WO2017075037A1

WO2017075037A1 - Primed growth factors and uses thereof

Info

Publication number: WO2017075037A1
Application number: PCT/US2016/058841
Authority: WO
Inventors: Michelle STRAUB
Original assignee: Scholar Rock Inc
Current assignee: Scholar Rock Inc
Priority date: 2015-10-27
Filing date: 2016-10-26
Publication date: 2017-05-04
Anticipated expiration: 2018-04-27

Abstract

Aspects of the present disclosure relate to substantially pure preparations of primed recombinant transforming growth factors and compositions (e.g., pharmaceutical compositions) comprising the same. Methods for modulating transforming growth factor receptors and for treating subjects having a condition associated with reduced transforming growth factor receptor activity (e.g., using any of the preparations described herein) are also provided. Antibodies that selectively bind primed recombinant transforming growth factors and methods of making the same are also provided.

Description

PRIMED GROWTH FACTORS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62/247, 179, filed October 27, 2015, and entitled "PRIMED GROWTH

FACTORS AND USES THEREOF", the contents of which are incorporated herein by reference for all purposes.

BACKGROUND OF INVENTION

Transforming growth factors interact with cell surface serine/threonine-specific protein kinase receptors and generate intracellular signals via a family of proteins called SMADs. They play fundamental roles in the regulation of basic biological processes such as growth, development, tissue homeostasis and regulation of the immune system. Thus, inhibitors and activators of transforming growth factors may be useful for regulating such pathways to treat diseases or conditions associated with transforming growth factor receptor activation.

SUMMARY OF INVENTION

Aspects of the disclosure relate to a recognition that, in some embodiments, enzymatic cleavage of a transforming growth factor (e.g., by a proprotein convertase and a tolloid protease) produces a form of the growth factor in which its prodomain remains non-covalently associated with its mature growth factor portion but does not prevent the growth factor from signaling; a form referred to herein as, "primed". In some embodiments, proteolytic cleavage events allow for the growth factor prodomain to sample an open, "active" conformation which allows for the growth factor to bind to cell surface signaling receptors. In some embodiments, methods and compositions are provided herein for producing preparations of substantially purified primed recombinant transforming growth factor (rTGF). In some embodiments, primed rTGFs have improved pharmaceutical properties. For example, in some embodiments, primed rTGFs have improved solubility at neutral pH compared with mature transforming growth factor, enabling formulation of pharmaceutical preparations at neutral pH compared with acidic preparations of mature growth factor. Furthermore, in some embodiments, it has been found that primed rTGF is a more potent stimulator of intracellular signaling (e.g., SMAD mediated signaling) than other forms of the growth factor, e.g. , mature rTGF, latent rTGF, or pro-rTGF. In some embodiments, it has been found that primed rTGF has improved bioavailability compared with the mature form.

Aspects of the disclosure relate to a substantially pure preparation of a primed recombinant transforming growth factor (rTGF), for example, primed GDF8 or primed GDF11. In some embodiments primed rTGF is prepared by cleaving, or otherwise separating, the proprotein from the mature growth factor, such that the proprotein remains associated with the mature growth factor non-covalently. In some embodiments, primed rTGF is prepared by contacting an rTGF with a proprotein convertase to produce a latent rTGF; contacting the latent rTGF with a tolloid protease to produce primed rTGF; and purifying the primed rTGF, thus producing a substantially pure primed rTGF. In some embodiments, the proprotein convertase is a furin or PCSK5 and in some embodiments, the tolloid protease is a mT112 protease or a BMP1 protease. In some embodiments, primed rTGF is prepared by a method comprising: contacting an rTGF with a proprotein convertase to produce a latent rTGF; contacting the latent rTGF with a tolloid protease to produce primed rTGF; and substantially separating the proprotein convertase and/or tolloid protease from the primed rTGF.

The preparation of a substantially pure primed rTGF may further include producing the rTGF in vitro in one or more engineered cells that express the rTGF. In some embodiments, the rTGF is produced in one or more engineered cells that inducibly express the rTGF. In some embodiments, the one or more engineered cells secrete the rTGF into a cell culture media comprising a proprotein convertase and/or a tolloid protease. In some embodiments, the one or more engineered cells further express a proprotein convertase and/or a tolloid protease. The engineered cells, in some embodiments may be co-cultured with cells that express a proprotein convertase and/or a tolloid protease.

The preparation of a substantially pure primed rTGF may be in a solution, or may be in a lyophilized form. As one example, the primed rTGF is in a solution at a concentration of greater than 0.1 mg/ml, greater than 1 mg/ml, greater than 10 mg/ml or greater than 100 mg/ml. In some embodiments, primed rTGF is in a solution at a concentration in a range of 0.1 mg/ml to 10 mg/ml, 1 mg/ml to 10 mg/ml, or 0.1 mg/ml to 100 mg/ml. In some embodiments, the primed rTGF is at least 90% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) pure. In some embodiments, the primed rTGF is at least 95% pure.

In some embodiments, a preparation of primed rTGF is provided that comprises a molar ratio of a primed rTGF to a proprotein form of the rTGF in a range of 10 1 : 1 to 102 : 1, 101:l to 10³: 1, 10²: 1 to 10⁴: 1, 10²: 1 to 10⁶: 1. In some embodiments, a preparation of primed rTGF is provided that comprises trace amounts of a proprotein form of the rTGF. In some embodiments, a preparation of primed rTGF is provided that comprises primed rTGF in solution and less than 0.5 %, less than 0.1 %, less than 0.05 %, less than 0.01 %, less than 0.005 %, or less than 0.001 % by weight (w/v) of a proprotein form of the rTGF in the solution.

In some embodiments, a preparation of primed rTGF is provided that comprises a molar

1 2 1 3 ratio of a primed rTGF to a latent form of the rTGF in a range of 10 : 1 to 10 : 1, 10 : 1 to 10 : 1, 10²: 1 to 10⁴: 1, 10²: 1 to 10⁶: 1. In some embodiments, a preparation of primed rTGF is provided that comprises trace amounts of a latent form of the rTGF. In some embodiments, a preparation of primed rTGF is provided that comprises primed rTGF in solution and less than 0.5 %, less than 0.1 %, less than 0.05 %, less than 0.01 %, less than 0.005 %, or less than 0.001 % by weight (w/v) of a latent form of the rTGF in the solution.

A primed rTGF, in some embodiments, includes an epitope tag. In some embodiments, the epitope tag is at the C-terminus of the rTGF. In some embodiments, the epitope tag is at the N-terminus of the rTGF In some embodiments, the epitope tag is between the C-terminus and the N-terminus of the rTGF.

In some embodiments, the primed rTGF in a substantially pure preparation of primed rTGF is AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7,

BMP8A, BMP8B, GDF1 , GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY 1, LEFTY2, MSTN, NODAL, NRTN, PSPN, TGFp l, TGFp2, or TGFp3 protein.

Aspects of the disclosure include pharmaceutical compositions with a therapeutically effective amount of any of the primed rTGF preparations provided herein and a

pharmaceutically acceptable carrier. Pharmaceutical compositions provided herein, in some embodiments, are useful for decreasing muscle mass, improving blood vessel volume, improving neurogenesis, reversing age-related cardiac hypertrophy or improving skeletal muscle function. In some embodiments, the pharmaceutical compositions provided herein contain an amount of primed rTGF effective for decreasing muscle mass, improving blood vessel volume, improving neurogenesis, reversing age-related cardiac hypertrophy, improving skeletal muscle function promoting bone growth, or modulating the immune system (e.g., increasing or decreasing an immune response). Aspects of the disclosure relate to methods for modulating transforming growth factor (TGF) receptors in cells. In some embodiments, methods provided herein include modulating SMAD signaling in cells. In some embodiments, methods for modulating TGF receptors in cells include delivering an effective amount of any of the preparations provided herein to a medium comprising cells expressing the TGF receptors. In some embodiments, the cells are in vitro. In other embodiments, the cells are in in vivo. In some embodiments, the disclosure provided methods for modulating non-canonical TGF signaling pathways. In some embodiments, the methods include modulating MAP kinase pathways, Rho-like GTPase signaling pathways, and/or phosphatidylinositol-3-kinase/AKT pathways. However, these non-canonical TGF signaling pathways are not meant to be limiting and additional non-canonical TGF pathways are within the scope of this disclosure, for example, those described in Zhang Y.E., "Non-Smad pathways in TGF-beta signaling", Cell Res. 200i Jan; 19(1): 128-39; the contents of which are hereby incorporated by reference.

Aspects of the disclosure relate to methods for treating a subject having a condition associated with reduced transforming growth factor receptor activity. In some embodiments, the method includes administering any of the pharmaceutical compositions provided herein to the subject. In some embodiments, the condition is age-related cardiac hypertrophy. In some embodiments, the condition includes decreased blood vessel volume. In some embodiments, the condition includes decreased neurogenesis. In some embodiments, the condition includes decreased skeletal muscle function.

Aspects of the disclosure relate to methods for producing a primed recombinant transforming growth factor (rTGF). In some embodiments, the methods include expressing a rTGF in a cell; contacting the rTGF with a proprotein convertase to produce a latent rTGF; contacting the latent rTGF with a tolloid protease to produce the primed rTGF; and isolating the primed rTGF. In some embodiments, the proprotein convertase is a furin protease and/or the tolloid protease is a mTl 12 protease. In some embodiments, the recombinant rTGF is contacted with the proprotein convertase and/or the tolloid protease in vitro. In some embodiments, the rTGF is contacted with the proprotein convertase and/or the tolloid protease in a cell culture media. In some embodiments, the cell culture media is a conditioned media comprising a proprotein convertase and/or a tolloid protease. In some embodiments, the rTGF and/or the proprotein convertase and/or the tolloid protease is expressed inducibly in a cell. In some embodiments, the cell culture media comprises one or more engineered cells that express a proprotein convertase and/or a tolloid protease.

