 22, 23, 24, or 25 through 130-134, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO: 2, optionally wherein the Fc is IgG, optionally wherein IgG is human IgG. In some aspects, an Ac! RU B Fc-Fusion protein comprises or consists of the sequence set forth in SEQ ID NO:6 or SEQ ID NO: 10. In some aspects, a protein inhibits at least two of activin, myostatin, and GDF-11. In some aspects, a protein inhibits activin, myostatin, and GDF-1 1. In some aspects, a protein inhibits activin.

[00105] Such compositions comprise a therapeutically or prophy!actieally effective amount of the polypeptide or protein in admixture with pharmaceutically acceptable materials, and physiologically acceptable formulation materials. The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotomcity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable fonnulation materials include, but are not hmrted to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring;

flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethan ine, lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides

(preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18 ^th Edition, A.R. Gennaro, ed.. Mack Publishing Company, 1990).

[00106] As used herein, the term, "buffer" is intended to mean a substance that stabilizes the pH of a liquid, either its acidity or alkalinity. The term as it is used herein is intended to refer to a solution having a buffering substance, such as an acid, in equilibrium with its conjugate base. Exemplary buffers useful in a formulation disclosed herein include a potassium phosphate buffer. Exemplary salt fonns of buffers that can be included in a buffer of the invention include, for example, sodium, potassium, calcium, organic amino or magnesium salt. The term "buffer" as it is used herein also is intended to include all buffers other than potassium phosphate buffer that are well known to those skilled in the art and applicable for use with biopharmaceuticals such as therapeutic polypeptides. Given the teachings and guidance provided herein, those skilled in the art will understand that buffers other than potassium phosphate buffer can be equally substituted in the formulations of the invention to maintain or enhance the stability of a therapeutic polypeptide. Any of a wide variety of buffer components well known in the art can be used in a formulation of the invention. Such buffer components include, for example, acetic acid, glutamic acid, succinic acid, propionic acid, maleic acid, gluconate, histidine or other amino acids, citrate, phosphate, or salt forms thereof. A wide variety of other buffers including, for example, other organic acids, are well known in the art and can similarly be used as a buffer component in a formulation of the invention. Given the teachings and guidance provided herein, those skilled in the art will known that any of the above buffer components or others well known in the art can be selected and used in a formulation of the invention given the desired pH of the formulation and excipients, if any, included in the formulation. The buffer component can be supplied to the buffering system in a variety of different forms. In some aspects, a formulation can include 1 -30, 5-20, 5-10, 10-20, 15-20, 1-5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 19, 20, or greater than 20 mM of buffer, inclusive.

[00107] As used herein, the term, "excipient" is intended to mean a therapeutically inactive substance. Excipients can be included in a formulation for a wide variety of purposes including, for example, as a diluent, vehicle, buffer, stabilizer, tonicity agent, bulking agent, surfactant, cryoprotectant, lyoprotectant, anti-oxidant, metal ion source, chelating agent and/or preservative. Excipients include, for example, polyols such as sorbitol or mannitol; sugars such as sucrose, lactose or dextrose: polymers such as polyethylene glycol; salts such as NaCl, KC1 or calcium phosphate, amino acids such as glycine, methionine or glutamic acid, surfactants, metal ions, buffer salts such as propionate, acetate or succinate, preservatives and polypeptides such as human serum albumin, as well as saline and water. Particularly useful excipients of the invention include sugars including sugar alcohols, reducing sugars, non-reducing sugars and sugar acids. Excipients are well known in the art and can be found described in, for example, Wang W., Int. J. Pharm. 185: 129-88 (1999) and Wang W., Int. J. Pharm. 203: 1-60 (2000). Non-reducing sugars contain an anomeric carbon that is an acetal and is not substantially reactive with amino acids or polypeptides to initiate a Maillard reaction. Sugars that reduce Febiing's solution or Tollen's reagent also are known as reducing sugars. Specific examples of non-reducing sugars include sucrose, trehalose, sorbose, sucralose, melezitose and raffinose. Buffer excipients maintain the pH of liquid formulations through product shelf-life and maintain the pH of lyophilized fonnulations during the lyophilization process and upon reconstitution, for example. In general, excipients can be chosen on the basis of the mechanisms by which they stabilize proteins against various chemical and physical stresses.

[00108] As described herein, certain excipients are beneficial to include so as to alleviate the effects of a specifi c stress or to regulate a particular suscepti bility of a specific polypeptide. Other excipients are beneficial to include because they have more general effects on the physical and covalent stabilities of proteins. Particularly useful excipients include those chemically and functionally innocuous or compatible with aqueous buffer solutions and polypeptides so as to optimize the stability properties of a formulation. Various such excipients are described herein as exemplary excipients exhibiting chemical compatibility with the aqueous formulations of the invention and functional compatibility with the polypeptide included in such formulations. Those skilled in the art will understand that the teachings and guidance provided herein with respect to the exemplified excipients are equally applicable to the use of a wide range of other excipients well known in the art. For example, optimal excipients chosen to enhance or confer stability of a polypeptide within a formulation include those that are substantially free from, reacting with functional groups on the polypeptide. In this regard, both reducing and non -reducing sugars can be used as an excipient in a formulation of the invention. However, because reducing sugars contain a hemiacetai group they can react and form adducts or other modifications with amino groups on amino acid side chains of polypeptides (i.e., glycosylation). Similarly, excipients such as citrate, succinate or histidme also can form adducts with amino acid side chains. Given the teachings and guidance provided herein, those skilled in the art will know that greater retention of stability for a given polypeptide can be achieved by choosing a non-reducing sugar over a reducing sugar or over other amino acid-reactive excipients such as those exemplified above. Optimal excipients also are chosen to enhance or provide stabilization with reference to the mode of administration for an aqueous formulation of the invention. For example, parenteral routes of intravenous (IV), subcutaneous (SC) or intramuscular (IM) administration can be more safe and efficacious when all components of the fonnulation maintain physical and chemical stability during manufacture, storage and administration. Those skilled in the art will know to employ one or more excipients that maintain maximal stability of the active form of a polypeptide given, for example, a particular manufacturing or storage condition or a particular mode of administration. The excipients exemplified herein for use in a formulation exhibit these and other characteristics. [00109] The amount or concentration of excipient to use in a formulation of the invention will var - depending on, for example, the amount of polypeptide included in the formulation, the amount of other excipients included in the desired formulation, whether a diluent is desired or needed, the amount or volume of other components of the formulation, the total amount of components within a formulation, the specific activity of the polypeptide and the desired tonicity or osmolality to be achieved. Specific examples for excipient concentrations are exemplified further below. Further, different types of excipients can be combined into an formulation. Accordingly, a formulation of the invention can contain an excipient, two, three or four or more different types of excipients. Combinations of excipients can be particularly useful in conjunction with a fonnulation that contains two or more different polypeptides. The excipients can exhibit similar or different chemical properties. Given the teachings and guidance provided herein, those skilled in the art will know what amount or range of excipient can be included in any particular formulation to achieve a formulation of the invention that promotes retention in stability of the polypeptide. For example, the amount and type of a salt to be included in a formulation of the invention can be selected based on to the desired osmolality (i.e., isotonic, hypotonic or hypertonic) of the final solution as well as the amounts and osmolality of other components to be included in the formulation. Similarly, by exemplification with reference to the type of polyol or sugar included in a formulation, the amount of such an excipient will depend on its osmolality. Inclusion of about 5% sorbitol can achieve isotonicity while about 9% of a sucrose excipient is needed to achieve isotonicity. Selection of the amount or range of concentrations of one or more excipients that can be included within a fonnulation of the invention has been exemplified above by reference to salts, polyols and sugars. However, those skilled in the art will understand that the considerations described herein and further exemplified by reference to specific excipients are equally applicable to all types and combinations of excipients including, for example, salts, amino acids, other tonicity agents, surfactants, stabilizers, bulking agents,

cryoprotectants, lyoprotectants, anti-oxidants, metal ions, chelating agents and/or preservatives.

[00110] Excipients can be included in a formulation of the invention at concentration ranges generally between about 1-40% (w/v), between about 5-35% (w/v), between about 8- 30% (w/v), between about 8-25% (w/v) or about 8% (w/v). Concentrations as high as about 45% (w/v), 50% (w/v) or more than 50% (w/v) in certain instances also can be employed in the formulations of the invention. For example, in some instances, it can be desirable to include concentrations up to 60% (w/v) or 75% (w/v) to produce a hypertonic formulation of the invention. Such hypertonic solutions can be diluted to produce an isotonic formulation prior to use if desired. Other useful concentration ranges include between about 1-20%, particularly between about 2-18% (w/v), more particularly between about 4-16% (w/v), even more particularly between about 6-14% (w/v) or between about 8-12% (w/v) or about 10% (w/v). Excipient concentrations and/or amounts less than, greater than or in between these ranges also can be used in a formulation of the invention. For example, one or more excipients can be included in a formulation which constitute less than about 1 % (w/v).

Similarly, a formulation can contain a concentration of one or more excipients greater than about 40% (w/v). Accordingly, a formulation of the invention can be produced that contains essentially any desired concentration or amount of one or more excipients including, for example, 1 -30, 5-20, 5-10, 10-20, 15-20, 1-5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20% (w/v) or more, inclusive.

[00111] A buffer component of a formulation of the invention can include one or more excipients. As described previously, one role of an included excipient is to provide stabilization of the polypeptide against stresses that can occur during manufacturing, shipping and storage. To accomplish this role, at least one excipient can function as a buffer, stabilizer, tonicity agent, bulking agent, surfactant, cryoprotectant, lyoprotectant, anti-oxidant, metal ion source, chelating agent and/or preservative. In addition, at least one excipient also can function as a diluent and/or vehicle or be employed to reduce viscosity in high concentration formulations in order to enable their delivery and/or enhance patient convenience. Similarly, at least one excipient additionally can confer more than one of the above functions onto a formulation of the invention. Alternatively, two or more excipients can be included in a formulation of the invention to perform more than one of the above or other functions. For example, an excipient can be included as a component in a formulation of the invention to change, adjust or optimize the osmolality of the formulation, thereby acting as a tonicifier. Similarly, a tonicity agent and a surfactant can both be included in a formulation of the invention to both adjust the osmolality and control aggregation. Excipients, their use, formulation and characteristics are well known in the art and can be found described in, for example, Wang W, Int. J. Pharm. 185: 129-88 (1999) and Wang W., Int. J, Pharm. 203: 1-60 (2000),

[00112] Tonicity agents and/or stabilizers included in liquid formulations can be used, for example, to provide isotonicity, hypotonicity or hypertonicity to a formulation such that it is suitable for administration. Such excipients also can be used, for example, to facilitate maintenance of a polypeptides' structure and/or to minimize electrostatic, solution protein- protein interactions. Specific examples of tonicity agents and/or stabilizers include polyols, salts and/or amino acids. Tonicity agents and/or stabilizers included in lyophilized formulations can be used, for example, as a cryoprotectant to guard polypeptides from freezing stresses or as a lyoprotectant to stabilize polypeptides in the freeze-dried state. Specific examples of such cryo- and lyoprotectants include polyols, sugars and polymers.

