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WO2017075037A1 - Facteurs de croissance amorcés et leurs utilisations - Google Patents

Facteurs de croissance amorcés et leurs utilisations Download PDF

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Publication number
WO2017075037A1
WO2017075037A1 PCT/US2016/058841 US2016058841W WO2017075037A1 WO 2017075037 A1 WO2017075037 A1 WO 2017075037A1 US 2016058841 W US2016058841 W US 2016058841W WO 2017075037 A1 WO2017075037 A1 WO 2017075037A1
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Prior art keywords
primed
rtgf
growth factor
transforming growth
preparation
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PCT/US2016/058841
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English (en)
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Michelle STRAUB
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Scholar Rock, Inc.
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Publication of WO2017075037A1 publication Critical patent/WO2017075037A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6454—Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)

Definitions

  • 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.
  • 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”.
  • 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.
  • primed rTGFs have improved pharmaceutical properties.
  • 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.
  • 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.
  • it has been found that primed rTGF has improved bioavailability compared with the mature form.
  • Primed rTGF transforming growth factor
  • primed GDF8 or primed GDF11 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.
  • 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.
  • the proprotein convertase is a furin or PCSK5 and in some embodiments, the tolloid protease is a mT112 protease or a BMP1 protease.
  • 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.
  • the rTGF is produced in one or more engineered cells that inducibly express the rTGF.
  • the one or more engineered cells secrete the rTGF into a cell culture media comprising a proprotein convertase and/or a tolloid protease.
  • 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.
  • 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.
  • 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.
  • 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.
  • a preparation of primed rTGF 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 10 3 : 1, 10 2 : 1 to 10 4 : 1, 10 2 : 1 to 10 6 : 1.
  • a preparation of primed rTGF is provided that comprises trace amounts of a proprotein form of the rTGF.
  • a preparation of primed rTGF 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.
  • a preparation of primed rTGF that comprises a molar
  • a preparation of primed rTGF that comprises trace amounts of a latent form of the rTGF.
  • a preparation of primed rTGF 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.
  • the epitope tag is at the C-terminus of the rTGF.
  • 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.
  • the primed rTGF in a substantially pure preparation of primed rTGF is AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7,
  • compositions provided herein are useful for decreasing muscle mass, improving blood vessel volume, improving neurogenesis, reversing age-related cardiac hypertrophy or improving skeletal muscle function.
  • 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).
  • TGF transforming growth factor
  • methods provided herein include modulating SMAD signaling in cells.
  • 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.
  • the cells are in vitro.
  • the cells are in in vivo.
  • the disclosure provided methods for modulating non-canonical TGF signaling pathways.
  • the methods include modulating MAP kinase pathways, Rho-like GTPase signaling pathways, and/or phosphatidylinositol-3-kinase/AKT pathways.
  • 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.
  • the method includes administering any of the pharmaceutical compositions provided herein to the subject.
  • the condition is age-related cardiac hypertrophy.
  • the condition includes decreased blood vessel volume.
  • the condition includes decreased neurogenesis.
  • the condition includes decreased skeletal muscle function.
  • aspects of the disclosure relate to methods for producing a primed recombinant transforming growth factor (rTGF).
  • 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.
  • the proprotein convertase is a furin protease and/or the tolloid protease is a mTl 12 protease.
  • 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.
  • the primed transforming growth factor is non-covalently linked to the solid substrate.
  • the primed transforming growth factor is linked to the substrate via an antibody.
  • the antibody selectively binds the primed transforming growth factor.
  • the antibody is any of the antibodies provided herein.
  • the solid substrate comprises beads or a resin.
  • the solid substrate is a surface of an affinity column.
  • 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.
  • the transforming growth factor is GDF8 or GDFl 1.
  • 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.
  • 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.
  • the antibody is bound to a primed transforming growth factor.
  • the primed transforming growth factor is recombinant.
  • the transforming growth factor is GDF8 or GDFl 1.
