WO2014000042A1 - Compositions et procédés pour modifier des ligands de la famille du tgf-β - Google Patents

Compositions et procédés pour modifier des ligands de la famille du tgf-β Download PDF

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WO2014000042A1
WO2014000042A1 PCT/AU2013/000698 AU2013000698W WO2014000042A1 WO 2014000042 A1 WO2014000042 A1 WO 2014000042A1 AU 2013000698 W AU2013000698 W AU 2013000698W WO 2014000042 A1 WO2014000042 A1 WO 2014000042A1
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propeptide
seq
amino acid
activin
acid sequence
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PCT/AU2013/000698
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Craig A HARRISON
Yogeshwar MAKANJI
Kelly L PEGGIE
Sara L ZANGANA
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Prince Henry's Institute Of Medical Research
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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
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag

Definitions

  • the present invention relates generally to the provision of propeptides of Transforming Growth Factor- ⁇ (TGF- ⁇ ) family ligands for use in therapy and processes of producing same.
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • the invention relates to modifying propeptides of TGF- ⁇ - ligands to confer higher affinity binding to TGF- ⁇ family ligands.
  • the invention provides compositions, methods and uses, including pharmaceutical compositions and therapeutic uses.
  • the TGF- ⁇ super family comprises a large number of structurally related proteins that have important roles in cellular signalling and tissue homeostasis.
  • TGF- ⁇ superfamily Members of the TGF- ⁇ superfamily are synthesised as large precursor molecules consisting of an N-terminal prodomain and a C-terminal mature domain.
  • the dimeric mature protein (ligand) is secreted from the cell non-covalently associated with its dimeric propeptide.
  • the affinity of the propeptide for the mature protein is greater than the affinity of the mature protein for its receptor.
  • the propeptide blocks receptor binding and the mature TGF- ⁇ protein is said to be "latent”.
  • the affinity of the propeptide for the mature protein is less than the affinity of the mature protein for its receptor.
  • the mature TGF- ⁇ protein is secreted in an "active" form able to engage its receptor and exert its biological effects.
  • the affinity of the ligand for its receptors is greater than the affinity of the Hgand for its propeptide and the ligand is secreted in an "active" form.
  • the affinity of the ligand for its receptor is less than the affinity of the ligand for its propeptide and these ligands are secreted in a "latent" form.
  • the subsequent mechanism of activation varies according to cell type and context, but all activating mechanisms directly target propeptides.
  • TGF- ⁇ and TGF- ⁇ 3 the most prominent mechanism of activation involves the binding of ⁇ 6 and ⁇ 8 integrins to RGD sequences in their respective propeptides.
  • these activators permit TGF- ⁇ ⁇ and ⁇ 7 - ⁇ 3 to engage their signalling receptors.
  • Latent myostatin and GDF- 1 1 are activated following cleavage of their propeptides by members of the BMP-l/tolloid family of metal loproteases.
  • Type I receptors act downstream of type II receptors (TpRII, ActRII, AclRIIB, BMPRJI and MISR1I) and determine the signalling specificity within the receptor complex.
  • type II receptors phosphorylate and activate type I receptors, which subsequently propagate the signal by phosphorylating specific receptor-regulated (R-) SMAD transcription factors.
  • R-SMADs form a complex with the co-activator, SMAD4, and the resulting SMAD oligomer migrates to the nucleus to regulate TGF-P-responsive genes.
  • TGF- ⁇ ligands have low affinity for type II receptors and require the presence of co-receptors to signal. Indeed, cell responsiveness to ⁇ - ⁇ 2 has been shown to be dependent upon the transmembrane protein, betaglycan, whereas the GPI-anchored protein Cripto mediates Nodal signalling. Co-receptor binding causes conformational changes within TGF-p2 and Nodal, respectively, leading to increased affinity for type II receptors.
  • follistatin a monomeric glycoprotein that binds activin-related molecules with high affinity.
  • TGF- ⁇ superfamily antagonists include noggin, chordin, twisted gastrulation and gremlin. Similar to follistatin, these antagonists bind their target ligands (BMPs and GDFs) extracellularly and prevent them interacting with their signalling receptors. In this manner, these BMP antagonists regulate osteogenesis.