[00113] As used herein, the term, "surfactant" is intended to mean a substance that functions to reduce the surface tension of a liquid in which it is dissolved. Surfactants can be included in a formulation for a variety of purposes including, for example, to prevent or control aggregation, particle formation and/or surface adsorption in liquid formulations or to prevent or control these phenomena during the lyophilization and/or reconstitution process in lyophilized formulations. Surfactants include, for example, amphipathic organic compounds that exhibit partial solubility in both organic solvents and aqueous solutions. General characteristics of surfactants include their ability to reduce the surface tension of water, reduce the interfacial tension between oil and water and also form micelles. Surfactants of the invention include non-ionic and ionic surfactants. Surfactants are well known in the art and can be found described in, for example, Randolph T. W. and Jones L. S., Surfactant-protein interactions. Pharm. Biotechnol. 13: 159-75 (2.002). Briefly, non-ionic surfactants include, for example, alkyl poly (ethylene oxide), alkyl polyglucosides such as octyl glucoside and decyl maltoside, fatty alcohols such as cetyl alcohol and oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA. Specific examples of non-ionic surfactants include the polysorbates including, for example, polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 and the like; the poloxamers including, for example, poioxamer 188, also known as poloxalkoi or poly(ethylene oxide)-poly(propylene oxide), poioxamer 407 or polyethylene-polypropylene glycol and the like, and polyethylene glycol (PEG). Polysorbate 2.0 is synonymous with TWEEN 20, sorbitan monolaurate and polyoxyethylenesorbitan monoiaurate.

[ 00114] Optimal surfactants to include in a formulation of the invention can be chosen, for example, to enhance or promote retention in stability of the polypeptide by preventing or reducing aggregation and/or adsorption. For example, sorbitan fatty acid esters such as the polysorbates are surfactants exhibiting with a wide range of hydrophilic and emulsifying characteristics. They can be used individually or in combination with other surfactants to cover a wide range of stabilization needs. Such characteristics are particularly suitable for use with polypeptides because they can be tailored to cover the wide range of hydrophobic and hydrophilic characteristics of polypeptides. Considerations for selecting a surfactant include those described previously with reference to excipients in general as well as the hydrophobic character and critical micellar concentration of the surfactant. The surfactants exemplified herein, as well as many others well known in the art can be used in a formulation of the invention.

[001 IS] Surfactant concentration ranges for a formulation of the invention include those described previously with reference to excipients in general with particularly useful concentrations being less than about 1% (w/v). In this regard, surfactant concentrations generally can be used at ranges between about 0.0001-0.10% (w/v), particularly between about 0.002-0.05% (w/v), more particularly between about 0.003-0.01% (w/v), even more particularly between about 0.004-0.008% (w/v) or between about 0.005-0.006% (w/v). Surfactant concentrations and/or amounts less than, greater than or in between these ranges also can be used in a formulation of the invention. For example, one or more surfactants can be included in a formulation which constitute less than about 0.001 % (w/v). Similarly, a formulation can contain a concentration of one or more surfactants greater than about 0.10% (w/v). Accordingly, a formulation of the invention can be produced that contains essentially any desired concentration or amount of one or more surfactants including, for example, 0.001, 0.002, 0,003, 0.004, 0.005, 0.006, 0,007, 0.008, 0.009, 0,010, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10% (w/v) or more, inclusive. Various surfactants useful as an excipient in a fonnulation of the invention have been described previously. Other surfactants useful in either a liquid or lyophilized formulation of the invention include, for example, sugar esters such as esters lauric acid (C12), palmitic acid (C16), stearic acid (CI 8), macrogol eetostearyl ethers, macrogol lauryl ethers, macrogol oleyl ether, macrogol oleate, macrogoi stearate, macrogol glycerol ricinoieate, macrogol glycerol hydroxystearate; alkyl polyglucosides such as octyl glucoside and decyl maltoside; fatty alcohols such as cetyl alcohol and oleyl alcohol, and cocamides such as cocamide MEA, DEA, TEA, other non- ionic surfactants and other ionic surfactants.

[ 00116] Stability of a formulation of the invention, including a liquid formulation of the in vention, refers to the retention of structure and/or function of a polypeptide within a formulation. A polypeptide in a formulation of the invention will exhibit attributes such as resistance to change or deterioration that affect stability or function and therefore maintain consistent functional characteristics over time.

[00117] A buffer component of a formulation of the invention also can include one or more surfactants as an excipient. As described previously, one role of surfactants in a formulation of the invention is to prevent or minimize aggregation and/or adsorption such as surface -induced degradation. At sufficient concentrations, generally about the surfactant's critical mi cellar concentration, a surface layer of surfactant molecules serve to prevent protein molecules from adsorbing at the interface. Thereby, surface-induced degradation is minimized. Surfactant, their use, formulation and characteristics for formulations are well known in the art and can be found described in, for example, Randolph and Jones, supra, (2002),

[ 00118] In another embodiment, the stability of a polypeptide within a formulation of the invention includes, for example, the retention of physical and/or chemical stability.

Polypeptide stability can be assessed by, for example, determining whether the polypeptide has been subjected to a physical degradation and/or chemical degradation pathway such as those described previously, including chemical modification of its structure. Retention in stability of a polypeptide in a formulation of the invention includes, for example, retention of physical or chemical stability between about 80-100%, 85-99%, 90-98%, 92-96% or 94-95% compared to the stability of the polypeptide at an initial time point or relative to an identical control kept at a lower temperature, e.g., -70 degrees Celsius. Accordingly, stability of a polypeptide within a formulation of the invention includes retention of stability greater than 99.5%, at least about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% compared to the stability of the polypeptide at an initial time point relative to an identical control kept at a lower temperature, e.g., -70 degrees Celsius, inclusive.

[00119] In a further embodiment, stability of a polypeptide within a formulation of the invention includes, for example, retention of activity. Polypeptide activity can be assessed using, for example, an in vitro, in vivo and/or in situ assay indicative of the polypeptide's function. Retention of stability of a polypeptide in a formulation of the invention includes, for example, retention of activity between about 50-100% or more, depending on the variability of the assay. For example, retention in stability can include retention of activity between about 60-90% or 70-80% compared to the activity of the polypeptide at an initial time point. Accordingly, stability of a polypeptide within a formulation of the invention includes retention of activity of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% and can include activity measurements greater than 100% such as 105%, 1 10%, 1 ! 5%>, 120%, 125% or 150% or more compared to the activity of the polypeptide at an initial time point, inclusive. Generally, an initial time point is selected to be the time that a polypeptide is first prepared in a formulation of the invention or first examined for quality (i .e., meets release specifications). An initial time point also can include the time at which a polypeptide is reformulated in a formulation of the invention. The reformulation can be, for example, at a higher concentration, lower concentration or at the same concentration of an initial preparation.

[00120] A formulation of the invention can be prepared to be isotonic with a reference solution or fluid (i.e., blood serum). An isotonic solution has a substantially similar amount of dissolved solute in it compared to the things around it so that it is osmotically stable. Unless expressly compared to a specific solution or fluid, isotonic or isotonicity is exemplary used herein by reference to human blood serum (e.g., 300 mOsmol/kg). Therefore, an isotonic formulation of the invention will contain a substantially similar concentration of solutes or exhibit substantially similar osmotic pressure as human blood. In general, an isotonic solution contains about the same concentration of solutes as normal saline for humans and many other mammals, which is about 0.9 weight percent (0.009 g/ml) salt in aqueous solution (e.g., 0.009 g/ml NaCl). Formulations of the invention also can include hypotonic or hypertonic solution preparations.

[00121] The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. See for example, Remington ^' s Pharmaceutical Sciences, supra. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the polypeptide. For example, suitable compositions may be water for injection, physiological saline solution for parenteral administration. Ionic surfactants include, for example, anionic, catiomc and zwitterionic surfactants. Anionic surfactants include, for example, suifonate-based or carboxylate-based surfactants such as soaps, fatty acid salts, sodium dodecyl sulfate (SDS), ammonium iauryl sulfate and other alkyl sulfate salts. Cationic surfactants include, for example, quaternary ammonium-based surfactants such as cetyl trimethylammoniiim bromide (CTAB), other aikyitrimethyiammonium salts, cetyl pyridinium chloride, polyethoxylated tallow amine (POEA) and benzalkonium chloride. Zwitterionic or amphoteric surfactants include, for example, dodecyl betaine, dodecy] dimethylamine oxide, cocamidopropy] betaine and coco ampho glycinate.

[00122] The primary vehicle or earner in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions compri se Tris buffers, or acetate buffers, which may further include sorbitol or a suitable substitute thereof. In one embodiment, compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, the therapeutic composition may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[00123] The formulations can be delivered in a variety of methods, for example, by inhalation therapy, orally, or by injection. When parenteral administration is contemplated, the therapeutic compositions may be in the form of a pyrogen-free, parente rally acceptable aqueous solution comprising the desired polypeptide in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a polypeptide is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodibie particles, polymeric compounds (polylactic acid, polygiycolic acid), beads, or liposomes that provides for the controlled or sustained release of the product which may then be delivered via a depot injection. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

[00124] In another aspect, pharmaceutical formulations suitable for injectable

administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks ^' solution. Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes, Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions. In another embodiment, a pharmaceutical composition may be formulated for inhalation. Inhalation solutions may also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions may be nebulized. Pulmonary administration is further described in PCX Application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.

[00125] In some aspects, proteins can be formulated as a sterile aqueous solution, containing 50-100, 60-80, 65-75, 60-70, 70-80, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, or 80 mg/ml, protein, 1-30, 5-20, 5-10, 10-20, 15-20, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, or greater than 20 mM potassium phosphate buffer, less than 1-10, 5-10, 5, 5, 6, 7, 8, 8.8, 9, 10, or greater than 10 % (w/v) sucrose, and/or less than 0.006, 0.006, or greater than 0.006% (w/v) polvsorbate 20 at pH 4- 12, 5-6, 5-7, 6-7, 5, 6, 6.7, 7, or 8, inclusive. In some aspects, a protein can be formulated as a sterile aqueous solution, containing protein, potassium phosphate buffer, sucrose, and/or polysorbate 20. In some aspects, a protein can be formulated with potassium phosphate buffer, sucrose, and/or polysorbate 20. In some aspects, a protein can be formulated for IV administration. In some aspects, a protein can be formulated at neutral pH. In some aspects, a protein can be formulated at a pH of about 4-12, 5-6, 5-7, 6-7, 5, 6, 6.7, 7, or 8, inclusive. In some aspects, a protein described herein can be formulated with a non-naturally occurring component such as, e.g., a non-naturally occurring excipient.

[00126] It is also contemplated that certain formulations may be administered orally. In one embodiment, molecules that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the therapeutic molecule. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed. Pharmaceutical compositions for oral administration can also be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

[00127] Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol: starch from, corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium earboxymethylceUulose; gums, including arable and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.

[00128] Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arable, talc, poly vinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

[00129] Pharmaceutical preparations that can be used orally also include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

[00130] Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving polypeptides in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT/US93/00829 that describes controlled release of porous polymeric microparticles for the delivery- of pharmaceutical compositions. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. 3,773,919, EP 58,481 ), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers, 22:547-556 (1983), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277, (1981 ); Langer et al,, Chem. Tech., 12:98-105(1982)), ethylene vinyl acetate (Langer et al., supra) or poly~D(~)-3-hydroxybutyric acid (EP

133,988). Sustained-release compositions also include liposomes, which can be prepared by any of several methods known in the art. See e.g., Eppstein et al , PNAS (USA), 82:3688 (1985); EP 36,676; EP 88,046; EP 143,949.