Aspects of the disclosure relate to a solid substrate to which is linked a primed transforming growth factor. In some embodiments, the primed transforming growth factor is non-covalently linked to the solid substrate. In some embodiments, the primed transforming growth factor is linked to the substrate via an antibody. In some embodiments, the antibody selectively binds the primed transforming growth factor. In some embodiments, the antibody is any of the antibodies provided herein. In some embodiments, the solid substrate comprises beads or a resin. In some embodiments, the solid substrate is a surface of an affinity column.

Aspects of the disclosure relate to methods for obtaining an antibody that binds to a primed transforming growth factor. In some embodiments, the method includes contacting a substantially pure preparation of the primed transforming growth factor with a plurality of candidate antibodies or antigen binding fragments thereof; selecting candidate antibodies or antigen binding fragments that bind to the primed transforming growth factor; contacting the selected antibodies or antigen binding fragments with a transforming growth factor that is not primed; eliminating the antibodies or antigen binding fragments that bind to the growth factor that is not primed; and selecting the antibodies or antigen binding fragments that bind to the primed transforming growth factor and do not bind to the growth factor that is not primed. In some embodiments, the transforming growth factor is GDF8 or GDFl 1. In some embodiments, the antibodies or antigen binding fragments are eliminated if they bind to the growth factor that is not primed with an affinity greater than or equal to the affinity that they bind to the growth factor that is primed. In some embodiments, the transforming growth factor that is not primed is the pro form of the transforming growth factor. In some embodiments, the transforming growth factor that is not primed is the latent form of the transforming growth factor.

Aspects of the disclosure relate to antibodies obtained by any of the methods provided herein. In some embodiments, the antibody is bound to a primed transforming growth factor. In some embodiments, the primed transforming growth factor is recombinant. In some

embodiments, the transforming growth factor is GDF8 or GDFl 1. In some embodiments, the antibody selectively binds a primed transforming growth factor. In some embodiments, the antibody inhibits the primed transforming growth factor.

Aspects of the disclosure relate to a pharmaceutical composition having a therapeutically effective amount of any of the antibodies provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is for use in preventing muscle wasting or increasing muscle mass. In some embodiments, the antibody selectively binds myostatin (GDF8). In some embodiments, the antibody inhibits myostatin (GDF8) activity (e.g. , functions as an antagonist anti-GDF8 antibody).

Aspects of the disclosure relate to primed recombinant transforming growth factor

(rTGF) comprising an epitope tag. In some embodiments, the epitope tag is on the C-terminus of the rTGF. In some embodiments, the epitope tag is between the C-terminus and the N- terminus. In some embodiments, the recombinant transforming growth factor is GDF8 or GDF11.

Each of the limitations of the disclosure can encompass various embodiments of the disclosure. It is, therefore, anticipated that each of the limitations of the disclosure involving any one element or combinations of elements can be included in each aspect of the disclosure. This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a schematic showing prodomain assemblies. TGFP family members are secreted as procomplexes. TGB-β Ι "closed procomplex" and BMP9 "open procomplex" are depicted.

FIG. 2 is a schematic outlining the activation of rTGFs (e.g. , GDF11 and GDF8) by proprotein convertases and tolloid proteases.

FIG. 3A is an image of a gel depicting three species (pro, latent and tolloid cleaved) of an exemplary GDF11 growth factor. The samples in the first three lanes on the left were run under non-reducing (nR) conditions while the last three lanes on the right were run under reducing (R) conditions. The lane in the middle contains size standards. The uncleaved proGDFl l species and its respective cleavage products are indicated using arrows.

FIG. 3B shows Coomassie-stained SDS PAGE gels of the three species (pro, latent and primed) of GDF8 used in SEC-MALS. The samples in the first three lanes on the left were run under non-reducing conditions while the last three lanes on the right were run under reducing conditions. The uncleaved proGDF8 species and its respective cleavage products are indicated using arrows. Protein bands consisted of the proMyostatin monomer (-50 kD reducing, -100 kDa non-reducing), prodomain (-37 kD) and growth factor (12.5 kD reducing, -25 kDa non- reducing). Following tolloid protease and pro-protein convertase cleavage, two fragments of the prodomain were generated at -18 kD and -16 kDa (Fragment 1 and 2). The identity of these bands as the expected fragments generated after tolloid protease and proprotein convertase cleavage was confirmed by N-terminal sequencing.

FIG. 3C shows a Coomassie-stained SDS PAGE gel of a substantially pure proGDF8 sample.

FIG. 3D shows a Coomassie-stained SDS PAGE gel of an efficiently cleaved primed GDF8 sample.

FIGs. 4A-4B show three species of Myostatin and GDF11 analyzed by size exclusion chromatography coupled to multiangle light scattering (SEC-MALS) traces of proGDFl l and latent GDFl lspecies. Individual samples are identified in Table 2, provided herein. FIG. 4A shows overlaid size exclusion chromatography coupled to multiangle light scattering (SEC- MALS) traces of GDFl 1 species. Individual samples are identified in Table 2, provided herein. The pro- and latent- conformations of GDF11 are indistinguishable based on SEC elution and calculated molecular mass. The primed version of GDFl 1 elutes earlier from SEC, with a similar molecular weight, indicating a potential conformational change of the protein. FIG. 4B shows overlaid size exclusion chromatography coupled to multiangle light scattering (SEC- MALS) traces of myostatin (GDF8) species. The pro- and latent- forms of GDF8 are comparable based on SEC elution and calculated molecular mass. The primed version of GDF8 elutes later from SEC, with a lower molecular weight, indicating a dissociation of the complex.

FIGs. 4C and 4D depict concentration-dependence of the SEC-MALS elution. In FIG. 4C, lOOug, 20ug, and lOug injections were analyzed for the three different GDF11 species. In contrast to proGDFl 1 and latent GDFl 1 (which stays associated as a dimer), primed GDFl 1 dissociates when diluted on the SEC column as indicated by later elution volumes and smaller calculated molecular weights. Calculated protein weights are shown in Table 2. In FIG. 4D, lOOug, 20ug, and lOug (primed only) injections were analyzed for the three different GDF8 species. In contrast to proGDF8 and latent GDF8 (which stays associated as a dimer), primed GDF8 dissociates when diluted on the SEC column. Calculated protein weights are shown in Table 3. FIG. 5 includes results showing the species of GDF11, separated by size exclusion chromatography and non-reducing gel electrophoresis, following cleavage by proprotein convertase and tolloid protease. GDFl 1 was treated with both proprotein convertase and tolloid protease and run on a SRT10 size exclusion chromatography (SEC) column in 20mM HEPES (pH8; 500mM NaCl; 5mM EDTA) buffer.

FIGs. 6A and 6B show results from a 293T cell in vitro CAGA luciferase reporter assay. No proteases were added to the recombinant proteins in this assay. The calculated EC50 for each of the species using the 293T cell in vitro reporter assay is shown in nM. FIG. 6A provides a graph showing that murine primed GDFl 1 elicits greater response than mature GDFl 1, latent GDFl 1 or proGDFl 1 using a 293T cell in vitro reporter assay. The calculated EC50 for each of the species using the 293T cell in vitro reporter assay is shown in Table 3. FIG. 6B provides a graph showing that primed Myostatin signals with equal efficacy as mature Myostatin.

DETAILED DESCRIPTION OF INVENTION

Myostatin (also referred to as GDF8) and GDF11, members of the TGFP superfamily, are both expressed as inactive precursor polypeptides (termed proMyostatin and proGDFl 1, respectively). GDF8 and GDFl 1, like many other members of the TGFP superfamily, exist as inactive precursor proteins in vivo, and their activation involves separation of the prodomain from the mature growth mature for biological activity. Activation and release of mature growth factor of the rTGF may be accomplished by several discrete protease cleavage events, as outlined in FIG. 2. The first cleavage step of proMyostatin and proGDFl 1 is carried out by a proprotein convertase, which cuts at a conserved RXXR site between the prodomain and mature growth factor. This cleavage produces a latent complex, in which the mature growth factor is shielded from binding to its receptors by the prodomain. Activation and release of the mature, active Myostatin growth factor is accomplished after cleavage by an additional protease from the BMP/tolloid family, such as mTLL-2 (FIG. 2).

The instant disclosure is based at least in part on the surprising discovery that transforming growth factors (e.g., GDF8 and GDF11) exist in a "primed" complex following enzymatic cleavage, e.g., by both a proprotein convertase and a tolloid protease. In this primed complex at least a portion of the prodomain remains non-covalently associated with the mature growth factor, which counters the view that the prodomain dissociates from the growth factor upon protease cleavage.

Furthermore, certain aspects of the disclosure are based on a surprising discovery that primed rTGF is a more potent stimulator of SMAD mediated signaling than mature rTGF, latent rTGF, or pro rTGF. In some embodiments, this potent stimulatory effect is exhibited in an in vitro cell based reporter assay, as described herein.

In some embodiments, primed transforming growth factors have improved

pharmaceutical properties since they have improved solubility properties (e.g., soluble in a neutral pH range, e.g. , pH 6.8 to pH 7.4) compared with mature transforming growth factor, which may improve the bioavailability of the growth factor. In some embodiments, a primed rTGF is soluble at concentrations (e.g., in a neutral pH range, e.g. , pH 6.8 to pH 7.4) of at least 0.1 mg/mL, at least 0.2 mg/mL, at least 0.5 mg/mL, at least 1.0 mg/mL, at least 1.5 mg/mL, at least 2.0 mg/mL, at least 2.5 mg/mL, at least 3 mg/mL, at least 3.5 mg/mL, or at least 4.0 mg/mL. In some embodiments, a primed rTGF is soluble at concentrations (e.g., in a neutral pH range, e.g. , pH 6.8 to pH 7.4) in a range of 0.1 mg/mL to 4.0 mg/mL or 0.1 mg/mL to 10 mg/mL or more. In some embodiments, a mature form of a transforming growth factor at neutral pH is less soluble than the primed form of a transforming growth factor at the same pH. Rather, in some embodiments, the mature form is comparably soluble at acidic pH (e.g., pH less than 6.8). Thus, in some embodiments, a mature form of a transforming growth factor is stored in an acidic solution (e.g. , an HCl solution, e.g. , a 1M to 5M solution of HCl). In some embodiments, the mature form of a transforming growth factor is stored in a 4M solution of HCl when the mature form of a transforming growth factor, e.g. , at a concentration of greater than 10 ng/mL.