  • the antibody selectively binds a primed transforming growth factor.
  • 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.
  • the pharmaceutical composition is for use in preventing muscle wasting or increasing muscle mass.
  • the antibody selectively binds myostatin (GDF8).
  • 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.
  • 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.
  • the recombinant transforming growth factor is GDF8 or GDF11.
  • FIG. 1 is a schematic showing prodomain assemblies. TGFP family members are secreted as procomplexes. TGB- ⁇ ⁇ "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.
  • rTGFs e.g. , GDF11 and GDF8
  • 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).
  • 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.
  • FIG. 4B shows overlaid size exclusion chromatography coupled to multiangle light scattering (SEC- MALS) traces of myostatin (GDF8) species.
  • SEC- MALS multiangle light scattering
  • GDF8 myostatin
  • FIGs. 4C and 4D depict concentration-dependence of the SEC-MALS elution.
  • lOOug, 20ug, and lOug injections were analyzed for the three different GDF11 species.
  • 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.
  • lOOug, 20ug, and lOug (primed only) injections were analyzed for the three different GDF8 species.
  • 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.
  • 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.
  • transforming growth factors e.g., GDF8 and GDF11
  • 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.
  • primed rTGF is a more potent stimulator of SMAD mediated signaling than mature rTGF, latent rTGF, or pro rTGF.
  • this potent stimulatory effect is exhibited in an in vitro cell based reporter assay, as described herein.
  • primed transforming growth factors have improved
  • a primed rTGF is soluble at concentrations (e.g., in a neutral pH range, e.g.
  • 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.
  • 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).
  • 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).
  • 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.
  • primed transforming growth factors have improved
  • 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.
  • a “primed recombinant transforming growth factor (rTGF)” or “primed rTGF” refers to recombinant form of a member of the TGF- ⁇ 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.
  • primed rTGF refers to recombinant form of a member of the TGF- ⁇ 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).
  • a primed rTGF is a primed recombinant form of a TGF- ⁇ superfamily protein that is regulated in a protease-dependent manner, e.g. , that is activated by one or more protease cleavage events.
  • prodomain that remains associated, non-covalently, with its mature growth factor domain does not substantially impede growth factor binding to receptors.
  • 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.
  • a primed rTGF elutes at a different volume than a corresponding pro- or latent- form of the rTGF in a gel filtration column.
  • a primed rTGF elutes earlier than a corresponding pro- or latent- form in a gel filtration column.
  • 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.
  • the calculated molecular weight remains substantially the same as the corresponding pro- or latent-form, which indicates a conformational change.
  • conformational state of primed rTGF facilitates access to the receptor by the mature growth factor.
  • 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.
  • enzymatic cleavage of a transforming growth factor produces a primed form of the growth factor in which its prodomain remains non-covalently associated with its mature growth factor portion.
  • the TGF- ⁇ superfamily of proteins include a large family of structurally related cell regulatory proteins that were named after the founding member, TGF- ⁇ ⁇ , 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.
  • TGF- ⁇ superfamily of proteins are found in a variety of species, including invertebrates as well as vertebrates.
  • members of the TGF- ⁇ 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).
  • members of the TGF- ⁇ 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,
  • the member of the TGF- ⁇ superfamily of proteins is myostatin (GDF8) or GDFl 1.
  • the myostatin (GDF8) or GDFl 1 is human or mouse. It should be appreciated that the TGF- ⁇ superfamily members described and referenced herein are exemplary and additional TGF- ⁇ superfamily members from humans as well as other species ⁇ e.g., mice) fall within the scope of this disclosure.
  • TGF- ⁇ superfamily may also include proteins that are homologous to one or more members of the TGF- ⁇ superfamily.
  • a protein is considered to be homologous to a member of the TGF- ⁇ 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- ⁇ superfamily.
  • Calculation of the percent identity of two amino acid sequences 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).
  • 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- ⁇ 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
  • 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)).
  • 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.