[00131] The pharmaceutical composition to be used for in vivo adm inistration typically must be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. The composition for parente al administration may be stored in lyophilized form or in solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[00132] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

[00133] In a specific embodiment, kits for producing an-dose administration unit are provided. The kits may each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g. , liquid syringes and lyosyringes).

[00134] An effective amount of a pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the polypeptide is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage may range from about O. lmg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. Polypeptide compositions may be preferably injected or administered intravenously (IV). Long-acting pharmaceutical compositions may be administered every three to four days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation. The frequency of dosing will depend upon the pharmacokinetic parameters of the polypeptide in the formulation used. Typically, a composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be administered as an dose, or as multiple doses (at the same or different concentrations/dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose-response data.

[00135] Dosages for use with the proteins and polypeptides described herein can be 0.1- 10, 0.1 -5, 0,25-5, 0.25-3, 1-3, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1 , 2, 3, 4, 5, or greater than 5 mg/kg, inclusive. In some aspects each dosage can be administered every less than 1-10, 2-8, 2-5, 1-4, 1, 1 , 2, 3, 4, 5, 6, 7, 8, or greater than 8 weeks, inclusive. In some aspects each dosage can be administered every less than 1-10, 2-8, 2-5, 1-4, 1, 1 , 2, 3, 4, 5, 6, 7 days, inclusive. In some aspects each dosage is administered IV.

[00136] The route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), mtracerebiOventricuiar, intramuscular, intra-ocuiar, intraarterial, intraportal, mtralesional routes, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, or intraperitoneal; as well as intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means, by sustained release systems or by implantation devices. Where desired, the compositions may be administered by bolus injection or continuously by infusion, or by implantation device. Alternatively or additionally, the composition may be administered locally via implantation of a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

[00137] In some cases, svActRIIB polypeptides can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide. Such cells may be animal or human cells, and may be autologous, heterologous, or xenogeneic. Optionally, the cells may be immortalized. In order to decrease the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues. The encapsulation materials are typically biocompatible, semi -permeable polymeric enclosures or membranes that allow the release of the polypeptide product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.

[00138] svActRIIB gene therapy in vivo is also envisioned wherein a nucleic acid molecule encoding svActRIIB, or a derivative of svActRIIB is introduced directly into the subject. For example, a nucleic acid sequence encoding a svActRIIB is introduced into target cells via local injection of a nucleic acid construct with or without an appropriate deliver}' vector, such as an adeno-associated virus vector. Alternative viral vectors include, but are not limited to, retroviruses, adenovirus, herpes simplex, viras and papilloma virus vectors. Physical transfer of the viras vector may be achieved in vivo by local injection of the desired nucleic acid construct or other appropriate delivery vector containing the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), or microparticle bombardment (gene-gun) .

[00139] The compositions of the present disclosure may be used alone or in combination with other therapeutic agents, e.g., to enhance their therapeutic effects or decrease potential side effects. In some aspects, doxorubicin can be administered in a combination. In some aspects, doxorubicin is liposomal doxorubicin. In some aspects, doxorubicin is given at 40 mg/m ² . In some aspects, doxorubicin is given at 5-200, 10-150, 25-100, 30-50, 35-45, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mg/m ² , inclusive.

[00140] A formulation of the invention also can include combinations of polypeptides in the formulation. For example, a formulation of the invention can include an polypeptide for treatment of one or more conditions. A formulation of the invention also can include two or more different polypeptides. Use of multiple polypeptides in a formulation of the invention can be directed to, for example, the same or different indications. Similarly, multiple polypeptides can be used in a formulation of the invention to treat, for example, both a pathological condition and one or more side effects caused by the primary treatment.

Multiple polypeptides also can be included in a formulation of the invention to accomplish different medical purposes including, for example, simultaneous treatment and monitoring of the progression of the pathological condition. Multiple, concurrent therapies such as those exemplified above as well as other combinations well known in the art are particularly useful for patient compliance because an formulation can be sufficient for some or all suggested treatments and/or diagnosis. Those skilled in the art will know those polypeptides that can be admixed for a wide range of combination therapies. Similarly, a formulation of the invention also can be used with small molecule pharmaceuticals and combinations of one or more polypeptides together with one or more small molecule pharmaceuticals. Therefore, the invention provides for a formulation of the invention containing 1 -10, 2-3, 1 , 2, 3, 4, 5 or 6 or more different polypeptides as well as for one or more polypeptides combined with one or more small molecule pharmaceuticals.

[00141] A fonnulation of the invention also can include one or more preservatives and/or additives well known in the art. Similarly, a formulation of the invention can further be formulated into any of various know delivery fonnulations. For example, a formulation of the invention can include lubricating agents, emulsifying agents, suspending agents, preserving agents such as methyl- and propylhydroxy-benzoates, sweetening agents and flavoring agents. Such optional components, their chemical and functional characteristics are well known in the art. Similarly well known in the art are formulations that facilitate rapid, sustained or delayed release of the polypeptide after administration. A formulation of the invention can be produced to include these or other formulation components well known in the art.

[00142] A formulation of the invention also can be produced, for example, in states other than an aqueous liquid. For example, as a lyophilized formulation. A lyophilized formulation will generally contain, for example, a bulking or caking agent and an amorphous stabilizer.

[00143] Once a formulation of the invention is prepared as described herein, stability of the one or more polypeptides contained within the formulation can be assessed using methods well known in the art. Several of such methods are exemplified further below in the

Examples and include size exclusion chromatography and particle counting. Any of a variety of functional assays including, for example, binding activity, other biochemical activity and/or physiological activity can be assessed at two or more different time points to determine the stability of the polypeptide in the buffered fonnulation of the invention.

[00144] A formulation of the invention will, in general, be prepared according to pharmaceutical standards and using pharmaceutical grade reagents. Similarly, a formulation of the invention will, in general, be prepared using sterile reagents in a sterile manufacturing environment or sterilized following preparation. Sterile injectable solutions can be prepared using well known procedures in the art including, for example, by incorporating one or more polypeptides in the required amount in an acetic acid, glutamic acid or succinic acid buffer or excipient of the invention with one or a combination of formulation components described herein followed by sterilization microfiltration. In the specific embodiment of sterile powders for the preparation of sterile injectable solutions, particularly useful methods of preparation include, for example, vacuum drying and freeze-drying (lyophilization) as described previously. Such drying methods will yield a powder of the one or more polypeptides together with any additional desired components from a previously sterile-filtered solution thereof.

Kits

[00145] Also described herein are kits comprising a vial comprising a protein disclosed herein and instructions for use. The protein can be in any suitable pharmaceutical composition as described herein, e.g., a liquid suspension suitable for IV infusion.

Alternatively, the protein can be in a lyophilized state suitable for re-suspension before use. Tire instructions for use can include storage instructions, patient selection, dosages, administration methods, time periods for use, clinical endpoints, and the like. In some aspects, the instructions include instructions to perform a method disclosed herein,

[00146] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

EXAMPLES

[00147] Below are examples of specific embodiments. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[00148] The practice of the present invention can employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et ai., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);

Remington's Pharmaceutical Sciences, 18th Edition (Eastern, Pennsylvania: Mack Publishing Company, 3990); Carey and Sundberg Advanced Organic Chemistry 3' ^"a Ed. (Plenum Press) Vols A and B( 1992).

Example 1: STM 434 Stability.

[00149] The stabilit ⁷ of formulated STM 434 stability was determined using various methods at various time points under various conditions. STM 434 was formulated as shown above (STM 434 Injection was formulated as 70 mg/mL STM 434, 10 mM potassium phosphate, 8.8% (w/v) sucrose, 0.006% (w/v) polysorbate 20, pH 6.7).

Methods

[00150] The analytic methods used for assessing stability were as follows:

[00151] Concentration by Ultraviolet (UV) Absorbance

[00152] The concentration of STM 434 in the drug product was measured by using a UV spectrophotometer. The absorbance of 280 nm light (A280) and the extinction coefficient at 280nm (ε280) of 1.7 mg-mL^-cm ^"1 were used to determine the concentration based on Beer's law.

[00153] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70°C when the concentration is 70.0 ± 7.0 mg/mL. The percent stability of a given protein at a given time point can also be calculated relative to this number.

[00154] Visual Appearance

[00155] This method provides for the visual assessment of STM 434 Injection. A vial was observed under ambient light, and the general appearance reported with respect to visible particles, degree of coloration, and clarity.

[00156] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70°C when the appearance is clear, colorless to slightly yellow liquid, and essentially free of particles.

[00157] Osmolality

[00158] The osmolality of STM 434 Injection was measured using a freezing-point depression osmometer, which was calibrated against an osmolality standard of 290 mOsm/kg.

[00159] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -7Q°C when the osmolality is 330.0 ± 50.0 mOsm/kg. The percentage stability of a given protein at a given time point can also be calculated relative to this number.

[00160] pH

[00161] The pH of STM 434 Injection was measured using a pH meter calibrated with

National Institute of Science and Technology (NIST)-traceable standards.

[00162] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70°C when the pH is 6.7 ± 0.3 at 25°C. The percent stability of a given protein at a given time point can also be calculated relative to this number.

[00163] Volume in Container

[00164] The volume of STM 434 Injection solution in vials was measured.

[00165] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70"C when the volume in the container is not less than 1.0 mL. The percent stability of a given protein at a given time point can also be calculated relative to this number.

[00166] Size Exclusion High Performance Liquid Chromatography (SE-HPLC)

[00167] SE-HPLC is a method of separating proteins by their hydrodynamic size, which is approximately correlated with molecular weight (MW). SE-HPLC allows for analysis of the molecular size distribution of STM 434 in the drug product, especially with respect to aggregation, fragmentation, and other impurities. Resolution of monomelic STM 434 from other species by SE-HPLC was performed on a TSKgel G3000SWxl HPLC column, using an isocratic mobile phase of 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8, at a flow rate of 0.5 mL/miii. Protein species were detected by A280 mm over a 35 -minute assay run time. The elution time of proteins was inversely proportional to the log of their MW. Results from the assay are reported as percent A280 peak area relative to the total A280 peak area,

[00168] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70°C when the SE-HPLC main peak is > 95.0%. The percent stability of a given protein at a given time point can also be calculated relative to this number.

[00169] Imaged Capillary Isoelectric Focusing (iclEF)

[00170] cIEF is a high-resolution protein separation technique based on differences in isoelectric point (pi) among proteins. The Protein Simple (formerly Convergent Bioscience) iCE280 IEF analyzer system uses a whole capillar}' zone imaging detector that enables analysis without the mobilization step required for conventional cIEF instruments. With respect to STM 434 Injection, icIEF was used as a purity test.

[00171] In this icIEF method, STM 434 samples were treated with siaiidase to reduce the substantial complexity imparted by the varying levels of sialic acid in the product. After desialylation, samples were mixed with earner ampholytes to generate a pH gradient, methyl cellulose to reduce electroosmotic flow, and markers of known pi which absorb 280 mm light. The mixture was introduced into an internally coated fused silica capillary with a UV~ transparent segment between inlet and outlet reservoirs assembled in a cartridge. When voltage was applied to the capillary segment, the solution forms a pH gradient, in which the ampholytes, pi markers, and protein species in the sample were focused at their respective pi. A whole capillary absorption image was taken with a charge -coupled device camera, allowing for monitoring of the focused zones in the capillary at 280 nm, corresponding to the various protein species and the pi markers. Inclusion of the pi markers allows for calibration of the pi gradient in the capillary, providing for reproducible determination of pi values of the various species in a sample. Comparable to HPLC, the icIEF results are provided as A280 peaks as a function of migration distance in the capillary. Results from the assay are reported as percent A280 peak area relative to the total A280 peak area for the protein species.