  • BMPs and GDFs target ligands
  • Activin A can positively or negatively regulate the growth of numerous adult tissues, including gonads, liver, stomach, adipose, muscle and bone. Together, with the more recent observations that activin A is involved in the pathogenesis of inflammatory and fibrotic human diseases, these findings indicate that inhibiting the activity of activin A may be an effective strategy for restoring homeostasis in disease-affected tissues,
  • TGF- ⁇ ligands play in the maintenance of tissue homeostasis in adult life and in the progression of many diseases, including cancer, fibrosis, autoimmune and vascular disorders, make them attractive therapeutic targets.
  • binding proteins, soluble receptors, propeptides and type I receptor kinase inhibitors have all been utilised successfully to block TGF- ⁇ superfamily signalling in murine disease models.
  • these molecules are pleiotropic and there are concerns regarding off- target effects when considering their appropriateness for treatment of disease. For example, the ability of follistatin to block myostatin signalling and thus increase muscle size and strength has been well documented and represents a promising therapeutic strategy for muscular dystrophy.
  • exogenous follistatin has also been shown to profoundly suppress FSH secretion by the pituitary, increase liver mass, impair neurogenesis, and attenuate wound healing.
  • soluble activin type II receptors which bind multiple TGF- ⁇ ligands, have been used variously to increase muscle mass, induce bone formation, and treat obesity and diabetes. Off-target effects resulting from the inhibition of TGF- ⁇ ligands could be minimised by the development of more specific antagonists.
  • the latent TGF- ⁇ ligand, myostatin is naturally inhibited by its own propeptide.
  • a modified propeptide that is resistant to metalloprotease activation it has been shown that adeno-associated viral (AAV) gene delivery enhances systemic skeletal muscle growth via hypertrophy.
  • AAV adeno-associated viral
  • myostatin propeptide also ameliorates the dystrophic phenotype in mdx mice, a murine model of Duchenne muscular dystrophy.
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID NO: l), ⁇ 400>2 (SEQ ID NO:2), etc.
  • a summary of sequence identifiers is provided in Table 2.
  • a sequence listing is provided after the claims.
  • the present specification discloses modified propeptides of TGFp family ligands which are suitably in isolated, synthetic, recombinant or purified form.
  • TGFp family propeptides are selected from those TGF- ⁇ propeptides that are considered to be “active” that is they are naturally produced having a lower affinity for their mature domain than between the mature domain and its receptor.
  • These "active" TGFp family peptides include without limitation activin B, activin A, activin C, activin E, BMP7, BMP5. BMP6, BMP8A, BMP8B, BMP2, BMP4, BMPI O, GDF2, GDF5, GDF6, GDF7, BMP3, BMP3B, lefty 1 , lefty2, GDF 1 , GDF3, NODAL, BMP15, GDF9, GDF15, MIS and inhibin. These molecules, in their naturally occurring (wild-type) form are unable to antagonise receptor signalling by binding to the mature protein.
  • the present specification discloses a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 1 , or a functional fragment thereof, the propeptide is covalently linked to a dimerization domain (see Figure 5A3).
  • the dimerization domain is an immunoglobulin Fc domain or at least a hinge domain thereof.
  • references to "functional fragments” includes propeptides without their native signal sequence.
  • the term also includes nucleic acid molecules encoding propeptides lacking their native signal sequences.
  • native signal sequences may be replaced with non-native (heterologous) signal sequences in order to optimise expression in a range of environments.
  • the present specification discloses a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, or a functional fragment thereof, the propeptide is covalently linked to a dimerization domain.
  • the present specification discloses a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, or a functional fragment thereof, the propeptide is covalently linked to a dimerization domain.
  • the specification provides a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 1 , or a functional fragment thereof, with the proviso that the amino acid includes SEQ ID NO: 8 or SEQ ID NO: 9 or a conservative variant thereof.
  • residue 64 of the propeptide is Ser or a conservative amino acid substitution thereof and residue 65 is Lys or a conservative amino acid substitution thereof.
  • the specification provides a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, or a functional fragment thereof, with the proviso that the amino acid sequence includes SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variants thereof.
  • residue 64 is Ser or a conservative amino acid substitution thereof and residue 65 is Lys or a conservative amino acid substitution thereof.
  • the specification provides a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, or a functional fragment thereof, with the proviso that the amino acid sequence includes. SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variants thereof.
  • nucleic acid constructs encoding a modified propeptide as described herein or a functional fragment thereof.
  • host cells comprising a nucleic acid construct encoding a modified propeptide as described herein, wherein the host cell expresses the propeptide.