  • a primed rTGF is a protein that has been designed to meet particular requirements or to have particular design features.
  • a primed rTGF comprises an epitope tag.
  • an "epitope tag” refers to a peptide sequence that may be genetically grafted onto a protein (e.g., a member of the TGF- ⁇ 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.
  • 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.
  • primed rTGFs provided herein may be used in research, experimental and/or diagnostic applications.
  • 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.
  • 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.
  • transforming growth factor e.g., a pro, a latent, a mature, or a primed form
  • 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.
  • the efficiency of protein secretion is determined by the signal peptide.
  • 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.
  • the signal peptide targets the rTGF for secretion.
  • the signal peptide comprises an Ig kappa chain (V-I region) signal peptide.
  • the signal peptide comprises the amino acid sequence
  • 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.
  • 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.
  • 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.
  • a GDFl 1 that has been cleaved by a proprotein convertase e.g., furin
  • latent GDFl 1 e.g., furin
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the tolloid protease site comprises the amino acid sequence QRD.
  • a tolloid protease site comprising the amino acid sequence QRD is cleaved between the R and the D residue by a tolloid protease.
  • the tolloid protease site comprises the amino acid sequence QGD.
  • 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.
  • 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).
  • a tolloid protease is homologous to a tolloid protease selected from the group consisting of: BMP-1, mTLD, mTLL-1 and mTLL-2.
  • 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.
  • substantially pure preparation of a primed recombinant transforming growth factor 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.
  • 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.
  • a substantially pure preparation of a primed rTGF has a relatively low amount of one or more impurities (e.g., other rTGFs).
  • 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.
  • 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.
  • 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 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).
  • antibodies are disclosed herein that specifically bind primed rTGF.
  • antibodies are disclosed herein that specifically bind primed GDF8 or primed GDF11.
  • an rTGF may be contacted with any of the proprotein convertases provided herein.
  • an rTGF is contacted with a furin.
  • an rTGF may be contacted with any of the tolloid proteases provided herein.
  • 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.
  • the rTGF is produced in one or more engineered cells that inducibly express the rTGF.
  • the one or more engineered cells secrete the rTGF into a cell culture media comprising a proprotein convertase and/or a tolloid protease.
  • 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.
  • engineered cell refers to a cell that does not occur in nature.
  • Engineered cells of the present disclosure 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).
  • exogenous nucleic acids i.e., nucleic acids that the cell would not normally contain
  • nucleic acids that do not occur in nature e.g., an engineered nucleic acid encoding a rTGF.
  • 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.
  • an engineered nucleic acid comprises nucleotide sequences from different organisms (e.g., from different species).
  • 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.
  • the primed rTGF is in a solution at a concentration from 1 mg/ml to 1000 mg/ml.
  • 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 1
  • the primed rTGF is in a solution at a concentration of greater than 100 mg/ml.
  • the solution comprises a buffer.
  • 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.
  • aspects of the disclosure relate to methods for modulating transforming growth factor (TGF) receptors in cells.
  • 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.
  • the cells are in vitro. In other embodiments, the cells are in in vivo.
  • 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).
  • in vivo refers to events that occur within an organism (e.g., an animal).
  • aspects of the disclosure relate to methods for producing a primed recombinant transforming growth factor (rTGF).
  • 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.
  • 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.
  • the proprotein convertase is a furin protease and/or the tolloid protease is a mTl 12 protease. In some embodiments, the
  • the recombinant rTGF is contacted with the proprotein convertase and/or the tolloid protease in vitro.
  • the rTGF is contacted with the proprotein convertase and/or the tolloid protease in a cell culture media.
  • the cell culture media is a conditioned media comprising a proprotein convertase and/or a tolloid protease.
  • the rTGF and/or the proprotein convertase and/or the tolloid protease is expressed inducibly in a cell.
  • 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.
  • the primed transforming growth factor is non-covalently linked to the solid substrate.