In some embodiments, primed transforming growth factors have improved

pharmaceutical properties because the prodomain fragments may localize the rTGF to the correct microenvironment in vivo, thereby potentially reducing off-target effects, potentially increasing potency, and/or potentially increasing the half-life of the primed rTGF. Thus, in some embodiments, primed transforming growth factors may be more effective for treating disease associated with decreased transforming growth factor receptor activity. Primed transforming growth factors may also be useful as antigens for inhibitory campaigns to specifically block signaling of transforming growth factors, such as, for example, GDF8 and GDF11. As used herein, a "primed recombinant transforming growth factor (rTGF)" or "primed rTGF" refers to recombinant form of a member of the TGF-β superfamily of proteins, or a homologous protein thereof, in which its prodomain is cleaved, or otherwise separated from the mature growth factor but remains associated, non-covalently, with the mature growth factor domain. In some embodiments, primed rTGF refers to recombinant form of a member of the TGF-β superfamily of proteins, or a homologous protein thereof, in which its prodomain is cleaved, or otherwise separated, at proprotein convertase cleavage site(s) and/or tolloid protease cleavage site(s). In some embodiments, a primed rTGF is a primed recombinant form of a TGF- β superfamily protein that is regulated in a protease-dependent manner, e.g. , that is activated by one or more protease cleavage events. In some embodiments, prodomain that remains associated, non-covalently, with its mature growth factor domain does not substantially impede growth factor binding to receptors. In some embodiments, the prodomain of a primed rTGF is conformationally open or otherwise unconstrained (in comparison with a pro-form of the rTGF) so as to permit the mature growth factor to bind to its cognate growth factor receptor. In some embodiments, a primed rTGF elutes at a different volume than a corresponding pro- or latent- form of the rTGF in a gel filtration column. In some embodiments, a primed rTGF elutes earlier than a corresponding pro- or latent- form in a gel filtration column. In some embodiments, a primed rTGF elutes earlier than a corresponding pro- or latent-form in a gel filtration column, while the calculated molecular weight remains substantially the same as the corresponding pro- or latent-form, which indicates a conformational change. In some embodiments, the

conformational state of primed rTGF facilitates access to the receptor by the mature growth factor. For example, in some embodiments, the conformational state of primed rTGF facilitates access of its mature growth factor to a cognate receptor, such that efficacy for signaling through the receptor by the primed rTGF is functionally similar to signaling by the mature growth in the absence of proteases, such as is depicted in FIG. 6.

In some embodiments, enzymatic cleavage of a transforming growth factor (e.g., by a proprotein convertase and a tolloid protease) produces a primed form of the growth factor in which its prodomain remains non-covalently associated with its mature growth factor portion. In some embodiments, the TGF-β superfamily of proteins include a large family of structurally related cell regulatory proteins that were named after the founding member, TGF-β Ι, whose growth factor structure was described in Schlunegger MP, et. ah , (July 1992). "An unusual feature revealed by the crystal structure at 2.2 A resolution of human transforming growth factor-beta 2". Nature 358 (6385): 430-4. Members of the TGF-β superfamily of proteins are found in a variety of species, including invertebrates as well as vertebrates. For example, members of the TGF-β superfamily have been described by Robertson LB. et ah, (August 2013), "Unchaining the beast; insights from structural and evolutionary studies on TGFP secretion, sequestration, and activation". Cytokine growth Factor Rev. 24 (4):355-72; Herpin A., et. ah, (May 2004), "Transforming "growth factor-beta-related proteins: an ancestral and widespread superfamily of cytokines in metazoans". Dev. Comp. Immunol. 28 (5): 461-85; Burt DW (April 1992). "Evolutionary grouping of the transforming growth factor-beta superfamily". Biochem. Biophys. Res. Commun. 184 (2): 590-5; and Burt DW, et. al, (1994). "Evolution of the transforming growth factor-beta superfamily". Prog. Growth Factor Res. 5 (1): 99-118; each of which is incorporated herein by reference. In some embodiments, members of the TGF-β superfamily of proteins include, without limitation, proteins encoded by the following human genes: AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDFl, GDF10, GDFl l, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY 1, LEFTY2, MSTN,

NODAL, NRTN, PSPN, TGFB 1, TGFB2, and TGFB3. In some embodiments, the member of the TGF-β superfamily of proteins is myostatin (GDF8) or GDFl 1. In some embodiments, the myostatin (GDF8) or GDFl 1 is human or mouse. It should be appreciated that the TGF-β superfamily members described and referenced herein are exemplary and additional TGF-β superfamily members from humans as well as other species {e.g., mice) fall within the scope of this disclosure.

Members of the TGF-β superfamily may also include proteins that are homologous to one or more members of the TGF-β superfamily. In some embodiments, a protein is considered to be homologous to a member of the TGF-β superfamily if it is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to one or more members of the TGF-β superfamily.

Calculation of the percent identity of two amino acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes {e.g., gaps can be introduced in one or both of a first and second nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence (e.g., the amino acid sequence of a member of the TGF-β superfamily). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using methods such as those described in

Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et ah, Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Atschul, S. F. et al, J. Molec. Biol, 215, 403 (1990)).

The primed transforming growth factors provided herein are recombinant. A primed recombinant transforming growth factor (rTGF) refers to a primed transforming growth factor that has been designed, produced, prepared, synthesized, and/or manufactured by a human using recombinant techniques. Accordingly, a primed recombinant transforming growth factor does not occur in nature. In some embodiments, a primed rTGF is a protein that has been designed to meet particular requirements or to have particular design features. For example, in some embodiments a primed rTGF comprises an epitope tag. As used herein, an "epitope tag" refers to a peptide sequence that may be genetically grafted onto a protein (e.g., a member of the TGF- β superfamily). Exemplary epitope tags include, but are not limited to, a FLAG-tag, a hexa- histidine-tag (His-tag), a V5-tag, a Myc-tag, an Fc-tag and an HA-tag. In some embodiments, the epitope tag comprises the amino acid sequence DYKDDDDK (SEQ ID NO: 9) or HHHHHH (SEQ ID NO: 10). The epitope tag may be a short peptide sequence chosen because high-affinity antibodies can be reliably produced in many different species. Such epitope tags may be useful for western blotting, immunofluorescence, ELISA and immuneprecipitation. Such epitope tags may improve the bioavailability of a primed rTGF by increasing its half-life in vivo.

In some embodiments, primed rTGFs provided herein may be used in research, experimental and/or diagnostic applications. For example, primed rTGFs provided herein may be used as a standard in a functional assay, such as, for example a western blot assay, an immunoprecipitation assay, an immunofluorescence assay, or an ELISA. As one example, a substantially pure preparation of a primed rTGF can be used as a standard control in an ELISA. Such ELISAs may be used for detecting pro, latent, mature, and/or primed forms of a transforming growth factor (e.g. , myostatin) to, for example, track processing in vivo, or to measure the half-life of a specific form of transforming growth factor (e.g., a pro, a latent, a mature, or a primed form) in vivo.

In some embodiments, a primed rTGF comprises a signal peptide. A signal peptide refers to a short peptide, typically having a length from 5 to 30 amino acids, that can direct a protein to the secretory pathway, localize a protein inside an organelle (e.g., the endoplasmic reticulum, golgi, or endosomes), and/or insert the protein into a cellular membrane. Signal peptides can be heterogeneous and many prokaryotic and eukaryotic signal peptides are functionally interchangeable even between different species. In some embodiments, the efficiency of protein secretion is determined by the signal peptide. In some embodiments, suitable signal peptides may be used, for example, peptides described in Kober L et. ah , (April 2013). "Optimized signal peptides for the development of high expressing CHO cell lines". Biotechnol. Bioeng. 110 (4): 1164-73; and von Heijne G (Jul 1985). "Signal sequences: The limits of variation". J Mol Biol 184 (1): 99-105; the contents of each is hereby incorporated by reference. In some embodiments, the signal peptide targets the rTGF for secretion. In some embodiments, the signal peptide comprises an Ig kappa chain (V-I region) signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence

MDMRVP AQLLGLLLLWFS G VLG (SEQ ID NO: 11).

In some embodiments, recombinant transforming growth factors provided herein are primed. A recombinant transforming growth factor is primed if it is cleaved at a proprotein convertase cleavage site and a tolloid protease site. For example, a full length GDFl 1 that has been cleaved by a proprotein convertase and has been cleaved by a tolloid protease is referred to as primed GDFl 1. A full length transforming growth factor that is not cleaved at a proprotein convertase cleavage site and is not cleaved at a tolloid protease site is referred to as a "pro" form. For example, a full length GDFl 1 that has not been cleaved by a proprotein convertase and has not been cleaved by a tolloid protease is referred to as proGDFl 1. A transforming growth factor that is cleaved at a proprotein convertase cleavage site and not cleaved at a tolloid protease site is referred to as a "latent" form. For example a GDFl 1 that has been cleaved by a proprotein convertase (e.g., furin) and not by a tolloid protease is referred to as latent GDFl 1. A schematic representation of the pro, latent, and primed forms of GDF8/11 is shown in FIG. 2. A "proprotein convertase cleavage site," as used herein, is an amino sequence that can be cleaved by a proprotein convertase, as described herein. Any appropriate methods may be used for identifying proprotein convertase cleavage sites and testing cleavage of the cleavage sites by their cognate proprotein convertase . See e.g., Duckert, P., et al., Prediction of proprotein convertase cleavage sites. Protein Engineering Design and Selection 2004; the contents of which are hereby incorporated by reference. In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence R-X-X-R, where R is arginine and X is any amino acid. In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence R-X-(K/R)-R, where R is arginine, K is lysine and X is any amino acid. In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence is R- V-R-R, where R is arginine and V is valine. Exemplary proprotein convertase cleavage sites for human and mouse myostatin and GDF11 are shown below, in bold, in SEQ ID NOs: 1-4.