  • the present invention provides a method of treating or preventing activin-induced conditions, such as muscle wasting, fibrosis, inflammation in a subject, the method comprising administering to the subject a modified propeptide as described herein or a nucleic acid construct encoding same which provides the modified propeptide to the subject.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: .19, or a functional fragment thereof, the propeptide is covalently linked to a dimerization domain.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, or a functional fragment thereof, the propeptide is linked to a dimerization domain.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29, or a functional fragment thereof, the propeptide is linked to a dimerization domain.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 1 , or a functional fragment thereof, with the proviso that the amino acid sequence includes SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variants thereof.
  • residue 64 of the propeptide comprising SEQ ID NO: 1 1 is Ser or a conservative amino acid substitution thereof and residue 65 is Lys or a conservative amino acid substitution thereof.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, or a functional fragment thereof, with the proviso that the amino acid sequence includes SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variants thereof.
  • residue 64 is Ser or a conservative amino acid substitution thereof and residue 65 is Lys or a conservative amino acid substitution thereof.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25 or SEQ ID NO: 29, or a functional fragment thereof, with the proviso that the amino acid sequence includes SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variants thereof.
  • the present therapeutic or prophylactic methods are suitable for treating or preventing cachexia-anorexia syndrome.
  • the present therapeutic or prophylactic methods are suitable for treating or preventing activin-induced inflammatory conditions.
  • the modified propeptide binds to activin A and the activity of activin A is reduced.
  • the activity of activin A and B is reduced.
  • the invention provides a method for down regulating the activity of endogenous activin A in a subject, the method comprising administering a modified activin A propeptide or a modified activin B propeptide as described herein or a nucleic acid construct encoding same.
  • the invention provides a method of down regulating the activity of endogenous activin A and B in a subject, the method comprising administering a modified activin B propeptide as described herein or a nucleic acid construct encoding same.
  • the invention provides a method of down regulating the activity of endogenous activin B in a subject, the method comprising administering a modified activin B propeptide as described herein or a nucleic acid construct encoding same.
  • modified activin propeptides and nucleic acid constructs including expression vectors comprising same, for use in therapy including in the manufacture of a medicament for use in therapy.
  • the specification describes a process for producing a modified "active" TGF- ⁇ family propeptide, the method comprising co-expressing the propeptide together with a dimerization domain directly or indirectly covalently linked to the N- terminus or C-terminus of the propeptide., wherein the presence of the dimerization domain enhances the affinity of the modified propeptide for its mature domain.
  • Recombinant propeptides for example, are conveniently produced by transfecting host cells with a nucleic acid molecules encoding the modified propeptide, culturing the host cell to express the recombinant propeptide and isolating or purifying the propeptide.
  • modified propeptides may be made by synthetic or semi-synthetic means known in the art.
  • the immunoglobulin Fc domain may be introduced by recombinant (as a fusion protein) or synthetic means.
  • Figure 1 is a diagrammatic representation showing synthesis, extracellular localisation and activation of activin A.
  • Activin A is synthesised as a precursor molecule consisting of an N-terminal prodomain and a C-terminal mature domain. During synthesis, the prodomain forms intimate contacts with the mature domain, facilitating folding and dimerisation of activin A. Dimeric precursors are cleaved by proprotein convertases and activin A is secreted from the cell non-covalently associated with its prodomain.
  • the prodomain targets mature activin A to the extracellular matrix ensuring: (i) its localisation within the vicinity of target cells; and (ii) its juxtaposition to activating molecules.
  • C Activation of activin A occurs following displacement of the prodomain by activin type II receptors (ActRIIA/ActRIIB).
  • FIG. 2 is a diagrammatic representation showing active and latent TGF- ⁇ proteins.
  • All TGF- ⁇ proteins are secreted from cells non-covalently associated with their prodomains, however, most, including activin A and activin B, are considered to be "active" (i.e. they have higher affinity for their signalling receptors than for their prodomains, and so the prodomain can be readily displaced).
  • B Five TGF- ⁇ ligands (TGF- ⁇ , ⁇ - ⁇ 2, ⁇ - ⁇ 3, myostatin and GDF1 1 ) are secreted in a latent form (i.e. they have higher affinity for their prodomains than for their signalling receptors).
  • Activation of the TGF- ⁇ isoforms occurs following integrin-mediated conformational changes within the prodomain, whereas activation of myostatin and GDF1 1 requires BMP 1 -mediated proteolytic cleavage of the prodomain .
  • FIG. 3 is a diagrammatic representation if the structure of pro-TGF- ⁇ .
  • the crystal structure of pro-TGF- ⁇ was recently solved (PDB ID:3RJR - Shi et al., 201 1 ).