[00172] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70°C when the icIEF main peak is 65.0 ± 10.0%. The percent stability of a given protein at a given time point can also be calculated relative to this number.

[00173] Sodium Dodecyl Sulfate Capillary Electrophoresis (CE-SDS), Non-reduced (nrCE-SDS)

[00174] CE-SDS, non-reduced was used as a purity test for STM 434 Injection. The methodology involves heat denaturation of proteins in the presence of the detergent SDS, which binds to polypeptides at a relatively constant ratio of SDS:polypeptide. The negatively charged SDS bound to proteins causes them to migrate in an electric field according to MW.

[00175] In this method, STM 434 samples were incubated at 70°C for 10 minutes with SDS, a free sulfhydryl blocking agent, and an internal MW standard under non-reducing conditions. After incubation, treated samples were loaded into an autosampler, which also carries solutions of rinses and a specialized SDS gel. The CE system was programmed for sequential rinses, gel loading, and electrokinetic sample injections into a bare fused silica capillary. For each sample, the injected proteins were subjected to an electric field for MW~ based separation, including mobilization through a detector window, where the capillary coating had been removed and light passed through the capillary to a photodiode array detector, which in this method was set to collect 220 nm absorbance (A220). Results from the assay are reported as percent A220 peak area relative to the total A280 peak area for the protein species.

[00176] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70°C when the nrCE-SDS main peak is > 88.0%. The percent stability of a given protein at a given time point can also be calculated relative to this numbe .

[00177] CE-SDS, Reduced (rCE-SDS)

[00178] CE-SDS, reduced was used as a purity test for STM 434 Injection. The methodology involves heat denaturation of a specified concentration of protein in the presence of the detergent SDS, and a reducing agent which cleaves disulfide bonds into free sulfhydryls, winch in the case of STM 434 causes the separation of the two polypeptide chains of the homodimer. The negatively charged SDS, which binds to polypeptide chains at a relatively constant ratio of SDS:polypeptide, causes the polypeptides to migrate in an electric field according to MVV.

[00179] In this method, STM 434 samples were incubated at 70°C for 10 minutes with SDS, the reducing agent, beta-mercaptoethanol, and an internal MW standard under non- reducing conditions. After incubation, treated samples were loaded into an autosampler, which also carried solutions of rinses and a specialized SDS gel. The CE system was programmed for sequential rinses, gel loading, and electrokinetic sample injections into a bare fused silica capillary. For each sample, the injected proteins were subjected to an electric field for MW-based separation, including mobilization through a detector window, where the capillary coating had been removed and light passed through the capillary to a photodiode array detector, which in this method was set to collect 220 nm absorbance (A220). Results from the assay are reported as percent A220 peak area relative to the total A280 peak area for the protein species.

[00180] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70"C when the rCE-SDS main peak is >90.0%. The percent stability of a given protein at a given time point can also be calculated relative to this number. [00181] Product-specific ELISA

[00182] The methodology utilized in the STM 434 Injection identity test was a solid-phase sandwich ELISA, Microtster strips were coated with an anti-STM 217 monoclonal antibody, which has high affinity for STM 434 as these proteins differ by a single amino acid (serine- 20 in STM 217, threonine-20 in STM 434). In sequence, samples or controls were added to the wells, followed by a biotinylated monoclonal antibody generated against human ActR2B, followed by Neutravidin-horse radish peroxidase (HRP) conjugate. STM 434 present in samples binds to the immobilized capture antibody and the biotinylated secondary antibody, and Neutravidin-HRP binds this complex through the high affinity of avidin for biotin. The HRP moiety of the complex catalyzes the conversion of the chromogenic substrate tetramethyl benzidine (TMB) to a product which absorbs 450 m light. Identity was confirmed in samples whose signal-to-noise ratio, equal to the sample's background- corrected A450 divided by the background-corrected A450 of blank wells, was no less than 10.0.

[00183] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70°C when the ELISA confirms the identity of the protein, e.g., relative to a positive control. The percent stability of a given protein at a given time point can also be calculated relative to the 450 nm number determined by ELISA.

[00184] Cell-Based Bioassay (Bioassay)

[00185] The potency of STM 434 Injection was measured in a reporter gene expression assay using the C2C12 pMARE clone #44 skeletal muscle cell line. Murine C2C12 cells were stably transfected with a human myostatin/activin-responsive luciferase construct. When the engineered cell line was incubated with the ligand myostatin, signal transduction occured following myostatin binding to the activin receptors. This resulted in the activation of the luciferase reporter gene and the resulting production of luciferase. The reaction of luciferase with luciferin resulted in luminescence that was measured in a luminometer. STM 434 inhibits this signaling in a dose -dependent manner. The data was analyzed using a 4- parameter curve fit model. Once parallelism/similarity to the reference curve was established, biological concentrations were interpolated from the curve and a relative potency of the sample against the reference standard was calculated.

[00186] A protein at a given time point and temperature is considered to have equivalent stability or activity to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70°C when the bioassay shows a relative potency of 60 to 140%. The percent stability or activity of a given protein at a given time point can also be calculated relative to this number.

[00187] Endotoxin

[00188] The method for quantifying bacterial endotoxin in STM 434 Injection utilized a kinetic chromogensc endotoxin detection system. Dilution series of endotoxin standard, STM 434 samples, and STM 434 samples spiked with endotoxin standard for system suitability were prepared, and loaded into a 96-well plate, along with water as a blank for system suitability. After incubating the plate at 37°C for 10 minutes, Limulus amebocyte lysate (LAL) reagent containing a peptide labeled with p-nitroaniline (pNA) was added to the wells. Endotoxin present in the samples and standards converted a proenzyme in LAL to an active enzyme which cleaved pNA from the colorless peptide to generate a signal at 405 nm absorbance (A405). The A405 from the endotoxin standards were plotted to generate a standard curve, and the endotoxin content of STM 434 samples was determ ined from their A405 readings against the standard curve. The results are reported in terms of endotoxin units per milligram (EU/mg) after determining the EU/mL of each sample and dividing by the sample's protein concentration in mg/mL.

[00189] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70°C when the endotoxin level is < 0.5 endotoxin units (EU)/mg. The percent stability of a given protein at a given time point can also be calculated relative to this number.

[00190] Sterility

[00191] The method for determining the sterility of STM 434 Injection utilized a membrane filtration technique to retain any microorganisms in the test articles.

[00192] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70"C when the sterility of the product meets the requirements of the United States Pharmacopeia. The percent stability of a given protein at a given time point can also be calculated relative to the numbers provided by the United States Pharmacopeia.

[00193] Subvisible Particles

[00194] The amount of subvisible particles present in STM 434 Injection was determined using light obscuration. The results are reported as the number of particles with diameters 10 μηι or larger, and with diameters 25 μιη or larger. [00195] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70"C when there are not more than 6,000 >10μηι particles per container and there are not more than 600 >25μηι particles per container. The percent stability of a given protein at a given time point can also be calculated relative to these numbers.

|00196] Polysorbate 20 Concentration

[00197] The concentration of polysorbate 20 (PS20) in SIM 434 Injection was determined by a method that utilizes a mixed-mode HPLC column run in reversed phase mode to separate PS20 from protein, with quantitative detection by charged aerosol detection (CAD). An injection sequence of PS20 standards provides a calibration curve based on the baseline- corrected CAD responses as a function of PS20 concentration, which fits a second-order polynomial. The reported PS20 concentration of an STM 434 sample was determined by entering its baseline-corrected CAD responses into the polynomial equation and solving for P S20 concen tration .

[00198] A protein at a given time point and temperature is considered to have equivalent stability to the protein at time zero (TO) or an identical protein, under otherwise identical conditions, at -70"C when the polysorbate 20 concentration is 0.006 ± 0.003% (w/v). The percent stability of a given protein at a given time point can also be calculated relative to this number.

Results

[00199] The tables below (Tables 18-39) show formulated STM 434 stability-related data generated using the above methods, at least in part, at the indicated time points and indicated conditions.

[00200] Table 18 shows that the tested characteristics of formulated 434 kept at 5°C for up to 54 months are similar to formulated 434 kept at -70°C for up to 54 months. Table 22 shows that the tested characteristics of formulated 434 kept at 5°C for up to 6 months are similar to those of formulated 434 kept at 5°C at 0 months. Tables 25 and 30 show that the tested characteristics of formulated 434 kept at 2-8°C for up to 12 months are similar to those of formulated 434 kept at 2-8°C at 0 months. Table 36 shows that the tested characteristics of formulated 434 kept in an upright position at 2-8°C for up to 6 months are similar to those of formulated 434 kept in an upright position at 2-8°C at 0 months. Table 37 shows that the tested characteristics of formulated 434 kept in an inverted position at 2-8°C for up to 6 months are similar to those of formulated 434 kept in an inverted position at 2-8°C at 0 months.

[00201] This data demonstrates that formulated STM 434 is highly stable for up to 54 months at refrigerated temperatures (e.g., 2-8°C (2, 3, 4, 5, 6, 7, 8°C); 5°C) and even at high concentrations suc as 70mg/mL. This level of stability is much greater than the stability expected of proteins kept at 4°C, which is typically in the range of 1 month. See Pierce Technical Resource TR0043.1 , Protein, Stability and Storage, at Table 1

(ht^://sites.bioindiana.edu/~chenlab/protocol_files/prote in_storage.pdf); and Webster et al., Predicting Long-Term Storage Stability of Therapeutic Proteins, Pharmaceuti cal

Technology. Volume 37, Issue 1 1 (Nov. 2, 2013). Thus formulated STM 434 is a highly stable composition that can be stored at refrigerated temperatures (e.g., 2-8°C) for an extended period of time greater than 1 month, e.g., up to 3, 6, 12, 18, 24, 36, 48, or 54 months or more.