Examples of proprotein convertases for use in accordance with the present disclosure include, without limitation, those listed in Table 1 : PCSK1, PCSK2, PCSK3/furin, PCSK4, PCSK5, PCSK6, PCSK7, PCSK8, PCSK9, subtilisin or kexin.

Table 1: List of mammalian proprotein convertases including alternative gene names.

In some embodiments, a proprotein convertase comprises (i) a catalytic domain that hydrolyzes a peptide bond of a protein containing a proprotein convertase cleavage site, and (ii) a binding pocket that binds to an rTGF containing a proprotein convertase cleavage site.

A proprotein convertase may be obtained from any mammal including, without limitation, humans or rodents (e.g. , mice, rats, hamsters). Examples of human proprotein convertases, without limitation, are listed in Table 1. In some embodiments, a proprotein convertase is homologous to a proprotein convertase selected from the group consisting of: PCSKl, PCSK2, PCSK3/furin, PCSK4, PCSK5, PCSK6, PCSK7, PCSK8, PCSK9, subtilisin, and kexin. For example a proprotein convertase may be at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least about 99.9% identical to PCSKl, PCSK2, PCSK3/furin, PCSK4, PCSK5, PCSK6, PCSK7, PCSK8, PCSK9, subtilisin or kexin.

A "tolloid protease site," as used herein, is an amino acid sequence that can be cleaved by a tolloid protease, as described herein. In some embodiments, appropriate tolloid protease sites are described, for example, in Hopkins D.R., et ah, "The Bone Morphogenetic Protein 1/Tolloid-like Metalloproteinases," Matrix Biol. 2007 Sep; 26(7): 508-523; the contents of which are hereby incorporated by reference. In some embodiments, the tolloid protease site comprises the amino acid sequence QRD. In some embodiments, a tolloid protease site comprising the amino acid sequence QRD is cleaved between the R and the D residue by a tolloid protease. In some embodiments, the tolloid protease site comprises the amino acid sequence QGD. In some embodiments, a tolloid protease site comprising the amino acid sequence QGD is cleaved between the G and the D residue by a tolloid protease. Examples of tolloid protease sites for human and mouse myostatin and GDF11 are shown below, in bold, in SEQ ID NOs: 1-4.

Examples of tolloid proteases for use in accordance with the present disclosure include, without limitation BMP-1, mTLD, mTLL-1 and mTLL-2, as well as any isoforms thereof. A tolloid protease may be obtained from any mammal including, without limitation, humans or rodents {e.g., mice, rats, hamsters).

In some embodiments, a tolloid protease is homologous to a tolloid protease selected from the group consisting of: BMP-1, mTLD, mTLL-1 and mTLL-2. For example a tolloid protease may be at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least about 99.9% identical to BMP-1, mTLD, mTLL-1 and mTLL-2.

Aspects of the disclosure relate to a substantially pure preparation of a primed recombinant transforming growth factor (rTGF). As used herein, the term "substantially pure preparation of a primed recombinant rTGF" refers a preparation containing a primed rTGF in which the relative amount of the primed rTGF in the preparation is greater than that of another form (e.g., a pro- or latent- form) of the rTGF in the preparation. In some embodiments, a substantially pure preparation of a primed rTGF contains at least 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, or 99.5% primed rTGF relative to a pro- rTGF form or a latent-rTGF form of the rTGF or mature form of the rTGF. In some

embodiments, a substantially pure preparation of a primed rTGF has a relatively low amount of one or more impurities (e.g., other rTGFs). In some embodiments, the substantially pure preparation of a primed rTGF is at least 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, or 99.5% pure.

In some embodiments, primed rTGF is prepared by contacting an rTGF with a proprotein convertase to produce a latent rTGF; contacting the latent rTGF with a tolloid protease to produce primed rTGF; and purifying the primed rTGF, thus producing a substantially pure primed rTGF. Purifying the primed rTGF can be performed using methods provided herein, for example. In some embodiments, the primed rTGF is purified by affinity purification methods, for example, using an antibody to an epitope tag that is bound to the primed rTGF or by using an antibody that specifically bonds the primed rTGF.

An antibody that "specifically binds" to a target antigen (e.g., a primed rTGF), binds to the target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to non-target antigens (e.g., a pro rTGF and/or a latent rTGF). In some embodiments, antibodies are disclosed herein that specifically bind primed rTGF. In some embodiments, antibodies are disclosed herein that specifically bind primed GDF8 or primed GDF11. To prepare a primed rTGF, an rTGF may be contacted with any of the proprotein convertases provided herein. In some embodiments, an rTGF is contacted with a furin. To prepare a primed rTGF, an rTGF may be contacted with any of the tolloid proteases provided herein. In some embodiments, an rTGF is contacted with a mT112 protease.

The preparation of a substantially pure primed rTGF may further include producing the rTGF in vitro in one or more engineered cells that express the rTGF. In some embodiments, the rTGF is produced in one or more engineered cells that inducibly express the rTGF. In some embodiments, the one or more engineered cells secrete the rTGF into a cell culture media comprising a proprotein convertase and/or a tolloid protease. In some embodiments, the one or more engineered cells further express a proprotein convertase and/or a tolloid protease. The engineered cells, in some embodiments, may be co-cultured with cells that express a proprotein convertase and/or a tolloid protease.

An "engineered cell," as used herein, refers to a cell that does not occur in nature.

Engineered cells of the present disclosure, in some embodiments, contain one or more exogenous nucleic acids (i.e., nucleic acids that the cell would not normally contain) or nucleic acids that do not occur in nature (e.g., an engineered nucleic acid encoding a rTGF).

Accordingly, an engineered cell can be a cell that has been designed, produced, prepared, synthesized, manufactured and/or manipulated by a human.

An "engineered nucleic acid," as used herein, is a nucleic acid that does not occur in nature. It should be understood, however, that while an engineered nucleic acid as a whole is not naturally-occurring, it may include nucleotide sequences that occur in nature. In some embodiments, an engineered nucleic acid comprises nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, an engineered nucleic acid includes a bacterial nucleotide sequence, a murine nucleotide sequence, a human nucleotide sequence, and/or a viral nucleotide sequence. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. A "recombinant nucleic acid" is a molecule that is constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids or a combination thereof) and, in some embodiments, can replicate in a living cell. A "synthetic nucleic acid" is a molecule that is amplified in vitro or chemically synthesized (e.g., using a nucleic acid automated synthesizer). A synthetic nucleic acid includes nucleic acids that are chemically modified, or otherwise modified, but can base pair with naturally- occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include nucleic acids that result from the replication of either of the foregoing.

The preparation of a substantially pure primed rTGF may be in a solution, or may be in a lyophilized form. In some embodiments, the primed rTGF is in a solution at a concentration from 1 mg/ml to 1000 mg/ml. For example the primed rTGF can be in a solution at a concentration from 1 mg/ml to 10 mg/ml, from 1 mg/ml to 50 mg/ml, from 1 mg/ml to 100 mg/ml, from 1 mg/ml to 200 mg/ml, from 1 mg/ml to 500 mg/ml, from 1 mg/ml to 600 mg/ml, from 1 mg/ml to 800 mg/ml, from 10 mg/ml to 50mg/mL, from 10 mg/ml to 100 mg/ml, from 10 mg/ml to 200 mg/ml, from 10 mg/ml to 500 mg/ml, from 10 mg/ml to 600 mg/ml, from 10 mg/ml to 800 mg/ml, from 100 mg/ml to 200 mg/ml, from 100 mg/ml to 500 mg/ml, from 100 mg/ml to 600 mg/ml, from 100 mg/ml to 800 mg/ml, from 200 mg/ml to 500 mg/ml, from 200 mg/ml to 600 mg/ml, from 200 mg/ml to 800 mg/ml, from 800 mg/ml to 1000 mg/ml, from 500 mg/ml to 800 mg/ml, from 500 mg/ml to 1000 mg/ml, from 600 mg/ml to 800 mg/ml, or from 600 mg/ml to 1000 mg/ml. As one example, the primed rTGF is in a solution at a concentration of greater than 100 mg/ml. In some embodiments, the solution comprises a buffer. In some embodiments, the primed rTGF is at least 90% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) pure. In some embodiments, the primed rTGF is at least 95% pure.

Modulating transforming growth factor receptors in cells

Aspects of the disclosure relate to methods for modulating transforming growth factor (TGF) receptors in cells. In some embodiments, methods for modulating TGF receptors in cells include delivering an effective amount of any of the preparations or compositions (e.g., a substantially pure preparation of a primed recombinant transforming growth factor) or antibodies specific for a primed rTGF provided herein to a medium comprising cells expressing the TGF receptors. In some embodiments, the cells are in vitro. In other embodiments, the cells are in in vivo. As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., an animal). As used herein, the term "in vivo" refers to events that occur within an organism (e.g., an animal). Methods for producing primed recombinant rTGF

Aspects of the disclosure relate to methods for producing a primed recombinant transforming growth factor (rTGF). In some embodiments, the methods include expressing a rTGF in a cell; contacting the rTGF with a proprotein convertase to produce a latent rTGF; contacting the latent rTGF with a tolloid protease to produce the primed rTGF; and isolating the primed rTGF. As used herein, the term "isolated" refers to a substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated substances are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. In some embodiments, the proprotein convertase is a furin protease and/or the tolloid protease is a mTl 12 protease. In some embodiments, the

recombinant rTGF is contacted with the proprotein convertase and/or the tolloid protease in vitro. In some embodiments, the rTGF is contacted with the proprotein convertase and/or the tolloid protease in a cell culture media. In some embodiments, the cell culture media is a conditioned media comprising a proprotein convertase and/or a tolloid protease. In some embodiments, the rTGF and/or the proprotein convertase and/or the tolloid protease is expressed inducibly in a cell. In some embodiments, the cell culture media comprises one or more engineered cells that express a proprotein convertase and/or a tolloid protease.