  • the two mature TGF- ⁇ monomers are shown in yellow and blue, while the prodomains are orange and green.
  • the orange prodomain would be linked to the yellow mature domain (and the green prodomain to the blue mature domain) prior to cleavage by proprotein convertases.
  • the prodomains primarily interact with their opposing mature domains.
  • the arrangement of prodomain secondary structure elements (a helix, ⁇ strand) is indicated and this structure is likely to be maintained across the TGF- ⁇ superfamily.
  • the two prodomains form the neck, shoulders and upper arms, and connect at the elbows to the crossed forearms formed by the two TGF- ⁇ monomers.
  • the prodomains via the al helix, also provide a strap to fasten the straight-jacket.
  • the arm regions of the prodomains form two anti- parallel, four-stranded ⁇ -sheets.
  • the bow is tied by reciprocal interchain disulfide bonds between Cys and Cys (Shi et al., 201 1 ) Residues within the al and a2 helices and the intervening latency lasso of the prodomains encircle the fingers of each TGF- ⁇ monomer and are likely to be important in directing the folding of the growth factor domain.
  • lie 46 , He 53 , Leu 54 , Leu 57 and Leu 59 on one surface of the amphipathic al helix interact with Tip 308 , Trp 310 and aliphatic side chains on one TGF- ⁇ monomer. These tryptophans, as well as other fingertip residues, are then contacted by prolines and hydrophobic residues within the
  • Val and Tyr within the a2 helix complete the encirclement by burying Val 367 and Val 376 on the outer convex surface of the TGF- ⁇ ⁇ finger (Shi et al., 201 1 ).
  • the TGF- ⁇ isoforms are secreted in a latent form inter alia because of two major adaptions within their prodomains: (i) cysteine residues (Cys 223 and Cys 225 ) within the bowtie region, which covalently link the two prodomain chains; and (ii) fastener residues (Lys 56 , Tyr 103 , Tyr 104 , Ala 105 and Arg 267 ), which intimately connect the straight-jacket and arm regions of the prodomains (Shi et al., 201 1 ). Mutation of these latency-conferring residues results in the spontaneous activation of TGF- ⁇ (Brunner et al., 1989 and Shi et al., 201 1).
  • Figure 4 is a diagrammatic representation showing the important 'fastener' residues within the TGF- ⁇ prodomain are conserved in other latent TGF- ⁇ ligands.
  • A Within the pro-TGF- ⁇ structure, a backbone hydrogen bond between the nitrogen of Ala 105 and the oxygen of Lys 56 caps the C-terminal end of the al helix. Moreover, the carbonyl oxygen of Ala 105 forms a hydrogen bond to Arg 267 in the a5 helix. Lys 56 is a key fastener residue.
  • FIG. 5 is a diagrammatic representation showing modified activin A and activin B prodomains.
  • the wild-type activin A and activin B prodomains (see construct 1A & IB, Figure 5) have low affinity for activin A and activin B, respectively. As such, they can not be used to inhibit activin activity. Therefore, a series of modifications were incorporated into the activin prodomains to increase their affinity for their mature ligands.
  • a FLAG tag see construct 2A, Figure 5
  • the Fc region promoted covalent dimerisation of the fusion protein and, thereby, mimicked the dimerisation observed between TGF- ⁇ prodomain chains. It also improved the stability and, therefore, in vivo half-life of the activin A prodomain.
  • the (Inhibin) activin A prodomain lacks the key ' fastener' residues (Tyr, Tyr/His and Ala; see Figure 4); it retains relatively high sequence homology with myostatin and GDF-1 1 in the surrounding regions (see below).
  • Myo i0 *SLEDDD YHATTETIITMPTES- 1 "
  • the non-conserved region of the activin B prodomain ( l23 IPHLDGHASPGADGQERVSE 142 ; shaded grey, above) was replaced with the 'fastener' region of the myostatin prodomain ( 107 EDDD YH ATTET 1 17 ) (see construct 4B, Figure 5).
  • FIG. 6 is a representation of data showing Pro-Activin A-Fc and Pro-Activin B-Fc are expressed as covalent dimers.
  • A HE 293F cells were transfected with Pro- Activin A-Fc (construct 5A, Figure 5) or Pro-Activin B-Fc (construct 4B, Figure 5). After 48h, conditioned medium was collected and analysed by SDS-PAGE and Western blot. Pro-Activin A-Fc was secreted as a covalent dimer of 120 kDa.