Table 18: 434 DP/DS Stability Sample Testing Results

Sample DP, 5°C DP, -20°C DP, -70°C DS, 5°C DS, -70°C

Lo†. # 001003698 001003696 001003696 00100351 001003510 5 5 5 06 6

Method Time

54 months 54 months 54 months 54 months 54 months point

Colorless, Colorless, Colorless, Colorless, Colorless,

Visual Clear, Clear, Clear, Clear, Clear,

Appearance No visible No visible No visible No visible No visible particles particles particles particles particles pH 6.7 6.7 6.7 6.8 6.7

Protein Cone. 71 .6 71 .3 76.0 71 .5

71 .8 mg/mL

(A280) mg/mL mg/mL mg/mL mg/mL

98.2%

98,2%

98.6% 98,8% Main Main Pk 98.8% Main Pk

Main Pk Pk 1 .5% Main Pk

SE-HPLC 1 .5%

1 .4% HMW 1 .2% HMW HMW 1 .2% HMW HMW

0.0% LMW 0.0% LMW 0.3% 0.0% LMW 0.3% LMW

LMW

60.8% 69.9% 65.8%

65.6%

Main Pk Main Pk 72.4% Main Main Pk

Main Pk

22,9% 14.8% Pk 20.6% ic!EF 24.7%

APG APG 15.5% APG APG

APG

16.4% 15.3% 12.2% BPG 13.7%

9.8% BPG

BPG BPG BPG

94.1 % 94.2% 94.7% Main 94.5% 94.4% Main Pk Main Pk Pk Main Pk Main Pk nrCE-SDS

5.9% Pre- 5.8% Pre- 5,3% Pre- 5.5% Pre- 5.6% Pre- peak peak peak peak peak

98.0% 98.0% 97.8% Main 97.7% 97.8% rCE-SDS

Main Pk Main Pk Pk Main Pk Main Pk

Cell Based 87% 99% 1 1 1 % 101 % 99%

Relative Relative Relative Relative Relative Bioassay

Potency Potency Potency Potency Potency

DP is drug product; DS is drug substance Table 19: STM 434 in ection Lot 001001 ½965 Stability E lata Summary: - -70°C

CL = colorless; SY = slightly yellow; HMW = high molecular weight; LMW = low molecular weight

Table 20: ST 434 Injection Lot 0010036965 Stability Data Summary: -20°C

CL = colorless; SY = slightly yellow; HMW = high molecular weight; LMW = low molecular weight; N.S. = not scheduled Table 21 : STM 434 Injection Lot 0010036965 Stability Data Summary: 5x Freeze/Thaw

CL = colorless; SY = slightly yellow; BMW = high rooiecular weight; LMW = low molecular weight

STM 434 Injection Lot 0010036965 Stability Data Summary: 5°C

CL = colorless; SY = slightly yellow; HMW = high molecular weight; LMW = low molecular weight; N.S. = not scheduled

3: STM 434 Injectioi a Lot 0010036965 St ability Data Summary: 25°C

CL = colorless; SY = slightly yellow; HMW = high moiecuiai' weight; LMW = low moiecuiai weight; N.S. = not scheduled

Table 24: STM 434 Injection Lot EG-13-0150 Stability Data Summary: -20°C (SPN- 643)

CLR L = clear liquid; CL = colorless; LY = light yellow; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; N.D. = not detected; N.S. = not scheduled; NDDi = no detectable dye ingress

*Three reported values due to deviation (anal st executed three injections; method calls for one) Table 25: STM 434 Injection Lot EG-13-0150 Stability Data Summary: 2-8°C (SPN- 644)

CLR L = clear liquid; CL = colorless; LY = light yellow; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; N.D. = not detected; N.S. = not scheduled; NDDi = no detectable dye ingress

*Three reported values due to deviation (anal st executed three injections; method calls for one) Table 26: STM 434 Injection Lot EG-13-0150 Stability Data Summary: 25°C

CLR L = cieai liquid; CL = colorless; LY = light yellow; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; .D. = not detected

Table 27: STM 434 Injection Lot EG-I3-0150 Stability Data Summary: 5* Freeze/Thaw

CLR L = cieai liquid; CL = colorless; LY = light yellow; EFOP = essensially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; .D. = not detected

STM 434 Injection Lot FG-13-0230 Stability Data Summary

657)

CLR L = clear liquid; CL = coloriess; LY = light yellow; EFOP = essesktially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low Eoolecttlar weight; APG = acidic peak group; BPG = basic peak group; N.D. = not detected; N.S. = uoi scheduled STM 434 Injection Lot FG-13-0230 Stability Data Summary

648)

CLR L = clear liquid; CL = colorless; LY = light, yellow; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight ;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; .D. = not detected; N.S. = uoi scheduled Table 30: STM 434 Injection Lot FG-13-0230 Stability Data Summary: 2-8°C

649)

CLR L = clear liquid; CL = colorless; LY = light yellow; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; N.D. = not detected; N.S. ~ aol scheduled Table 31: STM 434 Injection Lot FG-13-0230 Stability Data Summary: 25°C (SPN-650)

CLR L = clear liquid; CL = colorless, LY = light yellow; EFOP = essentially free of particles, NVP = no visible particles, I-IMW = high molecular weight;

LMW = Sow molecular weight; APG = acidic peak group; BPG = basic peak, group; N.D. = not detected; N.S. = not scheduled

Table 32: STM 434 Injection Lot FC*-14-O056 Stability Data Summary: -20°C (SPN- 654)

CLR L = dear liquid; CL = coioriess; LY = light yellow; EFOP = esseskiially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; N.D. = not detected; N.S. ~ aol sctkediiled STM 434 Injection Lot FC _* -14-O056 Stability Data Summary: 2-8°C

655)

CLR L = dear liquid; CL = coioriess; LY = light yellow; EFOP = esseskUaily free οΓ particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; .D. = not detected; N.S. = aot scheduled STM 434 Injection Lot FC _* -14-O056 Stability Data Summary: 25°C (SPN-656)

CLR L = clear liquid; CL = colorless; LY = light yellow; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low irioiecular weight; APG = acidic peak group; BPG = basic peak group; N.D. = not detected; N.S. = not scheduled STM 434 Injection Lot FG-14-0109 Stability Data Summary: -20°C Upright (SPN-676)

CLR L = cieai liquid; CL = colorless; LY = light yellow; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; N.D. = not detected; N.S. = uoi scheduled ½: STM 434 Injection Lot FG- 14-0109 Stability- Data 5 >umm ary: 2- 8°C \ \ (SPN-677)

CLR L = clear liquid; CL = coioriess; LY = light ye!lo ; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; .D. = not detected; N.S. = mt scheduled \7: STM 434 Injection Lot FG- 14-0109 Stability Data 5 >umm ary: 2- 8°C In (SPN-678)

CLR L = clear liquid; CL = coioriess; LY = light ye!lo ; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low !oolecular weight; APG = acidic peak group; BPG = basic peak group; NJD. = not detected; N.S. = uoi scheduled STM 434 Injection Lot FG-14-0109 Stability Data Summary: 25°C Upright (SPN-679)

CLR L = cieai liquid; CL = colorless; LY = light yellow; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight ;

LMW = low !oolecular weight; APG = acidic peak group; BPG = basic peak group; N.D. = not detected; N.S. = not scSkeduled; NDDI = no detectable dye ingress STM 434 Injection Lot FG-14-0109 Stability Data Summary: 25°C Inverted (SPN-680)

CLR L = cieai liquid; CL = colorless; LY = light yellow; EFOP = essentially free of particles; NVP = no visible particles; HMW = high molecular weight;

LMW = low molecular weight; APG = acidic peak group; BPG = basic peak group; N.D. = not detected; N.S. = uoi scheduled; NDDI = no detectable dye ingress administration alters human body composition.

A human subject was selected for treatment with STM 434 using the protocol in Example 4 of PCT/US2015/035818, herein incorporated by reference. The subject was a 62 year old African-American male with recurrent papillary' renal cell carcinoma metastatic to the contralateral kidney and pelvic lymph nodes. He had received 8 prior lines of antitumor therapy with Torisel, Pazopanib, and investigational agents including B7-H3 mAb, cMet inhibitors, a CHKl inhibitor, an Aurora kinase inhibitor, and recombinant human T terleukin 10.

[00203] STM 434 Formulation. Packing, and Storage

[00204] STM 434 was formulated as a sterile aqueous solution intended for IV administration, containing 70 mg/mL STM 434, 10 mM potassium phosphate buffer, 8.8% (w/v) sucrose, and 0.006% (w/v) polysorbate 20 at pH 6.7. Formulated STM 434 solution was packaged into 5-mL glass vials, with 13 mm fluoropoiymer stoppers and 13 mm seals. Vials of STM 434 were stored in a non -frost-free freezer at a temperature of -20°C (± 5°C). Prior to use, STM 434 was thawed overnight in a refrigerator at 2°C to 8°C.

[00205] STM 434 Administration and Monitoring

[00206] STM 434 was administered to the subject at 0.25 mg kg IV approximately every 4 weeks (Cohort 2).

[00207] First DXA scan was 30 Dec 2014; Dosing was 6 Jan 2015, 3 Feb 2015, and 3 Mar 2015; Follow up DXA scan was on 31 March 2015.

[00208] Changes from baseline in lean body mass, appendicular lean mass, and fat mass (visceral and subcutaneous)

[00209] Various body composition measurements were taken approximately three months apart to assess the impact of STM 434 on body composition, e.g., muscle and fat. Lean body- mass, appendicular lean mass, and fat mass, and fat distribution (visceral and subcutaneous) were determined by DXA scans. DXA scans were analyzed by the site radiologist and confirmed by the central radiology laborator ' using IBIS (Imaging Biomarker Information System) software to ensure accurate subject positioning and machine calibration.

[00210] Table 40 shows the results of the measurements for the subject. The results demonstrate substantial increases in lean body mass and substantial decreases in fat mass after only approximately 3 months of treatment at a relatively low dose of STM 434.

2015

% Δ 12.30% 8.40% -48% -99% ^■ 47.8% ^■ 97.8% 282% -1.3% N/A

TLBM= total lean body mass;

ALM= Appendicular lean mass;

TFM= total fat mass;

VFM= visceral fai mass;

SFM= subcutaneous fat mass;

TBMD= total bone mineral density;

TFDIS= total fat distribution;

VFDTS= visceral fat distribution;

SFDIS= subcutaneous fat distribution;

1] Procedures

on:

S 3] Fixed tissues from a large panel of patient derived xenograft models were subjected to mRNA transcript-specific in-situ hybridization for semi quantitative assessment of transcript positivity for INHBA and ACVR2B. This assay procedure, process, and resulting semi-quantitative scores for the entirety of the tissue panel are described in PCT/US2015/01 1396, filed January 14, 2015 and herein incorporated by reference, in its entirety, for all purposes. Tissues with high scores for INHBA and/or ACVR2B were selected for downstream in vivo efficacy assessment for STM 434.

[00214] Assessment:

[00215] Solid tumor xenograft models were generated from patient material that had been passaged exclusively in vivo as sub-cutaneous hind-flank implants in athymic mice. Study mice were engrafted, staged, and treated as described in Table 41. Prior to and throughout the study duration, animals were maintained as described in Table 42, with on-study body weight & tumor volumes assessed at least twice a week . Study animals were randomized into 4 treatment amis upon achieving >125 mm ³ tumor volumes, as described in the tables.

Treatment arms were distributed and treated once weekly with subcutaneous bolus injection of STM 434 or mock treatment as described in Table 43.

[00216] Based on established STM 434 exposure profiles in mouse and man, 1, 3, and 10 mg/kg subcutaneous doses in the mouse are calculated to result in similar exposures as would be expected for 1.05, 3.96, and 11.7 mg/kg intravenous doses in humans. The rationale for this equivalence is as follows: 1. A 1 mg/kg SC dose of 434 in mouse affords the following exposure: AUC = 2330 hr*mcg/mL and Cmax = 10.2 mcg/mL

2. A 3 mg/kg SC dose of 434 in mouse affords the following exposure: AUC = 8830 hr*mcg/mL and Cmax ^:=: 30.6 mcg/mL

3. A 10 mg/kg SC dose of 434 in mouse affords the following exposure: AUC = ^: 34800 hr*mcg/mL and Cmax = 117 mcg/mL

[00217] In humans, 0.75 mg/kg Q2W IV provides the following exposure on Cycle 1 :

AUC = 22,30 hr* mcg/mL and Cmax = 20.5 mcg/mL, Therefore, based on these exposures comparisons and relative dose:exposure ratios:

1. 1 mg/kg SC in mouse = 1.05mg/kg IV in human to match AUC values.

2. 3 mg/kg SC in mouse = 3.96 mg/kg IV in human to match AUC values.

3. 10 mg/kg SC in mouse = 11 ,7 mg/kg IV in human to match AUC values.