Aspects of the disclosure relate to methods for obtaining an antibody that binds to a primed transforming growth factor. In some embodiments, the method includes contacting a substantially pure preparation of the primed transforming growth factor with a plurality of candidate antibodies or antigen binding fragments thereof; selecting candidate antibodies or antigen binding fragments that bind to the primed transforming growth factor; contacting the selected antibodies or antigen binding fragments with a transforming growth factor that is not primed; eliminating the antibodies or antigen binding fragments that bind to the growth factor that is not primed; and selecting the antibodies or antigen binding fragments that bind to the primed transforming growth factor and do not bind to the growth factor that is not primed. In some embodiments, the transforming growth factor is GDF8 or GDFl 1. In some embodiments, the antibodies or antigen binding fragments are eliminated if they bind to the growth factor that is not primed with an affinity greater than or equal to the affinity that they bind to the growth factor that is primed. In some embodiments, the transforming growth factor that is not primed is the pro form of the transforming growth factor. In some embodiments, the transforming growth factor that is not primed is the latent form of the transforming growth factor. Aspects of the disclosure relate to antibodies obtained by any of the methods provided herein. In some embodiments, the antibody is bound to a primed transforming growth factor. In some embodiments, the primed transforming growth factor is recombinant. In some

embodiments, the transforming growth factor is GDF8 or GDF11. In some embodiments, the antibody selectively binds a primed transforming growth factor. In some embodiments, the antibody inhibits the primed transforming growth factor.

Aspects of the disclosure relate to primed recombinant transforming growth factor (rTGF) comprising an epitope tag. In some embodiments, the epitope tag is on the C-terminus of the rTGF. In some embodiments, the epitope tag is between the C-terminus and the N- terminus. In some embodiments, the recombinant transforming growth factor is GDF8 or GDF11.

Aspects of the disclosure provide nucleic acids comprising a sequence encoding a recombinant transforming growth factor and an epitope tag. In some embodiments, the sequence encoding the epitope tag is 5' to the sequence encoding the recombinant transforming growth factor. In some embodiments, the sequence encoding the epitope tag is 3' to the sequence encoding the recombinant transforming growth factor. In some embodiments, the nucleic acid is in a vector. In some embodiments, the nucleic acid further comprises one or more heterologous promoters. In some embodiments, the nucleic acid further comprises a sequence encoding a proprotein convertase and/or a tolloid protease.

Aspects of the disclosure relate to an engineered cell comprising any of the nucleic acids provided herein. In some embodiments, the engineered cell further comprises a nucleic acid encoding a proprotein convertase and/or a tolloid protease.

Aspects of the disclosure relate to a conditioned medium, comprising a proprotein convertase and/or a tolloid protease. In some embodiments, the proprotein convertase comprises furin and/or the tolloid protease comprises mT112. In some embodiments, the medium further comprises a transforming growth factor. In some embodiments, the transforming growth factor is recombinant. In some embodiments, the transforming growth factor comprises primed transforming growth factor.

Aspects of the disclosure relate to a cell culture system comprising one or more of the engineered cells provided herein. In some embodiments, the cell culture system further comprises any of the conditioned media provided herein. Pharmaceutical Compositions

pharmaceutically acceptable carrier. The pharmaceutical compositions, in some embodiments, are for use in decreasing muscle mass, improving blood vessel volume, improving neurogenesis, reversing age-related cardiac hypertrophy or improving skeletal muscle function. In some embodiments, the pharmaceutical compositions provided herein contain an amount of primed rTGF effective for decreasing muscle mass, improving blood vessel volume, improving neurogenesis, reversing age-related cardiac hypertrophy or improving skeletal muscle function.

Aspects of the disclosure relate to a pharmaceutical composition having a therapeutically effective amount of any of the antibodies provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is for use in preventing muscle wasting or increasing muscle mass. In some embodiments, the antibody selectively binds myostatin (GDF8). In some embodiments, the antibody inhibits myostatin (GDF8).

One or more of the substantially pure compositions of primed rTGF and/or one or more of the antibodies that specifically binds a primed rTGF can be mixed with a pharmaceutically acceptable carrier (excipient), including buffer, to form a pharmaceutical composition for use in alleviating a disease or disorder that is associated with myopathy. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.

Pharmaceutically acceptable excipients (carriers) including appropriate buffers. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. In one example, a pharmaceutical composition described herein contains one or more primed rTGF or one or more anti-primed rTGF antibodies. In some embodiments, the more than one anti-primed rTGF antibodies recognize different

epitopes/residues of the primed rTGF.

The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

In some examples, the pharmaceutical composition described herein comprises liposomes containing one or more of the substantially pure compositions of primed rTGF and/or one or more of the antibodies that specifically binds a primed rTGF, which can be prepared by appropriate methods, Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.

Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized

phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

One or more of the substantially pure compositions of primed rTGF and/or one or more of the antibodies that specifically binds a primed rTGF may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. In other examples, the pharmaceutical composition described herein can be formulated in sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(v nylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administration must be sterile.

This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally 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.

The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present disclosure, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 mg to about 500 mg of the active ingredient of the present disclosure. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate. Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g. TweenTM 20, 40, 60, 80 or 85) and other sorbitans (e.g.

SpanTM 20, 40, 60, 80 or 85). Compositions with a surface- active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%.

The emulsion compositions can be those prepared by mixing an anti-proMyostatin antibody with IntralipidTM or the components thereof (soybean oil, egg phospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are

administered by the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

Treating disorders associated with altered transforming growth factor receptor activity Aspects of the disclosure relate to methods for treating a subject having a condition associated with reduced transforming growth factor receptor activity. In some embodiments, the method includes administering any of the pharmaceutical compositions provided herein to the subject. In some embodiments, the method includes administering a preparation comprising a primed rTGF(e.g., primed GDF11). In some embodiments, the condition is age-related cardiac hypertrophy. In some embodiments, the condition includes decreased blood vessel volume. In some embodiments, the condition includes decreased neurogenesis. In some embodiments, the condition includes decreased skeletal muscle function. In some embodiments, the

pharmaceutical composition comprises a primed rTGF. In some embodiments, the

pharmaceutical composition comprises a primed GDF11 or a protein homologous to a primed GDF11.

Aspects of the disclosure relate to methods for treating a subject having a condition associated with increased transforming growth factor receptor activity. In some embodiments, the method includes administering any of the antibodies that specifically bind a primed rTGF, provided herein to the subject. In some embodiments, the antibodies that specifically bind a primed rTGF inhibit the transforming growth factor. In some embodiments, the condition is myopathy.

As used herein, the term "myopathy" refers to a muscular disease in which the muscle fibers do not function properly, typically resulting in muscular weakness. Myopathies include muscular diseases that are neuromuscular or musculoskeletal in nature. In some embodiments, the myopathy is an inherited myopathy. Inherited myopathies include, without limitation, dystrophies, myotonias, congenital myopathies (e.g.,nemaline myopathy, multi/minicore myopathy, and centronuclear myopathy), mitochondrial myopathies, familial periodic myopathies, inflammatory myopathies and metabolic myopathies (e.g., glycogen storage diseases and lipid storage disorder). In some embodiments, the myopathy is an acquired myopathy. Acquired myopathies include, without limitation, external substance induced myopathy (e.g., drug-induced myopathy and glucocorticoid myopathy, alcoholic myopathy, and myopathy due to other toxic agents), myositis (e.g.,dermatomyositis, polymositis and inclusion body myositis), myositis ossificans, rhabdomyolysis, myoglobinurias, and disuse atrophy. In some embodiments, the myopathy is disuse atrophy, which may be caused by bone fracture (e.g. a hip fracture) or by nerve injury (e.g., spinal cord injury (SCI)). In some embodiments the myopathy is related to a disease or disorder such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA),cachexia syndromes due to renal failure, AIDS, cardiac conditions and/or cancer. In some embodiments the myopathy is related to ageing.

As used herein, the term "subject" refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). In some embodiments, the subject is a patient. In some embodiments, the subject is a healthy volunteer.

To practice the method disclosed herein, an effective amount of any of the

pharmaceutical compositions described above can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal,

intracerebrospinal, subcutaneous, intra- articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, an anti-primed rTGF antibody or preparation of primed rTGF can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a disease/disorder associated with an increase or decrease in transforming growth factor receptor activity, such as those noted above. A subject having disease or disorder associated with an increase or decrease in transforming growth factor receptor activity can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds. A subject suspected of having any of such disease/disorder might show one or more symptoms of the disease/disorder. A subject at risk for the disease/disorder can be a subject having one or more of the risk factors for that disease/disorder.

"An effective amount" as used herein refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

Empirical considerations, such as the half-life, generally will contribute to the

determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a disease/disorder associated with myopathy. Alternatively, sustained continuous release formulations may be appropriate.

In one example, dosages for an anti-primed rTGF antibody as described herein may be determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of the antagonist. To assess efficacy of the antagonist, an indicator of the disease/disorder can be followed. In another example, dosages for a substantially pure preparation of a primed rTGF as described herein may be determined empirically in individuals who have been given one or more administration(s) of the preparation. Individuals are given incremental dosages of the agonist. To assess efficacy of the agonist, an indicator of the disease/disorder can be followed.

Generally, for administration of any of the pharmaceutical compositions described herein, an initial candidate dosage can be about 2 mg/kg. For the purpose of the present disclosure, a typical daily dosage might range from about any of 0.1 g/kg to 3 g/kg to 30 μg/kg to 300 g/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a disease or disorder associated with transforming growth receptor activity, or a symptom thereof. An exemplary dosing regimen comprises administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the antibody, or followed by a maintenance dose of about 1 mg/kg every other week. However, other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. For example, dosing from one- four times a week is contemplated. In some embodiments, dosing ranging from about 3 g/mg to about 2 mg/kg (such as about 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg, about 300 μg/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) can vary over time.