  • Pro-activin B-Fc was secreted in various forms: (i) a 120 kDa covalent dimer; (ii) a 105 kDa form (presumed to be generated by proteolysis of one prodomain chain); and (iii) a 90 kDa form (presumed to be generated by proteolysis of both prodomain chains).
  • the 90 and 105 kDa forms of Pro- Activin B-Fc are not expected to have any inhibitory activity as they lack the prodomain regions (al and a2 helicies) that contact the mature domain.
  • Pro-Activin B-Fc was secreted as a covalent dimer of 120 kDa.
  • COS7 cells do not proteolytically cleave Pro-Activin B-Fc.
  • Figure 7 is a graphical representation of data showing potency and specificity of Pro-Activin A-Fc and Pro-Activin B-Fc.
  • A HEK293T cells were transfected with a Smad2/3 -responsive luciferase reporter and 24 h later were stimulated with 200 pM activin A ( ⁇ ), activin B ( ⁇ ), myostatin ( ) or GDFl l ( ⁇ ), in the absence or presence of increasing concentrations of Pro-Activin A-Fc (construct 5A, Figure 5).
  • HEK293T cells were transfected with a Smad2/3- responsive luciferase reporter and 24 h later were stimulated with 200 pM activin A ( ⁇ ), activin B ( ⁇ ), myostatin ( A ) or GDF l l ( ⁇ ), in the absence or presence of increasing concentrations of Pro-Activin B-Fc (construct 4B, Figure 5). After 24 h, cells were harvested in solubilization buffer [1% Triton X-100, 25 mM glycylglycine (pH 7.8), 15 mM MgS04, 4 mM EGTA, and 1 mM dithiothreitol], and the luciferase activity was measured.
  • solubilization buffer 1% Triton X-100, 25 mM glycylglycine (pH 7.8), 15 mM MgS04, 4 mM EGTA, and 1 mM dithiothreitol
  • Pro-Activin B-Fc antagonised both activin A (IC50 0.4nM) and activin B (IC50 0.65nM) activity, but had no effect on myostatin or GDFl l signalling.
  • Pro- Activin B-Fc is a more potent activin antagonist than Pro-Activin A-Fc, however, it is less specific as it blocks the activity of both activin isoforms.
  • C The potency of Pro-Activin B-Fc to inhibit activin B activity (IC50 0.65nM) was 3-fold lower than the common activin antagonist, follistatin (IC50 0.22nM), but was greater than the soluble activin type II receptors (ActRIIA and ActRIIB).
  • follistatin, sActRIIA and sActROB antagonise the actions of multiple TGF- ⁇ proteins, including activin A, activin B, myostatin and GDF- 1 1.
  • Figure 8 illustrates initial testing of the modified propeptides of the present invention in mice.
  • AAV6-modified activin A prodomain 10 viral genomes
  • AAV6 vector 10 10 or 10" viral genomes
  • mice received empty AAV6 vector alone ( 10 viral genomes) in their left TA. Mice were culled after 4 weeks and the mass of the left and right TA muscles were measured. Results are presented graphically: Activin A caused the anticipated 30% decrease in muscle mass in the left TA, but the activin effect in the right TA was inhibited by co-expression of the modified activin A prodomain (at the 10 1 0 viral dose). At the higher viral titre ( 10 1 ' ), the modified activin A prodomain not only inhibited the activin response in the injected right TA, but also reduced activin-induced muscle wasting in the left TA.
  • Figure 9 provides amino acid and nucleotide sequences of wild-type Pro- Activin A/FLAG/FC.
  • Figure 10 provides amino acid and nucleotide sequences of modified Pro- Activin A/FLAG/FC-Fastener.
  • Figure 11 provides amino acid and nucleotide sequences of wild-type Pro- Activin B/FLAG/FC.
  • Figure 12 provides amino acid and nucleotide sequences of modified Pro- Activin B/FLAG/FC-Fastener.
  • FIG. 13 illustrates the potency and specificity of Pro-Activin A-Fc and Pro- Activin B-Fc in vivo.
  • AAV6 adeno-associated viral vector
  • TA right tibialis anterior
  • Table 1 provides a description of the SEQ ID NOs provided herein.
  • Table 2 provides exemplary amino acid substitutions. DETAILED DESCRIPTION OF EMBODIMENTS
  • Reference herein to amino acid sequences or nucleic acid sequences of TGF- ⁇ family propeptide means propeptide sequences from any organism, though preferably mammalian or human, including wild-type or polymorphic sequences that are naturally occurring (native).