Table 41 : PdX Model Protocol Tablature

42: Animal Handling and Measurement

Animal j Charles River Feeding/Drinki 1

Ad Libitum Supplier: j Laboratories ngTime: j

Acclimatizati Room j

>24 Hours 70-74°F on Period: j Temperature: identification ! Relative

Ear Notch 30-60%

Humidity:

Sea!safe ^® Pius

Caging Type: Light Period: 12 Hours

Tec niplast, USA

Corncob bedding, }

Environment Data Capture

nesting sheets, Direct Electronic Enrichment: Method:

plastic housing

Teklad 2919

(Irradiated) Data Capture Digital Scale /

Feed Type:

19% protein, 9% fat,; instrumentfs): Digital Caliper

4% fiber

Drinking Reverse Osmosis, I Data Storage Redundant Cloud

Water: ] 2ppm C: ₂ Method: S rver

Table 43: Group and Treatment Scheme

Dose

Treatment ROA. / Schedule

roup N- (mg/kg)

PBS s.c. / Ixwkiy

-15

ST 34 lmg/kg s.c. / Ixwkiy

-15

STM434 3mg/kg s.c. / Ixwkiy

-15

ST 434 lOmg/kg s.c. / Ixwkiy

-15

Notes: ST 1584, STl 185 and ST1587 to have 10/group ST1145 and ST1725 to have 15/group.

[00219] ST1587 Pancreatic Adenocarcinom : Resulting raw body weights and tumor weights for the STl 587 model are reported in Tables 44 and 45. Mean tumor volumes, whole body weights, change in body weights and change in host-specific body weights for ST1587 are also shown in Figure 1.

[00220] Figure 1A: Tumor growth curves associated with study groups is shown. No significant difference between groups. B: Mean gross body weights for all groups are shown. Mice receiving 10 mg/kg STM-434 show significant and consistent body weight gains over vehicle treated mice. C: Change in body weight from start of study is depicted for each treatment group. Mice receiving 10 mg/kg STM-434 show significant and consistent body weight gains over vehicle treated mice. D: Tumor-corrected, host-specific change in body weights are shown for each treatment group over the course of study. STl 587 model induces significant loss of body weight as a function of tumor growth. All treatment groups demonstrate mitigation and/or reversal of body weight loss, with 10 mg/kg STM-434 having the greatest effect.

[00221] Host-specific body weights were calculated as the total body weight measured for each individual minus the calculated tumor weight for each individual, using a 1,000 mm ^J = Ig conversion. This calculation isolates host body weight from the calculated weight of the tumor that the host is bearing.

[00222] End of study analysis of body weights, including comparative total and host specific body weight changes on the final, fully enrolled study day are shown in Figure 2.

[00223] Figure 2A&B: Mean body weight (A) and change in body weight (B) at the end of study is shown. STM-434 treatment resulted in statistically significant increases in body weight over vehicle treated animals. C: Host-specific change in body weight at end of study is shown for each treatment group. STM434 treatment resulted in dose dependant mitigation of cancer-associated cachexia, with 10 ltig/kg STM-434 treatment showing statistically significant reversal of cachexia in this model.

[00224] ST1584 Pancreatic Adenocarcinoma: Resulting raw body weights and tumor weights for the ST1584 model are reported in Tables 46 and 47. Mean tumor volumes, whole body weights, change in body weights and change in host-specific body weights for ST 1584 are also shown in Figure 3.

[00225] End of study analysis of body weights, including comparative total and host specific body weight changes on the final, fully enrolled study day are shown in Figure 4.

[00226] Figure 3 A: Tumor growth curves associated with study groups is shown. No significant difference between groups. B: Mean gross body weights for all groups are shown. Mice receiving 10 mg/kg STM-434 show significant and consistent body weight gains over vehicle treated mice. C: Change in body weight from start of study is depicted for each treatment group. Mice receiving 10 mg/kg STM-434 show significant and consistent body- weight gains over vehicle treated mice. D: Tumor-corrected, host-specific change in body weights are shown for each treatment group over the course of study. Treatment with 10 mg/kg STM-434 demonstrates increased host body weight over vehicle groups.

[00227] Figure 4 A&B: Mean body weight (A) and change in body weight (B) at the end of study is shown. STM-434 treatment resulted in statistically significant increases is body weight over vehicle treated animals. C: Host-specific change in body weight at end of study is shown for each treatment group. STM434 treatment resulted in dose dependent increase in host body weight, with 10 mg/kg STM-434 treatment showing statistically significant gains over the course of study.

[00228] ST1185 Pancreatic Adenocarcinoma: Resulting raw body weights and tumor weights for the ST1185 model are reported in Tables 48 and 49. Mean tumor volumes, whole body weights, change in body weights and change in host-specific body weights for STl 185 are also shown in Figure 5.

[00229] End of study analysis of body weights, including comparative total and host specific body weight changes on the final, fully enrolled study day are shown in Figure 6.

[00230] Figure 5A: Tumor growth curves associated with study groups is shown. No significant difference between groups, B: Mean gross body weights for all groups are shown. Consistent body weight gains are demonstrated for mice in ail 4 treatment groups, C: Change in body weight from start of study is depicted for each treatment group. Mice receiving 1 , 3 and 10 mg kg STM-434 show significant and consistent body weight gains over vehicle treated mice. D: Tumor-corrected, host-specific change in body weights are shown for each treatment group over the course of study. Treatment with 1, 3 and 10 mg/kg STM-434 demonstrates increased host body weight over vehicle groups.

[00231] Figure 6A&B: Mean body weight (A) and change in body weight (B) at the end of study is shown. STM-434 treatment resulted in statistically significant increases is body weight over vehicle treated animals from start of study. C: Host-specific change in body weight at end of study is shown for each treatment group. STM434 treatment resulted in dose dependent increase in host body weight, with 3 and 10 mg/kg STM-434 treatment showing statistically significant gams over the course of study.

[00232] ST1145 Squamous Cell Head & Neck Carcinoma: Resulting raw body weights and umor weights for the STl 145 model are reported in Tables 50 and 51. Mean tumor volumes, whole body weights, change in body weights and change in host-specific body- weights for STl 145 are also shown in Figure 7.

[00233] End of study analysis of body weights, including comparative total and host specific body weight changes on the final, fully enrolled study day are shown in Figure 8, [00234] Figure 7 A: Tumor growth curves associated with study groups is shown. No significant difference between groups. B: Mean gross body weights for all groups are shown. Consistent body weight gains are demonstrated for mice in all 4 treatment groups; however STM-434 treatment demonstrates dose -dependent increases in overall bodyweight gain over the course of study. C: Change in body weight from start of study is depicted for each treatment group. Mice receiving 1, 3 and 10 mg/kg STM-434 show significant and consistent body weight gains over vehicle treated mice. D: Tumor-corrected, host-specific change in body weights are shown for each treatment group over the course of study. Treatment with 1, 3 and 10 mg/kg STM-434 demonstrates increased host body weight over vehicle groups. [00235] Figure 8A&B; Mean body weight (A) and change in body weight (B) at the end of study is shown. STM-434 treatment resulted in dose-dependent and statistically significant increases is body weight over vehicle treated animals from start of study. C: Host-specific change in body weight at end of study is shown for each treatment group. STM434 treatment resulted in dose dependent increase in host body weight, with 3 and 10 mg/kg STM-434 treatment showing statistically significant gains over the course of study versus vehicle control.

Table 44: ST1S87 Pancreatic Adenocarcinoma Body Weights

Table 45: ST1587 Pancreatic Adenocarcinoma Tumor Volumes

Table 46: ST1584 Pancreatic Adenocarcinoma Body Weights Table 47: ST1S84 Pancreatic Adenocarcinoma Tumor Volumes

48: ST1185 Pancreatic Adenocarcinoma Body Weights

Treatment _f 434 3 mg/kg 43 10 mg/kg

Table 49: ST1185 Pancreatic Adenocarcinoma Body Weights

SO: ST114S Squamous Head & Neck Tumor Volumes ST1145 Squamous Head & Neck Tumor Volumes (continued)

Table 51 : ST1145 Squamous Head & Neck Body Weights

22

4.6 3.7 6.9 8.6 9.7 8.1 5.8 5.6 5.8 7.2 5.4 7.3 8.7 5.5 2.7

25

4.5 4.0 8.2 9.1 0.5 9.5 5.8 6.3 6.5 8.1 5.8 7.7 9.4 5.8 3.3

28

4.5 5.0 9.3 8.5 0.5 9.7 6.2 5,5 6,5 7.5 6.6 7,5 8,6 5.4 3.0

Example 4: BMP9 binding by STM 434 and STM 217.

[00236] STM 217 contains one mutation at position 28 and STM 434 contains two mutations in the receptor portion of the protein at positions 28 and 44. Like the wild type ActRllB, both STM 217 and STM 434 can bind to multiple TGFb family members. To support the understanding of the pharmacology of the compounds, binding of human BMP9 to STM 217 and STM 434 was characterized using Biacore.

[00237] Methods

[00238] BIAcore Solution Equilibrium Binding Analysis: Binding of STM 217 and STM 434 to human BMP9 were compared in a solution equilibrium binding assay using BIAcore 3000. STM 217 and STM 434 were immobilized with high surface density on the second and the fourth flow cells of a CMS chip by amine coupling. The first and the third flow cells were used as surface background controls. 2x serial diluted receptor (STM 217 and STM 434, from 100 nM to 0.05 nM) were mixed with 1 nM huBMP9 in PBS plus O. lmg/ml BSA, and 0.005% P20. The mixtures were then injected over the immobilized receptor (STM 217 and STM 434) as capture surfaces, and the binding response of the free BMP9 in the mixed solutions were measured. The binding response of BMP9 in the absence of the receptors in solution was defined as the 100% BMP9 binding signal, which represented 1 nM free BMP9 in solution , A decreased BMP9 binding response with increasing conce trations of the receptors in solution indicated that BMP9 bound to the receptors in solution, leading to the decrease of free BMP9 in solution that can bind to the immobilized receptor surfaces.

Plotting the BMP9 binding signal versus the receptor concentrations, EC50 values were obtained from nonlinear regression of the curves using a one-site competitive binding model provided in the GraphPad software (GraphPad Software, San Diego, California).

[00239] BlAcore Kinetic binding Analys s: Binding affinity of STM 434 to human BMP9 was determined in kinetic binding assay using BlAcore 3000. STM 434 was captured at a low density,— 100 RU, on an anti-huFc antibody surface. Various concentrations (3.125 to 0.1 nM in 2x dilution) of huBMP9 were used to determine the binding kinetics of huBMP9/STM 434 interaction. K0 was calculated from global fittings using 1 : 1 kinetic binding model in BlAevaluation software (GE Healthcare, Piscataway, NJ).

100240] Results

[00241] The interaction of STM 217 and STM 434 to human BMP9 were compared in solution equilibrium binding assay using BlAcore 3000. EC5 values were calculated using a one-site competitive binding model in the GraphPad software. The data indicate that, within the resolution of the detection, STM 217 and STM 434 bind to human BMP9 with a similar affinity as shown in Figure 9. Average ECK values were determined as being 0.36 nM with 95% Confidence Intervals from 0.24 nM to 0.56 nM for STM 434, and as 0.45 nM, with 95% Confidence Intervals from 0.29 nM to 0.72 nM for STM 217 binding with huBMP9.

[00242] Biacore kinetic binding analysis was implemented to determine the binding constants of STM 434 interaction with human BMP9 as shown in Figure 10. The kinetic parameters were determined as ka-1.4e7 1/Ms, kd-1.7e-4 1/s, and Kd— 12 pM for STM 434 binding with huBMP9.