In some embodiments, for an adult patient of normal weight, doses ranging from about 0.3 to 5.00 mg/kg may be administered. The particular dosage regimen, e.g., dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent, and other considerations).

For the purpose of the present disclosure, the appropriate dosage of an anti-primed rTGF antibody or preparation of primed rTGF will depend on the specific antibody or preparation (or compositions thereof) employed, the type and severity of the disease/disorder, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antagonist, and the discretion of the attending physician. In some embodiments, a clinician will administer an anti-primed rTGF antibody or preparation of primed rTGF, until a dosage is reached that achieves the desired result. Administration of an anti-primed rTGF antibody or preparation of primed rTGF can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors. The administration of an anti- primed rTGF antibody or preparation of primed rTGF may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g. , either before, during, or after developing a disease or disorder associated with increased or decreased transforming growth factor receptor activity. As used herein, the term "treating" refers to the application or administration of a composition including one or more active agents to a subject, who has a disease/disorder associated with altered transforming growth factor receptor activity (e.g., myopathy), a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease/disorder.

Alleviating a disease/disorder associated with an increase or decrease in transforming growth factor receptor activity includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a disease/disorder associated with an increase or decrease in transforming growth factor receptor activity means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

"Development" or "progression" of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using suitable clinical techniques. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. "Development" includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of a disease/disorder associated with myopathy includes initial onset and/or recurrence.

In some embodiments, conventional methods can be used to administer the

pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g. , administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial,

intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

Injectable compositions may contain various carriers such as vegetable oils,

dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer' s solution or other suitable excipients. Intramuscular preparations, e.g. , a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for- Injection, 0.9% saline, or 5% glucose solution.

In one embodiment, an anti-primed rTGF antibody or preparation of primed rTGF is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the anti-primed rTGF antibody or preparation of primed rTGF or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g. , PCT Publication No. WO 00/53211 and U.S. Pat. No. 5,981,568.

Targeted delivery of therapeutic compositions containing a polynucleotide, or expression vector can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11 :202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621 ; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

Therapeutic compositions containing a polynucleotide (e.g. , those encoding the anti- primed rTGF antibody or rTGF) are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA or more can also be used during a gene therapy protocol. The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1 :51 ; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1 : 185; and Kaplitt, Nature Genetics (1994) 6: 148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of the coding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide (e.g., encoding any polypeptide disclosed herein) and expression in a desired cell may be used in some

embodiments. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g. , PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and

4,777, 127; GB Patent No. 2,200,651 ; and EP Patent No. 0 345 242), alphavirus-based vectors (e.g. , Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR- 1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR- 923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g. , PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191 ; WO 94/28938; WO 95/11984 and WO 95/00655).

Non-viral delivery vehicles and methods can also be employed. For example, in some embodiments, naked DNA can be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422, 120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91 : 1581.

The particular dosage regimen, e.g. , dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history.

Each of the limitations of the disclosure can encompass various embodiments of the disclosure. It is, therefore, anticipated that each of the limitations of the disclosure involving any one element or combinations of elements can be included in each aspect of the disclosure. This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. EXAMPLES

Example 1. Methods and Materials

Production of primed forms of Myostatin and GDF11

To produce precursor forms of myostatin {i.e., GDF8) and GDF11, stable cell lines overexpressing proMyostatin (murine and human), proGDFl 1 (murine and human), furin, BMP-1, PCSK5 and T112 were established. Protein constructs were stabiv integrated into FLP-IN™ T-REX™ 293 ceils (Life Technologies, Carlsbad, CA). Ceil lines were adapted to suspension growth in F17 media (Life Technologies, Carlsbad, CA) and were expressed according to manufacturer's instructions. A substantially pure proGDF8 sample was generated by expressing proGDF8 in the presence of 30 μΜ Decanoyl RVKR-CMK. Filtered supernatant was collected and the protein of interest purified by Ni-NTA chromatography (Qiagen). The protein was further purified by size exclusion chromatography (SEC). An additional FLAG resin purification step was applied as needed for removal of the Flag-tagged proteases from the latent and primed preparations of GDF8. ProMyostatin and proGDFl 1 were purified directly from cell culture supernatants.

Latent myostatin was produced via in-vitro cleavage of purified proMyostatin by furin protease which was expressed in cells and purified. Latent GDF11 was produced by adding stable cells overexpressing PCSK5 (a proprotein convertase) to the GDF11 expressing stable cells and purifying latent GDF11 from the cell supernatants. In both cases, the material was >95% latent, with the proteolysis reaction proceeding almost to completion under the conditions used. Primed myostatin was produced by in-vitro cleavage of proMyostatin utilizing conditioned media from mT112-overexpresing cells and purified furin protease. Primed myostatin was also generated by in vitro cleavage of latent Myostatin with

recombinant BMP-1 (R&D systems) (with a 30: 1 mass ratio of latent myostatin:BMP-l in the reaction mixture). All cleavage reactions occurred at 30 C for 16-24 hours, in some cases extra protease was added to the reaction mixture after 6-8 hours. Primed GDF11 was produced via co-culture of stable cell lines overexpressing proGDFl l, BMP-1, and PCSK5 as described above. SDS-PAGE analysis of purified myostatin and GDF11 forms produced by these methods are shown in FIGs. 3A-3D.

Primed GDFll and GDFS form, stable complexes in vitro The primed forms of GDFS and GDF11 remained associated as a non-covalent complex following purification by Ni-NTA and gel filtration. Fig5 shows an SDS-PAGE analysis of murine primed GDFl l which is partially cleaved by tolloid proteases and separated by size exclusion chromatography. This partially primed GDFl l ran on a SRT!O size exclusion chromatography (SEC) column in 20mM HEPES (pH8; 500mM NaCl; 5mM EDTA) buffer and showed separation of the primed GDF11 species, latent GDF11, and dissociated complex.

The purified forms of GDFS and GDF11 were analyzed for oligomerization and association between the growth factor domain and prodomain with multiangle light scattering coupled to gel filtration (SEC-MALS). FIGs. 4A-D shows the results of these experiments. Molecular weights and elution volumes of murine proGDFl 1, latent GDF11 and primed

GDFl l were analyzed on a superose 6 column pre -equilibrated in 20 mM Hepes pH 7.5, 500 niM NaCl. Molecular weights and elution volumes of human proGDFS, latent GDFS and primed GDFS were analyzed on a s200 column pre-equilibrated in 20 mM Hepes pH 7.5, 150 mM NaCl. For each set of data, the molecular weight (MW) and level of glycosylation is established and the data is analyzed as described in (Folta-Stogniew E. Methods Mol Biol. 2006;328:97-112).

The pro- and latent- conformations of GDFl l are indistinguishable based on SEC elution and calculated molecular mass. The primed version of GDF11 elutes earlier from SEC (FIG. 4A), with a similar molecular weight, indicating a potential conformational change of the protein. At lower concentrat ons of protein, primed GDF11 shows e vidence of dissociation (FIG. 4C), indicative of a weakened interaction between the growth factor domain and prodomain fragments. The pro- and latent- forms of GDF8 are indistinguishable based on SEC elution and calculated molecular mass (FIG. 4B). The primed version of GDFS elutes later from SEC, with a lower molecular weight, indicating a dissociation of the complex. This dissociation is also evident at lower concentrations of primed GDFS (FIG. 4D). Reporter cell assays

Samples of proMyostatin, latent myostatin, primed myostatin, latent GDFl l, proGDFl l or primed GDF11 were incubated with 293T cells containing a stably integrated pGL4 plasmid (Promega, Madison, WI) with a promoter comprising SMAD- responsive CAGA sequences. Cells were incubated at 37°C for 6 hours before detection of luciferase expression using

BRIGHT-GLO™ reagent (Promega, Madison, WI) according to manufacturer's instructions. The primed forms of myostatin and GDF11 signaled as well, or even better, than recombinant mature GDF8 and GDFl l (FIGs. 6A-6B). Uses for primed GDF8 and GDFll

In some embodiments, primed GDF8 and GDFl l are used as antigens in an inhibitory campaign to selectively block signaling of GDF8 or GDF11. In other embodiments, primed GDF8 or primed GDF11 is used to establish the specificity of an antibody to primed GDF8 or primed GDF11 as compared to the pro-form or the latent-form of GDF8 or GDF11. Primed GDF11 can be administered to aged patients to reverse age-related cardiac hypertrophy, improve blood vessel volume, improve neurogenesis, or improve skeletal muscle function. Primed forms of GDF8 and GDF11 can be manufactured to improve the stability and also the yield of growth factor preparations for research, or to manipulate the differentiation and proliferation of stem cells.

SEQUENCES

In the following sequences, tolloid cleavage sites are indicated in brackets only, proprotein convertase cleavage sites are indicated in brackets and underlining, modified human Ig kappa chain (V-I region) signal peptides are italicized only, His-tags are underlined only, Flag-tags are in brackets and italicized, and 3C Protease cleavage sites are underlined and italicized.

The amino acid sequence set forth in SEQ ID NO: 1 provides a non-limiting example of a recombinant human myostatin.

>FL His wt myostatin, human

J> ^y A( LLGLLLLW5GyLGHHHHHHNENSEQKENVEKEGLCNACTWRQNTKSSRI EAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDV[QRD]DSSDGSLEDDDYHA TTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQI LRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDE NGHDLAVTFPGPGEDGLNPFLEVKVTDTPKrRSRRlDFGLDCDEHSTESRCCRYPLTVDF EAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPI NMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 1)

The amino acid sequence set forth in SEQ ID NO: 2 provides a non-limiting example of a recombinant murine myostatin.