  • TGF- ⁇ family ligands/propeptides includes TGF- ⁇ ⁇ , TGF- 2, TGF-P3 , activin B, activin A, activin C, activin E, myoslatin, GDF 1 1 , BMP7, BMP5, BMP6, BMP8A, BMP8B, BMP2, BMP4, BMP10, GDF2, GDF5, GDF6, GDF7, BMP3, BMP3B, lefty 1 , lefty2, GDF 1 , GDF3, NODAL, BMP 15, GDF9. GDF 1 5, MIS and inhibin.
  • a non exhaustive list of active TGF- ⁇ family propeptides include activin B, activin A, activin C, activin E, BMP7, BMP5, BMP6, BMP8A, BMP8B, BMP2, BMP4, BMP 10, GDF2, GDF5, GDF6, GDF7, BMP3, BMP3B, lefty 1 , lefty2, GDF 1 , GDF3, NODAL, BMP15, GDF9, GDF15, MIS and inhibin.
  • Various standard approaches may be used by the skilled addressee to obtain, synthesis, or manipulate polypeptides and nucleic acid sequences defining these molecules or function fragments thereof as discussed further herein.
  • the subject invention is not limited to particular screening procedures for agents, specific formulations of agents and various medical methodologies, as such may vary.
  • the inventors determined that biologically active propeptides of TGF- ⁇ ligands can be produced independently of their mature domains. Further, modification of an active propeptide, activin A to include the N- terminal domain of TGF- ⁇ 1 results in a polypeptide that antagonises activin A signalling and retains the binding specificity of activin A propeptide for mature activin A.
  • the chimeric propeptide also bound to highly related mature activin B but failed to bind related mature myostatin or GDF 1 1 , to which TGF- ⁇ ⁇ propeptide binds.
  • Example 1 the inventors generated a modified activin B propeptide having an Fc domain (construct 3, Figure 5) and demonstrated its ability to potently and specifically inhibit activin A and activin B but not myostatin or GDF1 1 ( Figure 7).
  • the inventors also replaced the non-conserved region of the activin A prodomain ( 107 IGRRAEMNELMEQTSE 122 ) with the "fastener region" of the myostatin prodomain (' I0 DYHATTET 1 17 ) (SEQ ID NO: 8) (see construct 4A, Figure 5).
  • Corresponding modifications were made to the activin B propeptide (4B).
  • a FLAG tag (see construct 2B, Figure 5) was added to the C-terminus of the activin B prodomain, followed by the Fc domain of murine IgG2A (see construct 3B, Figure 5).
  • modified propeptides of TGF family ligands which are suitably in isolated, synthetic, recombinant or purified form.
  • the modified propeptides unlike the wild-type propeptide is able to down regulate the receptor binding activity of its nature protein. Accordingly, the modified propeptide will find broad application as compositions including pharmaceutical compositions.
  • the present invention provides modified propeptides of "active" ⁇ family ligands wherein the propeptides are modified by direct or indirect covalent linkage to a dimerization domain.
  • the modified propeptide comprising a dimerization Fc domain has a greater affinity for the mature domain relative to the affinity of the propeptide without the dimerization domain.
  • the dimerization domain is covalently linked to the C- terminal end of the propeptide.
  • the propeptide- dimerization domain is produced as a fusion protein.
  • the present specification discloses a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 1 , or a functional fragment thereof, the propeptide is linked to a dimerization domain.
  • the present specification discloses a propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 1 , or a functional fragment thereof, the propeptide is covalently linked to an immunoglobulin Fc domain.
  • the phrase "functional fragment”, as used herein, means a portion of the referenced propeptide that retains at least 80% of the activity including binding or antagonist activity of the parent molecule. Function can be assessed by assays known to those skilled in the art, such as those described in the Detailed Description and Examples section.
  • Reference to "functional fragments” includes propeptides without their native signal sequence.
  • the term also includes nucleic acid molecules encoding propeptides without their native signal sequences. As known in the art, native signal sequences may be, if required, replaced with non-native (heterologous) signal sequences in order to optimise expression in a range of environments. In order to avoid confusion and in art recognised procedure, the residue numbering of amino acid residues or nucleic acid bases remains constant between full length propeptide sequences and functional fragments or other parts thereof.
  • the dimerization domain is an exogenous/heterologous dimerization domain several of which are known in the art.
  • a suitable dimerization domain may be selected from the group comprising: immunoglobulin-fc hinge region/dimerization domain: LexA; yeast GCN4; bacteriophage Mu Gin invertase; E. coli NTRC; HSV- 1 ICP4; CH3; Zinc finger; fos; and a Jun leucine zipper.