  • the primed transforming growth factor is linked to the substrate via an antibody.
  • the antibody selectively binds the primed transforming growth factor.
  • the antibody is any of the antibodies provided herein.
  • the solid substrate comprises beads or a resin.
  • the solid substrate is a surface of an affinity column.
  • 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.
  • the transforming growth factor is GDF8 or GDFl 1.
  • 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.
  • the transforming growth factor that is not primed is the pro form of the transforming growth factor.
  • 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.
  • the antibody is bound to a primed transforming growth factor.
  • the primed transforming growth factor is recombinant. In some
  • the transforming growth factor is GDF8 or GDF11.
  • the antibody selectively binds a primed transforming growth factor.
  • the antibody inhibits the primed transforming growth factor.
  • rTGF primed recombinant transforming growth factor
  • the epitope tag is on the C-terminus of the rTGF.
  • the epitope tag is between the C-terminus and the N- terminus.
  • the recombinant transforming growth factor is GDF8 or GDF11.
  • nucleic acids comprising a sequence encoding a recombinant transforming growth factor and an epitope tag.
  • the sequence encoding the epitope tag is 5' to the sequence encoding the recombinant transforming growth factor.
  • the sequence encoding the epitope tag is 3' to the sequence encoding the recombinant transforming growth factor.
  • the nucleic acid is in a vector.
  • the nucleic acid further comprises one or more heterologous promoters.
  • 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.
  • the engineered cell further comprises a nucleic acid encoding a proprotein convertase and/or a tolloid protease.
  • a conditioned medium comprising a proprotein convertase and/or a tolloid protease.
  • the proprotein convertase comprises furin and/or the tolloid protease comprises mT112.
  • the medium further comprises a transforming growth factor.
  • the transforming growth factor is recombinant.
  • 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.
  • the cell culture system further comprises any of the conditioned media provided herein.
  • 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.
  • 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.
  • the pharmaceutical composition is for use in preventing muscle wasting or increasing muscle mass.
  • the antibody selectively binds myostatin (GDF8).
  • the antibody inhibits myostatin (GDF8).
  • 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.
  • a pharmaceutically acceptable carrier excipient
  • “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.
  • 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
  • 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.
  • pharmaceutically acceptable carriers excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.
  • 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,
  • 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.
  • the pharmaceutical composition described herein can be formulated in sustained-release format.
  • 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.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(v nylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and 7 ethyl-L-glutamate 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.
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • sucrose acetate isobutyrate sucrose acetate isobutyrate
  • poly-D-(-)-3-hydroxybutyric acid poly-D-(-)-3-hydroxybutyric acid.
  • compositions to be used for in vivo administration must be sterile.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a phospholipid e.g. egg phospholipids, soybean phospholipids or soybean lecithin
  • 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).
  • 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.
  • the compositions are
  • 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 relate to methods for treating a subject having a condition associated with reduced transforming growth factor receptor activity.
  • the method includes administering any of the pharmaceutical compositions provided herein to the subject.
  • the method includes administering a preparation comprising a primed rTGF(e.g., primed GDF11).
  • the condition is age-related cardiac hypertrophy.
  • the condition includes decreased blood vessel volume.
  • the condition includes decreased neurogenesis.
  • the condition includes decreased skeletal muscle function.
  • composition comprises a primed rTGF.
  • the primed rTGF comprises a primed rTGF.
  • 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.
  • the method includes administering any of the antibodies that specifically bind a primed rTGF, provided herein to the subject.
  • the antibodies that specifically bind a primed rTGF inhibit the transforming growth factor.
  • the condition is myopathy.
  • 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.
  • 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).
  • 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.
  • 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)).
  • bone fracture e.g. a hip fracture
  • nerve injury e.g., spinal cord injury (SCI)
  • 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.
  • 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.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and humans.
  • the subject is a patient. In some embodiments, the subject is a healthy volunteer.
  • 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,
  • a suitable route such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal,
  • 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.