>Flag-His FL myostatin, Mouse

MDMRVPAOLLGLLLLWFSGVLGfDYKDDDDKi i i iLEVLFOGPNEGSEREEN

VEKEGLCNACAWRQNTRYSRIEAIKIQILSKLRLETAPNISKDAIRQLLPRAPPLR

ELIDQYDV[QRD]DSSDGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFKF

SSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMS

PGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPF

LEVKVTDTPKrRSRRlDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYK

ANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQII

YGKIPAMVVDRCGCS (SEQ ID NO: 2)

The amino acid sequence set forth in SEQ ID NO: 3 provides a non-limiting example of a recombinant human GDF11.

>Flag-His human GDF11 FL

MDMRVPAOLLGLLLLWFSGVLGfDYKDDDDKimimimiLEVLFOGPAEGPAAAAAAAA A A A A AG VGGERS S RPAPS V APEPDGCP VC VWRQHS RELRLES IKS QILS KLRLKE APNIS REVVKQLLPKAPPLQQILDLHDF[QGD]ALQPEDFLEEDEYHATTETVISMAQETDPAVQ TDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGG GGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVT SLGPGAEGLHPFMELRVLENTKrRSRRlNLGLDCDEHSSESRCCRYPLTVDFEAFGWDW IIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFND KQQIIYGKIPGMVVDRCGCS (SEQ ID NO: 3)

The amino acid sequence set forth in SEQ ID NO: 4 provides a non-limiting example of a recombinant murine GDF11. >Flag-His FL GDF11, Mouse

MDMRVPAOLLGLLLLWFSGVLGfDYKDDDDKimimimiLEVLFOGPAEGPAAAA AAAAAAAGVGGERSSRPAPSAPPEPDGCPVCVWRQHSRELRLESIKSQILSKLRL KEAPNISREVVKQLLPKAPPLQQILDLHDF[QGD]ALQPEDFLEEDEYHATTETVI SMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQ ILRLKPLTGEGT AGGGGGGRRHIRIRS LKIELHSRS GHWQS IDFKQVLHS WFRQP QSNWGffilNAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKrRSRRlNLGLDC DEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTH LVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS (SEQ ID NO: 4)

The amino acid sequence set forth in SEQ ID NO: 5 provides a non-limiting example of the tolloid protease BMP1.

>BMP1 C-Flag His

MPGVARLPLLLGLLLLPRPGRPLDLADYTYDLAEEDDSEPLNYKDPCKAAAFLG

DIALDEEDLRAFQVQQAVDLRRHTARKSSIKAAVPGNTSTPSCQSTNGQPQRGA

CGRWRGRSRSRRAATSRPERVWPDGVIPFVIGGNFTGSQRAVFRQAMRHWEKH

TC VTFLERTDEDS YIVFT YRPC GCC S Y VGRRGGGPQ AIS IGKNCDKFGIV VHELG

HVVGFWHEHTRPDRDRHVSIVRENIQPGQEYNFLKMEPQEVESLGETYDFDSIM

HYARNTFSRGIFLDTIVPKYEVNGVKPPIGQRTRLSKGDIAQARKLYKCPACGET

LQDS TGNFS S PE YPNG YS AHMHC VWRIS VTPGEKIILNFTS LDLYRS RLC W YD Y V

E VRDGFWRKAPLRGRFC GS KLPEPIVS TDS RLW VEFRS S S NW VGKGFFA V YE AI

CGGDVKKDYGHIQSPNYPDDYRPSKVCIWRIQVSEGFHVGLTFQSFEIERHDSCA

YDYLEVRDGHSESSTLIGRYCGYEKPDDIKSTSSRLWLKFVSDGSINKAGFAVNF

FKEVDECSRPNRGGCEQRCLNTLGSYKCSCDPGYELAPDKRRCEAACGGFLTKL

NGSITSPGWPKEYPPNKNCrWQLVAPTQYRISLQFDFFETEGNDVCKYDFVEVRS

GLTADSKLHGKFCGSEKPEVITSQYNNMRVEFKSDNTVSKKGFKAHFFSEKRPA

LQPPRGRPHQLKFRVQKRNRTPQLEyL QG HHHHHH £> YKDDDDK] (SEQ ID

NO: 5)

The amino acid sequence set forth in SEQ ID NO: 6 provides a non-limiting example of the tolloid protease mTLL2. >mTLL2 C-Flag His

MPRATALGALVSLLLLLPLPRGAGGLGERPDATADYSELDGEEGTEQQLEHYHD

PCKAAVFWGDIALDEDDLKLFHIDKARDWTKQTVGATGHSTGGLEEQASESSP

DTTAMDTGTKEAGKDGRENTTLLHSPGTLHAAAKTFSPRVRRATTSRTERIWPG

GVIPYVIGGNFTGSQRAIFKQAMRHWEKHTCVTFIERTDEESFIVFSYRTCGCCSY

VGRRGGGPQAISIGKNCDKFGIVAHELGHVVGFWHEHTRPDRDQHVTIIRENIQP

GQE YNFLKME AGE VS S LGET YDFDS IMH Y ARNTFS RG VFLDTILPRQDDNG VRP

TIGQRVRLSQGDIAQARKLYKCPACGETLQDTTGNFSAPGFPNGYPSYSHCVWR

ISVTPGEKIVLNFTSMDLFKSRLCWYDYVEVRDGYWRKAPLLGRFCGDKIPEPL

VSTDSRLWVEFRSSSNILGKGFFAAYEATCGGDMNKDAGQIQSPNYPDDYRPSK

ECVWRITVSEGFHVGLTFQAFEIERHDSCAYDYLEVRDGPTEESALIGHFCGYEK

PEDVKSSSNRLWMKFVSDGSINKAGFAANFFKEVDECSWPDHGGCEHRCVNTL

GSYKCACDPGYELAADKKMCEVACGGFITKLNGTITSPGWPKEYPTNKNCVWQ

V V AP AQ YRIS LQFE VFELEGND VC KYDF VE VRS GLS PD AKLHGRFC GS ETPE VIT

S QS NNMR VEFKS DNT VS KRGFRAHFFS DKDEC AKDNGGC QHEC VNTFGS YLCR

CRNG YWLHENGHDC KE AGC AHKIS S VEGTLAS PNWPDKYPS RRECTWNIS S T A

GHRVKLTFNEFEIEQHQECAYDHLEMYDGPDSLAPILGRFCGSKKPDPTVASGSS

MFLRFYS DAS VQRKGFQ A VHS TEC GGRLK AE VQTKELYS H AQFGDNN YPS EAR

CDWVIVAEDGYGVELTFRTFEVEEEADCGYDYMEAYDGYDSSAPRLGRFCGSG

PLEEIYSAGDSLMIRFRTDDTINKKGFHARYTSTKFQDALHMKKLEyL QG HHH

H HfDYKDDDDK] (SEQ ID NO: 6)

The amino acid sequence set forth in SEQ ID NO: 7 provides a non-limiting example of the proprotein convertase furin.

>Furin 1-595 C-Flag-His

MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKH

GFLNLGQIFGDYYHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAKRRT

KRDVYQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEK

NHPDLAGNYDPGASFDVNDQDPDPQPRYTQMNDNRHGTRCAGEVAAVANNG

VCGVGVAYNARIGGVRMLDGEVTDAVEARSLGLNPNHIHIYSASWGPEDDGKT

VDGPARLAEEAFFRGVSQGRGGLGSIFVWASGNGGREHDSCNCDGYTNSIYTLS

IS S ATQFGN VPW YS E AC S S TLATT YS S GNQNEKQIVTTDLRQKCTES HTGTS ASA PLAAGIIALTLEANKNLTWRDMQHLVVQTSKPAHLNANDWATNGVGRKVSHS

YGYGLLDAGAMVALAQNWTTVAPQRKCIIDILTEPKDIGKRLEVRKTVTACLGE PNHITRLEHAQARLTLSYNRRGDLAIHLVSPMGTRSTLLAARPHDYSADGFNDW AFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVLYGTAPEGLPVPPESS GCKTLTS S OALEVLFOGPHHHH HiDYKDDDDKl (SEQ ID NO: 7)

The amino acid sequence set forth in SEQ ID NO: 8 provides a non-limiting

example of proprotein convertase PCSK5.

>PCSK5 secreted isoform C-Flag-His

MGWGSRCCCPGRLDLLCVLALLGGCLLPVCRTRVYTNHWAVKIAGGFPEANRI

AS KYGFINIGQIG ALKD Y YHF YHS RTIKRS VIS S RGTHS FIS MEPKVE WIQQQ V VK

KRTKRD YDFS RAQS T YFNDPKWPS M W YMHC S DNTHPC QS DMNIEG A WKRG YT

GKNIVVTILDDGIERTHPDLMQNYDALASCDVNGNDLDPMPRYDASNENKHGT

RCAGEVAAAANNSHCTVGIAFNAKIGGVRMLDGDVTDMVEAKSVSFNPQHVHI

YSASWGPDDDGKTVDGPAPLTRQAFENGVRMGRRGLGSVFVWASGNGGRSKD

HCSCDGYTNSIYTISISSTAESGKKPWYLEECSSTLATTYSSGESYDKKIITTDLRQ

RCTDNHTGTSASAPMAAGIIALALEANPFLTWRDVQHVIVRTSRAGHLNANDW

KTNAAGFKVSHLYGFGLMDAEAMVMEAEKWTTVPRQHVCVESTDRQIKTIRPN

S A VRS IYKAS GC S DNPNRH VN YLEH V V VRITITHPRRGDLAIYLTS PS GTRS QLLA

NRLFDHS MEGFKNWEFMTIHC WGERA AGD W VLE V YDTPS QLRNFKTPGKLKE

WS LVLYGTS VQP YS PTNEFPKVERFR YS RVEDPTDD YGTED Y AGPCDPECS E VG

CDGPGPDHCNDCLHYYYKLKNNTRICVSSCPPGHYHADKKRCRKCAPNCESCF

GSHGDQCMSCKYGYFLNEETNSCVTHCPDGSYQDTKKNLCRKCSENCKTCTEF

HNCTECRDGLSLQGSRCSVSCEDGRYFNGQDCQPCHRFCATCAGAGADGCINC

TEGYFMEDGRCVQSCSISYYFDHSSENGYKSCKKCDISCLTCNGPGFKNCTSCPS

GYLLDLGMCQMGAICKDATEESWAEGGFCMLVKKNNLCQRKVLQQLCCKTCT

FQGLEVLFOGPHHHHHHfD YKDDDDK1 (SEQ ID NO: 8)