  • Linker sequences may also be employed together with dimerization domains. Suitable linker sequences are discussed in review articles by George et. al, Protein Eng. 15:871 -9, 2002, and Argos, P., J. Mol. Biol.
  • Gly and Ser may consist of up to 20 amino acid residues such as Gly and Ser, and include, and comprise amino acids selected from the sequence group consisting of Gly, Ser, Ala, Thr and Arg, more particularly Gly- and Ser-Ser-Gly (GSSG).
  • Suitable linker sequences include, by way of example, the sequences: (Gly)2-Ala-(Gly) 2 , (Gly) 5 or (Gly) 8 , (Gly) 6 , (Gly) 7 or (Gly) 10 , Gly-Ser-Gly-Ser-Gly, (Gly) 4 , Gly-Ala-Gly, (Gly) 2 -Arg- (Gly) 2 -Ser, Ser-(Gly) 2 -Ser-Gly.
  • Reference herein to an "immunoglobulin Fc domain" and “Fc domain” includes a portion of an immunoglobulin heavy chain comprising at least a hinge region.
  • the Fc domain comprises at least hinge region and all or part of the CHI , CH2 and CH3 domains or all or part of the CH2 and CH3 domains or CH2 or CH3 domains of an IgG l , lgG2, IgG3 or IgG4 molecule based upon human, mammalian or other vertebrate sequences, and variants thereof known in the art.
  • the selection of a particular isotype or variant from known isotypes or variants is made to enhance pharmacological activity.
  • the skilled person may select a form or variant selected based upon its ability not to activate complement or to bind to certain Fc receptors. Residues in the hinge and CH2 domain are known to be important for Fc binding and complement binding. Plasmid vectors (for example, pFUSE-Fc from InvivoGen) are available for producing Fc fusion proteins using a range of species IgG of various isotype. All such ' naturally occuring sequences or variants known in the art are encompassed.
  • the present specification discloses a propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, or a functional fragment thereof, the propeptide is linked to a dimerization domain.
  • SEQ ID NO: 19 illustrates a modified activin A propeptide modified by inclusion of fastener residues as described in Example 1.
  • SEQ ID NO: 18 illustrates a nucleic acid sequence encoding a modified activin A propeptide modified by inclusion of fastener residues.
  • the amino acid sequence of an illustrative modified activin A propeptide including an immunoglobulin Fc dimerization domain and FLAG peptide is set out in SEQ ID NO: 23.
  • An illustrative encoding nucleic acid sequence is set out in SEQ ID NO: 22 (See Table 2).
  • the amino acid sequence of the propeptide comprises SEQ ID NO: 8 or a sequence comprising conservative amino acid substitutions thereof, or a functional fragment thereof.
  • the amino acid residue at position 65 of activin A is a basic amino acid (eg Lys, Arg or His).
  • the amino acid at residue 65 is Lys.
  • the amino acid residue at position 64 is a small amino acid residue (eg Ser or Thr or Pro).
  • the amino acid residue at position 64 is Ser.
  • the immunoglobulin Fc domain is an IgG2 Fc.
  • the propeptide comprising the amino acid sequence of SEQ ID NO: 1 1 and SEQ ID NO: 15 or of SEQ ID NO: 19 and SEQ ID NO: 15.
  • the present specification discloses a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, or a functional fragment thereof, wherein the propeptide is covalently linked to a dimerization domain.
  • the present specification discloses a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, or a functional fragment thereof, the propeptide is linked to an immunoglobulin Fc domain.
  • propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 29, or a functional fragment thereof, the propeptide is linked to a dimerization domain.
  • the present specification discloses a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 29, or a functional fragment thereof, the propeptide is covalently linked to an immunoglobulin Fc domain.
  • the amino acid sequence comprises SEQ ID NO: 9 or a sequence comprising one or more conservative amino acid substitution thereof, or a functional fragment thereof.
  • modified activin B propeptide comprises SEQ ID NO: 9.
  • Tyr 103 , Tyr 104 and Ala 105 are invarient among TGF- ⁇ isoforms whereas myostatin and GDF1 1 use "Tyr, His, Ala”.
  • SEQ ID NO: 8 and SEQ ID NO: 9 employ "Tyr, His, Ala” although “Tyr Tyr Ala” are contemplated together with flanking "Tyr, Tyr, Glu, Tyr” and "Glu, Asp, Asp, Asp” or "Glu, Glu, Asp, Glu” sequences.