  • 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 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.
  • 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.
  • sustained continuous release formulations may be appropriate.
  • 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.
  • 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.
  • an initial candidate dosage can be about 2 mg/kg.
  • a typical daily dosage might range from about any of 0.1 g/kg to 3 g/kg to 30 ⁇ g/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.
  • 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.
  • 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 ⁇ g/mg, about 10 ⁇ g/mg, about 30 ⁇ g/mg, about 100 ⁇ g/mg, about 300 ⁇ g/mg, about 1 mg/kg, and about 2 mg/kg) may be used.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • transforming growth factor receptor activity e.g., myopathy
  • 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.
  • compositions 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.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial,
  • intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques 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.
  • compositions may contain various carriers such as vegetable oils,
  • 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.
  • an anti-primed rTGF antibody or preparation of primed rTGF is administered via site-specific or targeted local delivery techniques.
  • 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.
  • compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol.
  • concentration ranges of about 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g 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
  • 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
  • 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)
  • AAV adeno-associated virus
  • Non-viral delivery vehicles and methods can also be employed.
  • 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.
  • proMyostatin murine and human
  • proGDFl 1 murine and human
  • furin BMP-1
  • PCSK5 PCSK5
  • T112 Protein constructs were stabiv integrated into FLP-INTM T-REXTM 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 ⁇ Decanoyl RVKR-CMK.
  • 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
  • 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.
  • SEC SRT!O size exclusion chromatography
  • FIGs. 4A-D shows the results of these experiments.
  • 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.
  • MW molecular weight
  • 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.
  • 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
  • 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
  • primed GDF8 and GDFl l are used as antigens in an inhibitory campaign to selectively block signaling of GDF8 or GDF11.
  • 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.
  • 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
  • 3C Protease cleavage sites are underlined and italicized.
  • amino acid sequence set forth in SEQ ID NO: 1 provides a non-limiting example of a recombinant human myostatin.
  • amino acid sequence set forth in SEQ ID NO: 2 provides a non-limiting example of a recombinant murine myostatin.
  • amino acid sequence set forth in SEQ ID NO: 3 provides a non-limiting example of a recombinant human GDF11.
  • 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
  • amino acid sequence set forth in SEQ ID NO: 5 provides a non-limiting example of the tolloid protease BMP1.
  • amino acid sequence set forth in SEQ ID NO: 6 provides a non-limiting example of the tolloid protease mTLL2. >mTLL2 C-Flag His
  • amino acid sequence set forth in SEQ ID NO: 7 provides a non-limiting example of the proprotein convertase furin.
  • amino acid sequence set forth in SEQ ID NO: 8 provides a non-limiting amino acid sequence set forth in SEQ ID NO: 8

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Abstract

Des aspects de la présente invention concernent des préparations pratiquement pures de facteurs de croissance transformants de recombinaison amorcés et de compositions (par ex. des compositions pharmaceutiques) comprenant celles-ci. L'invention concerne également des procédés permettant de moduler des récepteurs de facteurs de croissance transformants et de traiter des sujets présentant un état associé à une activité réduite des récepteurs de facteurs de croissance transformants réduite (par ex. en utilisant l'une quelconque des préparations décrites ici). L'invention concerne également des anticorps qui se lient sélectivement à des facteurs de croissance transformants de recombinaison amorcés et leurs procédés de fabrication.
PCT/US2016/058841 2015-10-27 2016-10-26 Facteurs de croissance amorcés et leurs utilisations WO2017075037A1 (fr)

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WO2020016425A1 (fr) * 2018-07-20 2020-01-23 Association Institut De Myologie Compositions pour le traitement de la sarcopénie ou de l'atrophie par inaction
US11759476B2 (en) 2020-12-14 2023-09-19 Regeneron Pharmaceuticals, Inc. Methods of treating metabolic disorders and cardiovascular disease with Inhibin Subunit Beta E (INHBE) inhibitors

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