Table 2. Samples sent for SEC-MALS (size exclusion chromatography with inline multi-angle light scattering). Data shows that the calculated protein weights of proGDFl 1, latent GDFl 1 and tolloid cleaved GDFl 1 are equal. Calculation of the theoretical monomer was 45.6 kDa and calculation of the theoretical dimer was 91.2 kDa. Species Identifier Mw polypeptide (kDa) grams of sugar per

gram of polypeptide

Pro Al 81 0.30

Pro A2 90 0.21

Pro A3 74 0.37

Average 81 0.29

StDev 8 0.08

Latent Bl 79 0.30

Latent B2 78 0.32

Latent B3 79 0.31

Average 79 0.31

StDev 0 0.01

Tolloid CI 82 0.42

Tolloid C2 74 0.33

Tolloid C3 75 0.39

Average* 77 0.38

StDev 5 0.05

Table 3. Samples sent for SEC-MALS (size exclusion chromatography with inline multi-angle light scattering). Data shows that the calculated protein weights of proGDF8, latent GDF8 and tolloid cleaved GDF8 are equal. Calculation of the theoretical monomer was 42.75 kDa and calculation of the theoretical dimer was 85.5 kDa.

Table 4. Murine Primed GDF11 Elicits Greater Response than Mature GDF11 (FIG. 8)

Table 5. Human Primed GDF8 Elicits similar Response as Mature GDF8 (FIG. 8)

Claims

CLAIMS What is claimed is:

1. A substantially pure preparation of a primed recombinant transforming growth factor (rTGF).

2. The preparation of claim 1, wherein the primed rTGF is prepared by a method comprising:

i. contacting an rTGF with a proprotein convertase to produce a latent rTGF; ii. contacting the latent rTGF with a tolloid protease to produce primed rTGF; and iii. substantially purifying the primed rTGF.

3. The preparation of claim 1, wherein the primed rTGF is prepared by a method comprising:

i. contacting an rTGF with a proprotein convertase to produce a latent rTGF; ii. contacting the latent rTGF with a tolloid protease to produce primed rTGF; and iii. substantially separating the proprotein convertase and tolloid protease from the primed rTGF.

4. The preparation of claim 2 or 3, wherein the proprotein convertase is a furin

5. The preparation of any one of claims 2 to 4, wherein the tolloid protease is a mT112 protease.

6. The preparation of any one of claims 2 to 5, wherein the preparation method further comprises:

iv. producing the rTGF in vitro in one or more engineered cells that express the rTGF.

7. The preparation of claim 6, wherein the one or more engineered cells inducibly express the rTGF.

8. The preparation of any one of claims 5 to 7, wherein the one or more engineered cells secrete the rTGF into a cell culture media comprising a proprotein convertase and/or a tolloid protease.

9. The preparation of any one of claims 5 to 8, wherein the one or more engineered cells further express a proprotein convertase and/or a tolloid protease.

10. The preparation of any one of claims 5 to 9, wherein the engineered cells are co- cultured with cells that express a proprotein convertase and/or a tolloid protease.

11. The preparation of any one of claims 1 to 10, wherein the primed rTGF is in a solution.

12. The preparation of any one of claims 1 to 10, wherein the primed rTGF is in a lyophilized form.

13. The preparation of any one of claims 5 to 11, wherein the primed rTGF is in solution at a concentration of greater than 1 mg/ml.

14. The preparation of any one of claims 1 tol3, wherein the primed rTGF is at least 90% pure.

15. The preparation of any one of claims 1 to 14, wherein the primed rTGF is at least 95% pure.

16. The preparation of any one of claims 1 to 15, wherein the primed rTGF comprises an epitope tag.

17. The preparation of claim 16, wherein the epitope tag is at the C-terminus of the rTGF.

18. The preparation of claim 14, wherein the epitope tag is between the C-terminus and the N-terminus of the rTGF.

19. The preparation of any one of claims 1-18, wherein the primed rTGF is a AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDFl, GDF10, GDFl l, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY 1, LEFTY2, MSTN, NODAL, NRTN, PSPN, TGFpl, TGFp2, or TGFp3 protein.

20. A pharmaceutical composition comprising (i) a therapeutically effective amount of the primed rTGF preparation of any one of claims 1-19, and (ii) a pharmaceutically acceptable carrier.

21. A pharmaceutical composition for use in decreasing muscle mass, improving blood vessel volume, improving neurogenesis, reversing age-related cardiac hypertrophy or improving skeletal muscle function comprising a therapeutically effective amount of the primed rTGF preparation of any one of claims 1-19.

22. A method for modulating transforming growth factor (TGF) receptors in cells, the method comprising

delivering an effective amount of the preparation of any one of claims 1-19 to a medium comprising cells expressing the TGF receptors.

23. The method of claim 22, wherein the cell is in vitro.

24. The method of claim 22, wherein the cell in in vivo.

25. A method for treating a subject having a condition associated with reduced transforming growth factor receptor activity, the method comprising

administering the pharmaceutical composition of claim 20 to the subject.

26. The method of claim 25, wherein the condition is age-related cardiac hypertrophy.

27. The method of claim 25, wherein the condition comprises decreased blood vessel volume.

28. The method of claim 25, wherein the condition comprises decreased

neurogenesis.

29. The method of claim 25, wherein the condition comprises decreased skeletal muscle function

30. A method for producing a primed recombinant transforming growth factor (rTGF), the method comprising

i. expressing a rTGF in a cell;

ii. contacting the rTGF with a proprotein convertase to produce a latent rTGF;

iii. contacting the latent rTGF with a tolloid protease to produce the primed rTGF; and

iv. isolating the primed rTGF.

31. The method of claim 30, wherein the proprotein convertase is a furin protease and/or the tolloid protease is a mTl 12 protease.

32. The method of claim 29 or 30, wherein the recombinant rTGF is contacted with the proprotein convertase and/or the tolloid protease in vitro.

33. The method of any one of claims 29-31, wherein the rTGF is contacted with the proprotein convertase and/or the tolloid protease in a cell culture media.

34. The method of claim 32, wherein the cell culture media is a conditioned media comprising a proprotein convertase and/or a tolloid protease.

35. The method of any one of claims 29-33, wherein the rTGF and/or the proprotein convertase and/or the tolloid protease is expressed inducibly in a cell.

36. The method of any one of claims 32-34, wherein the cell culture media comprises one or more engineered cells that express a proprotein convertase and/or a tolloid protease.

37. A solid substrate to which is linked a primed transforming growth factor.

38. The solid substrate of claim 36, wherein the primed transforming growth factor is non-covalently linked to the solid substrate.

39. The solid substrate of claim 36 or 37, wherein the primed transforming growth factor is linked to the substrate via an antibody.

40. The solid substrate of claim 38, wherein the antibody selectively binds the primed transforming growth factor.

41. The solid substrate of claim 38 or 39, wherein the antibody is the antibody of any one of claims 49-52.

42. The solid substrate of any one of claims 36-40, wherein the solid substrate comprises beads or a resin.

43. The solid substrate of any one of claims 36-41, wherein the solid substrate is a surface of an affinity column.

44. A method for obtaining an antibody that binds to a primed transforming growth factor, comprising

(i) contacting a substantially pure preparation of the primed transforming growth factor with a plurality of candidate antibodies or antigen binding fragments thereof; (ii) selecting candidate antibodies or antigen binding fragments that bind to the primed transforming growth factor;

(iii) contacting the antibodies or antigen binding fragments selected in (ii) with a transforming growth factor that is not primed;

(iv) eliminating the antibodies or antigen binding fragments of (iii) that bind to the growth factor that is not primed; and

(v) selecting the antibodies or antigen binding fragments that bind to the primed transforming growth factor and do not bind to the growth factor that is not primed.

45. The method of claim 43, wherein the transforming growth factor is GDF8 or GDF11.

46. The method of claim 43 or 44, wherein the antibodies or antigen binding fragments of (iii) are eliminated if they bind to the growth factor that is not primed with an affinity greater than or equal to the affinity that they bind to the growth factor that is primed.

47. The method of any one of claims 43-45, wherein the transforming growth factor that is not primed is the pro form of the transforming growth factor.

48. The method of any one of claims 43-45, wherein the transforming growth factor that is not primed is the latent form of the transforming growth factor.

49. An antibody obtained by the method of any one of claims 43-47.

50. The antibody of claim 48, wherein the antibody is bound to a primed

transforming growth factor.

51. The antibody of claim 49, wherein the primed transforming growth factor is recombinant.

52. The antibody of claim 49 or 50, wherein the transforming growth factor is GDF8 or GDF11.

53. An antibody, of any one of claims 48-51, that selectively binds a primed transforming growth factor.

54. An antibody, of any one of claims 48-52, that inhibits the primed transforming growth factor.

55. A pharmaceutical composition comprising (i) a therapeutically effective amount of the antibody of any one of claims 48-53, and (ii) a pharmaceutically acceptable carrier.

56. A pharmaceutical composition for use in preventing muscle wasting or increasing muscle mass, comprising a therapeutically effective amount of the antibody of any one of claims 48-53.

57. A primed recombinant transforming growth factor (rTGF) comprising an epitope tag.

58. The primed rTGF of claim 56, wherein the epitope tag is on the C-terminus of the rTGF.

59. The primed rTGF of claim 56, wherein the epitope tag is between the C-terminus and the N-terminus.

60. The primed rTGF of claim 56, wherein the recombinant transforming growth factor is GDF8 or GDF 11.