[00243] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[00244] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Example 5: Impact of STM 434 on solid tumors, cachexia., asid muscle.

[00245] Human subjects (n=25) were selected for treatment with STM 434 using the protocol in the Examples of PCT/US2015/035818, herein incorporated by reference. [00246] STM 434 was formulated as a sterile aqueous solution intended for IV administration, containing 70 mg/mL STM 434, 10 mM potassium phosphate buffer, 8,8% (w/v) sucrose, and 0,006% (w/v) polysorbate 20 at pH 6.7.

[00247] Subjects received STM 434 IV at doses of 0.25 (Q4W), 0.5 (Q4W or Q2W), 0.75

(Q2W), 1 (Q2W), and 2 mg/kg (Q2W). (Table Al).

[00248] Table Al : STM-434 Subject Baseline Characteristics

*Qm sech eft C ^Foaarssma, Kitosy, Leismya ssres s, PWHSWJSC, Thymic, Umehe!

[00249] Results:

[00250] The most common TEAEs were fatigue (n = 13), epistaxis (n ^:=: 7), a nd abdominal pain (n = ^: 6); Adverse Events (AEs) were generally Grade 1-2 (data not shown) AEs of interest are an expected consequence of STM 434 inhibition of bone morphogenetic protein 9, a vascular quiescence protein at the mucosa. The mean Tl/2 was 5-7 days, and PK was linear between 0.25 mg/kg and 1 mg/kg (data not shown).

[00251] STM 434 administration resulted in endocrine PD response to Activin A inhibition with reciprocal decreases in follicle stimulating hormone (FSH) in a majority of patients. (Figure 1 1A)

[00252] Increasing doses of STM 434 were associated with modulation of cachexia as assessed by changes in lean body mass (LBM) (p=0.005, based on a linear contrast by- cohort) and 6-minute walk distance (MWD) , (Figure 1 1B-C).

[00253] LBM (body weight minus body fat mass) was assessed by DEXA scan. Percent change in LBM represents the last available time point relative to pre-dose baseline. For each dose level, post-treatment LBM was measured at the following last available time points after first dose of STM 434.

0.25 mg/kg Q4W: 2 and 6 months

0.5 mg/kg Q4W: 1, 2, 3 and 12 months

0.5 mg/kg Q2W: 1, 1.5, 3, and 6 months

0.75 mg kg Q2W: 2 months

1 mg/kg Q2W: 1 and 2 months

g/kg Q2W: 1.5 and 2 months

The LBM-related dosing schedule for Figure 1 IB is shown in the table below.

Cohort !Dose !Subject Date 1st Dose ! Date Last Dose :# of Di Dses Diiraiion of TX fdaysj !

1 i0.25 mg/kg Q4W! 201-00002 ! 29-Oct-2014! 18-Feb-2015: 5! 112!

1 jO.25 mg/kg Q4WI203-00001 i 17-OG -2014 15-Dec-2014! 3! 59!

2 0.5 mg/kg Q4W ! 200-00001 6-jan-2015: 3-Mar-2015! 3! 56!

2 i0.5 mg/kg Q4W ! 201-00003 i ll~Mar-2Q15! 1-Ju 1-2015! 5! 112!

2 i0.5 mg/kg Q4W : 203-00005 i 15-Apr-2015! 15-May-2015! 2! 30!

2 io.5 mg/kg Q.4W ! 200-00002 i 28-Jan-2015! 29-Dec-2015! 15! 335!

2B 0.5 mg/kg Q2W ! 201-00005 i lO-jun -2015 8-Jul-2015! 3! 28!

2B 0.5 mg/kg Q2W ! 200-00004 i ll-Jun-2015! 7-Aug-2015! 3! 57!

2B 10.5 mg/kg Q2W ! 200-00005 i 25-]im-2015: 4-Aug-2015! 4! 40!

2B i0.5 mg/kg Q2W ! 203-00006 i 16-.!υπ-2015! 28-Ju 1-2015! 4! 42!

3B iO.75 mg/kg Q2W! 201-00006 i 20-Aug-2015! 15-Oct-2015! 5! 56!

3B 0.75 mg/kg Q2W! 200-00007 i .11- Se -2015 6- Nov-2015! 5! 56!

4B ! 1.0 mg/kg Q.2W ! 200-00008 i 27-Oct-2015: 9-Dec-2015! 4! 43!

4B i 1.0 mg/kg Q2W ! 201-00008 3-Dec-2015: 31-Dec-2015! 3! 28!

4B i 1.0 mg/kg Q2W ! 201-00007 i .18- ov- 2015 30-Dec-2015! 4! 42!

5B i 2.0 mg/kg Q2W 200-00009 i 12-Jan-2016! 9-Feb-2016! 2! 28!

SB ! 2.0 mg/kg Q2W ! 202-00001 i 20-Jan -2016! 2 ^» Mar-2016! 4! 42!

[00255] The 6-MWD measuring distance covered by a subject in 6 minutes over a flat surface was performed in accordance with guidelines from the American Thoracic Society. Absolute change represents the last available time point relative to pre-dose baseline. For each dose level, post-treatment 6MWD was measured at the following last available time points after first dose of STM 434.

0.25 mg kg Q4W: 1 and 5 months

0.5 mg/kg Q4VV: 1, 2, 3, and 6 months

0.5 mg/kg Q2W: 1 and 1.5 months

0.75 mg/kg Q2W: 2 and 5.5 months

1 mg/kg Q2W: 1 and 2 months 2 mg/kg Q2W: 1 and 1.5 months

[00256] The 6MWD~related dosing schedule for Figure 11C is shown in the table below.

[00257] Stable disease (SD) up to 12 months (thymic; n=l) and up to 6.1 months (granulosa; n=4) in duration has been observed in 5/25 (20%). The SD rate in granulosa tumors was 4/10 (40%). (Figure 12)

[00258] Conclusions:

[00259] Single agent STM 434 showed an acceptable safety profile in patients with advanced solid tumors and evidence of clinical activity in granulosa ovarian cancer.

[00260] STM 434 exhibits linear PK that support an every other week dosing.

[00261] Increasing doses of STM 434 were associated with decreased FSH suggestive PD target coverage.

[00262] Increasing doses of STM 434 resulted in modulation of cancer cachexia as assessed by increased LBM and 6-MWD.

sv ActRIIB-Fc (E28W, S44T) polynucleotide without signal sequence svActRI!B-Fc (E28W, S44T) polypeptide without signal sequence (STM 434) svActRIIB (E28Y, S44T) polynucleotide with signal sequence

svActRIIB (E28Y, S44T) polypeptide with signal sequence

svActRIIB (E28Y, S44T) polynucleotide without signal sequence

svActRIIB (E28Y, S44T) polypeptide without signal sequence

svActRIlB-Fc (E28Y, S44T) polynucleotide with signal sequence

sv ActRIIB-Fc (E28Y, S44T) polypeptide with signal sequence

sv ActRIIB-Fc (E28Y, S44T) polynucleotide without signal sequence svActRIIB-Fc (E28Y, S44T) polypeptide without signal sequence

ActRIIB (E28W) polypeptide, without signal sequence

ActRIIB-Fc (E28W) polynucleotide, without signal sequence

ActRIIB-Fc (E28W) polypeptide, without signal sequence

IgG2Fc_polype_ptide sequence

IgG lFc polypeptide sequence

IgG4 Fc polypeptide sequence

Linker amino acid sequence

Hinge linker #1 polynucleotide sequence

Hinge linker #1 peptide sequence

SEQUENCE

ATGACGGCGC CCTGGGTGGC CCTCGCCCTC CTCTGGGGAT CGCTGTGCGC CGGCTCTGGG CGTGGGGAGG CTGAGACACG GGAGTGCATC TACTACAACG CCAACTGGGA GCTGGAGCGC ACCAACCAGA GCGGCCTGGA GCGCTGCGAA GGCGAGCAGG ACAAGCGGCT GCACTGCTAC GCCTCCTGGC GCAACAGCTC TGGCACCATC GAGCTCGTGA AGAAGGGCTG CTGGCTAGAT GACTTCAACT GCTACGATAG GCAGGAGTGT GTGGCCACTG AGGAGAACCC CCAGGTGTAC TTCTGCTGCT GTGAAGGCAA CTTC GCAAC GAGCGCTTCA CTCATTTGCC AGAGGCTGGG GGCCCGGAAG TCACGTACGA GCCACCCCCG ACAGCCCCCA CC

MT.APWVALAL LWG3LCAGSG RGEAETRECI YYNANWELER TNOSGLERCE GEQDKRLHCY ASWRNSSGTI ELV GCWLD DFNCYDRQEC VATEE PQVY FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPT

ATGGAGTTTG GGCTGAGCTG GGTTTTCCTC GTTGCTCTTT AAG GGTGT CCAGTGTGAG ACACGGTGGT GCATCTACTA CAACGCCAAC TGGGAGCTGG AGCGCACCAA CCAGACCGGC CTGGAGCGCT GCGAAGGCGA GCAGGACAAG CGGCTGCACT GCTACGCCTC CTGGCGCAAC AGCTCTGGCA CCATCGAGCT CGTGAAGAAG GGCTGCTGGC T.AGATGACTT CAACTGCTAC GATAGGCAGG AGTGTGTGGC CACTGAGGAG AACCCCCAGG TGTACTTCTG CTGCTGTGAG GGCAACTTCT GCAACGAGCG CTTCACTCAT TTGCCAGAGG CTGGGGGCCC GGAAGTCACG TACGAGCCAC CCCCGACAGC CCCCACC

MEFGL3WVFL VALLRGVQCE TRWCIYYNAN WELERTNQTG LERCEGEQDK RLHCYASWRN SSGTIELVKK GCWLDDFNCY DRQECVATES NPQVYFCCCE GNFCNERFTH LPEAGGPEVT YEPPPTAPT

GAGACACGGT GGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGACC GGCCTGGAGC GCTGCGAAGG CGAGCAGG.AC AAGCGGCTGC ACTGCT.ACGC CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT GGCTAGATGA CTTCAAC GC TACGATAGGC AGGAGTGTGT GGCCACTGAG GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAGGGCAACT TCTGCAACGA GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC

ETRWCIYYNA NWELERTNQT GLERCEGEQD RLHCYA5WR NSSGTIELVK GCWLDDFNC YDROECVATE ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT

ATGGAGTTTG GGCTGAGCTG GGTTTTCCTC GTTGCTCTTT TAAGAGGTGT CCAGTGTGAG ACACGGTGGT GCATCTACTA CAACGCCAAC TGGGAGCTGG AGCGCACCAA CCAGACCGGC CTGGAGCGCT GCGAAGGCGA GCAGGACAAG CGGCTGCACT GCTACGCCTC CTGGCGCAAC AGCTCTGGCA CCATCGAGCT CGTGAAGAAG GGCTGCTGGC TAGATGACTT CAACTGCTAC GATAGGCAGG AGTGTGTGGC CACTGAGGAG AACCCCCAGG TGTACTTCTG CTGCTGTGAG GGCAACTTCT GCAACGAGCG CTTCACTCAT TTGCCAGAGG CTGGGGGCCC GGAAGTCACG T.ACGAGCC.AC CCCCGACAGC CCCCACCGGA GGGGGAGGAT CTGTCGAGTG CCCACCGTGC CCAGCACCAC CTGTGGCAGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGA CT CCCGGACCCC TGAGGTCACG TGCGTGGTGG TGGACGTGAG C C AC GAAGAC CCCGAGGTCC AGTTCAACTG GTACGT GGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCACGGGAGG AGCAGTTCAA CAGCACGTTC CGTGTGGTCA GCGTCCTCAC CGTTGTGCAC CAGGACTGGC TGAACGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGG CCTCCCAGCC C C CAT C GAGA AAACCATCTC C AAAAC C AAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGAGGA GATGACCAAG AACCAGGTCA GCCTGACCTG C C T G G T C AAA GGCTTCTATC C C AG C G AC AT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CACCTCCCAT GCTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GAT GC AT GAG GCTCTGCACA ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA. A

MEFGLSWVFL VALLRGVQCE TRWCI YYNAN WELERTNQTG LERCEGEQDK RLKCYASWRN S S GTI ELVKK GCWLDDFNCY DRQECVATEE NPQVYFCCCE GNFCNERFTH LPEAGGPEVT YEP P PTAPTG GGGSVECP PC PAP PVAGP SV FLFP PKPKDT LMI 3RT PEVT CVWDV3HED PEVQFNWYVD GVEVHNAKT K P R E E Q FN S T F RWSVLTWH Q DW LN G KEY K C K VS K G L PA P I EKT I S KTK GQPREPQVYT LP P 3REEMTK NQVS LTCLVK GFYP S D I AVE WE3NGQPENN YKTT P PMLDS DGS FFLYS L TVDKS RWQQG NVFS C SVMHE ALHNHYTQKS LS LS PGK

GAGACACGGT GGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGACC GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC CTCCTGGCGC AACAGCTCTG G C AC CAT C GA GCTCGTGAAG

A.AGGGCTGCT GGCTAGATGA CTTCAACTGC AC GAT AG G C AGGAGTGTGT GGCCACTGAG GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAGGGCAACT TCTGCAACGA GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC CACCCCCGAC AGCCCCCACC G GAG G G G GAG GATCTGTCGA GTGCCCACCG TGCCCAGCAC CACCTGTGGC AGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC AC C C T CAT G A TCTCCCGGAC CCCTGAGGTC ACGTGCGTGG TGGTGGACGT GAG C C AC GAA GACCCCGAGG TCCAGTTCAA CTGGTACGTG GACGGCGTGG AGG GCATAA TGCCAAGACA AAGCCACGGG AGGAGCAGTT CAACAGCACG TTCCGTGTGG TCAGCGTCCT CACCGTTGTG CACCAGGACT GGCTGAACGG CAAGGAGTAC AA.GT GCAAG G TCTCCAACAA AGGCCTCCCA GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGG GCAGC CCCGAGAACC ACAGGT GT AC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAAC C AG G TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT AT C C C AG C GA. CATCGCCGTG GAGTGGGAGA GCAAT GGGCA GCCGGAGAAC AACTACAAGA CCACACCTCC CAT GCT G GAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTGG AC AAGAG C AG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAA

ETRWCI YYNA NWELERTNQT GLERCEGEQD KRLHCYAS R N33 GT I ELVK KGCWLDDFNC YDRQECVATE ENPQVY FCCC EGNFCNERFT HLPEAGGPEV TYEP P PTAPT GGGG5VECP P CPAP PVAGP 3 VFLFP PKPKD TLMI S RT PEV TCVWDVSHE DPEVQFNW V D GVEVHNAKT KPREEQFN ST FRWSVLTVV HQDWLNG EY KCKVSNKGLP AP I EKT I S KT KGQPREPQVY TLP P S REEMT KNQVS LTCLV KGFYP S DIAV E ESNGQPEN NYKTT P PMLD S DGS FFLYS K LTVDKS RWQQ GNVFS C SVMH EALHNHYTQK S LSLS PGK

ATGGAGTTTG GGCTGAGCTG GGTTTTCCTC GTTGCTCTTT TAAG.AGGTGT C C AG T GT GAG ACACGGTACT G CAT C T AC T A CAACGCCAAC TGGGAGCTGG AG C G C.AC C AA CCAGACCGGC CTGGAGCGCT GCGAAGGCGA GCAGGACAAG CGGCTGCACT GCTACGCCTC CTGGCGC.AAC AGCTCTGGCA C CAT C GAG C T CGT GAAG.AAG GGCTGCTGGC T.AGAT GAC T T CAACTGCTAC GATAGGCAGG AGTGTGTGGC CACTGAGGAG AACCCCCAGG TGTACTTCTG CTGCTGTGAG GGCAACTTCT G CA AC G AG C G CTTCACTCAT TTGCCAGAGG CTGGGGGCCC GGAAGTCACG T AC GAG C C AC CCCCGACAGC CCCCACC

MEFGLSWVFL VALLRGVQCE TRYCI YYNAN WELERTNQTG LERCEGEQDK RLKCYASWRN S S GTI ELVKK GCWLDDFNCY DRQECVATEE NPQVYFCCCE GNFCNERFTH LPEAGGPEVT YEP P PTAPT

GAGACACGGT ACT GCAT CTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGACC GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT GGC AGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAGGGCAACT TCTGCAACGA GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC CACCCCCGAC AGCCCCCACC

E RYCIYYNA NWELERTNQT GLERCEGEQD RLHCYAS R N3SGTIELVK KGCWLDDFNC YDRQECVATE ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT

ATGGAGTTTG GGCTGAGCTG GGTTTTCCTC GTTGCTCTTT TAAGAGGTGT CCAGTGTGAG ACACGGTACT GCATCTACTA CAACGCCAAC TGGGAGCTGG AGCGCACCAA CCAGACCGGC CTGGAGCGCT GCGAAGGCGA GCAGGACAAG CGGCTGCACT GCTACGCCTC CTGGCGCAAC AGCTCTGGCA CCATCGAGCT CGTGAAGAAG GGCTGCTGGC TAGATGACTT CAACTGCTAC GATAGGCAGG AGTGTGTGGC CACTGAGGAG AACCCCCAGG TGTACTTCTG CTGCTGTGAG GGCAACTTCT GCAACGAGCG CTTCACTCAT TTGCCAGAGG CTGGGGGCCC GGAAGTCACG TAGGAGCCAC CCCCGACAGC CCCCACCGGA GGGGGAGGAT CTGTCGAGTG CCCACCGTGC CCAGCACCAC CTGTGGCAGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC TGAGGTCACG TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCCGAGGTCC AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCACGGGAGG AGCAGTTCAA CAGCACGTTC CGTGTGGTCA GCGTCCTCAC CGTTGTGCAC CAGGACTGGC TGAACGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGG CCTCCCAGCC CCCATCGAGA AAACCATCTC CAAAACCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGAGGA GATGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CACCTCCCAT GCTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A

MEFGLSWFL VALLRGVQCE TRYCIYYNAN WELERTNQTG LERCEGEODK RLHCYAS RN SSGTIELVKK GCWLDDFNCY DRQECVATEE NPQVYFCCCE GNFCNERFTH LPEAGGPEVT YEPPPTAPTG GGGSVECPPC PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CWVDVSHED PEVQFNWYVD GVEVHNAKT PREEQFNSTF RVVSVLTVVK QDWLNGKEYK CKVSNKGLPA PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGOPENN YKTTPPMLDS DGSFFLYSKL TVD SRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

GAGACACGGT ACTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGACC GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAGGGCAACT TCTGCAACGA GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC CACCCCCGAC AGCCCCCACC GGAGGGGG G GATCTGTCGA GTGCCCACCG TGCCCAGCAC CACCTGTGGC AGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACGTGCGTGG TGGTGGACGT GAGCCACGAA GACCCCGAGG TCCAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCA A TGCCAAGACA AAGCCACGGG AGGAGCAGTT CAACAGCACG TTCCGTGTGG TCAGCGTCCT CACCGTTGTG CACCAGGACT GGCTGAACGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGGCCTCCCA GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGGGCAGC CCCGAGAACC AC GGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACACCTCC CATGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTGG ACAAG GCAG GTGGCAGC G GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAA

ETRYCIYYNA NWELERTNQT GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE E PQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGGSVECPP CPAPPVAGPS VFLFPPKPKD TL I SRTPEV TCVWDVSHE DPEVQFNWYV DGVEVHNAKT KPREEOFNST FRWSVLTW HQD LNGKEY KCKVSNKGLP APIE TISKT KGQPREPQVY TLPP3 EEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKS RWQQ GNVFSC5VMH EALHNHYTQK SLSLSPGK

ETRWCIYYNA NWELERTNQS GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC Y D RQ E C VAT E ENPQVYFCCC E G F C N E R FT HLPEAGGPEV TYEPPPTAPT

GAGACACGGT GGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC GAACCAGAGC GGCCTGGAGC GCTGCGAAGG C GAG C AG G AC AAGCGGCTGC ACTGCTACGC CTCCTGGCGC AACAGCTCTG G C AC CAT C G A GCTCGTGAAG AAGGGCTGCT GGCTAGATGA CTTCAACTGC T AC GAT AG G C AGGAGTGTGT G G C C AC T GAG GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAGGGCAACT TCTGCAA.CGA GCGCTTCACT CAT T T G CC AG AGGCTGGGGG C C C GG AAGT C AC GT AC GAG C CACCCCCGAC AGCCCCCACC G GAG GAG GAG GAT C T GT C GA GTGCCCACCG TGCCCAGCAC CACCTGTGGC AGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC AC C C T CAT G A TCTCCCGGAC CCCTGAGGTC ACGTGCGTGG TGGTGGACGT GAG C C AC GA A G AC C C C GAG G TCCAGTTCAA CTGGTACGTG GACGGCGTGG AGG GCA AA TGCCAAGACA AAGCCACGGG AGGAGCAGTT CAACAGCACG TTCCGTGTGG TCAGCGTCCT CACCGTTGTG CACCAGGACT GGCTGAACGG CAAGGAGTAC AAGT GCAAG G TCTCCAACAA AGGCCTCCCA GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGG GCAGC CCCGAGAACC ACAGGT GTAC ACCCTGCCCC CATCCCGGGA G GAGAT G AC C AAGAAC C AG G TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT AT C C C AG C GA. CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AAC T ACAAGA CCACACCTCC CATGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG CT C AC C GT G G ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC ACAACCACTA C AC G C AGAAG AGCCTCTCCC TGTCTCCGGG TAAA

ETRWCIYYNA NWELERTNQS GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC Y D RQ E C VAT E ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGGSVECPP CPAPPVAGPS VFLFPPKPKD TLMI SRTPEV TCVWDVSHE DPEVQF WYV DGVEVHNAKT KPREEQFNST FRWSVLTW HQDWLNGKEY KCKVSNKGLP APIEKTISKT KGQPREPQVY TLP SREEMT KNQVS LTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKS RWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

APPVAGPSVF LFPPKPKDTL MI SRTPEVTC WVDVSHEDP EVQF WYVDG VEVHNAKTKP REEOFN3TFR W3VLTWHQ DWLNGKEYKC KVSNKGLPAP IEKTI SKT G QPREPQVYTL P P S R E EMT KN QVSLTCLVKG FYPSDIAVEW

E5NGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK

APELLGGPSV FLFPPKPKDI LMISRTPEVT CVWDV3HED PEV FNWYVG GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WE3NGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQ 5 LSLSPGK

APEFLGGPSV FLFPPKPKDT LMISRTPEVT CWVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS

3IEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFY SDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTOKS LSLSLGK

GGGGS

GGAGGGGGAG GATCTGTCGA GTGCCCACCG TGCCCA

GGGGSVECPP CP