  • the modified propeptide comprises the amino acid sequence of SEQ ID NO: 29.
  • the propeptide comprises the amino acid sequence of SEQ ID NO: 25 and SEQ ID NO: 15 or SEQ ID NO: 29 and SEQ ID NO: 15. in other embodiments, one or more cleavage sites are silenced as shown in the art or as described herein.
  • the specification provides a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 1 , or a functional fragment thereof, with the proviso that the amino acid residues include SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variant thereof.
  • the modified propeptide comprises an amino acid sequence that is: at least 95% identical to SEQ ID NO: 1 1 , SEQ ID NO: 19, SEQ ID NO: 25 or SEQ ID NO: 29 or a functional fragment thereof; at least 98% identical to SEQ ID NO: 1 1 , SEQ ID NO: 19, SEQ ID NO: 25 or SEQ ID NO: 29 or a functional fragment thereof; or at least 99% identical to SEQ ID NO: 1 1 , 19, 25 or 29 or a functional fragment thereof.
  • the propeptide comprises an amino acid sequence of one of SEQ ID NO: 1 1 , SEQ ID NO: 19, SEQ ID NO: 25 and SEQ ID NO: 29 or a functional fragment thereof.
  • Conservative amino acid variants include conservative amino acid substitutions and these are known to those skilled in the art. They can be naturally-occurring or artificially introduced. Exemplary conservative amino acid substitutions are described in Table 3. In the context of SEQ ID NO: 8 or SEQ ID NO: 9, these include the important "fastener residues" from myostatin and GDF1 1 , and conservative variants of SEQ ID NO: 8 and SEQ ID NO: 9 extend to corresponding residues from other active TGF- ⁇ propeptide such as ⁇ - ⁇ , -2 or -3 as shown in Figure 4.
  • residue 64 of the propeptide is Ser or a conservative amino acid substitution thereof and residue 65 is Lys or a conservative amino acid substitution thereof.
  • the specification provides a modified propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, or a functional fragment thereof, with the proviso that the amino acid sequences includes SEQ ID NO: 8 or SEQ ID NO: 9 or a conservative variant thereof.
  • residue 64 is Ser or a conservative amino acid substitution thereof and residue 65 is Lys or a conservative amino acid substitution thereof.
  • the specification provides a propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, or a functional fragment thereof, with the proviso that the amino acid sequence includes SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variants thereof.
  • the specification provides a propeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 29, or a functional fragment thereof, with the proviso that the amino acid sequence includes SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variants thereof.
  • variant peptides encompassed by the present invention are biologically active, that is, they continue to possess the antagonistic biological activity of the propeptide or the ability to confer higher affinity binding as described herein. Amino acid modifications are preferably conservative amino acid substitutions.
  • amino acid sequence variants of peptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad. Set. USA. 82: 488-492, 1985; Kunkel et al . Methods in Enzymol 154: 367-382, 1987; U.S. Patent No. 4,873, 192; Watson et al., "Molecular Biology of the Gene", Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987 and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al, Atlas of Protein Sequence and Structure, Natl. Biomed. Res. Found., Washington, D.C., 1978.
  • Variant propeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to a parent (e.g. , naturally-occurring or reference) amino acid sequence.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:
  • Acidic The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Amino acids having an acidic side chain include glutamic acid and aspartic acid.
  • Basic The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g. , histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Amino acids having a basic side chain include arginine, lysine and histidine.
  • the residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e. , glutamic acid, aspartic acid, arginine, lysine and histidine).
  • amino acids having acidic or basic side chains i.e. , glutamic acid, aspartic acid, arginine, lysine and histidine.
  • Hydrophobic The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.
  • Neutral/polar The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.
  • amino acids having a small side chain include glycine, serine, alanine and threonine.
  • the gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains.
  • proline differs from all the other natural-occurring amino acids in that its side chain is bonded to the nitrogen of the a-amino group, as well as the oc-carbon.
  • amino acid similarity matrices e.g. , PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al, (1978), A model of evolutionary change in proteins. Matrices for determining distance relationships In M. O. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp.
  • proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.
  • Amino acid residues can be further sub-classified as cyclic or non-cyclic, and aromatic or non-aromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large.
  • the residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not.
  • Small residues are, of course, always non-aromatic.
  • amino acid residues may fall in two or more classes.
  • Conservative amino acid substitution also includes groupings based on side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity as described herein.
  • amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains.
  • the first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains;
  • the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine;
  • the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).
  • a predicted non-essential amino acid residue in a propeptide is typically replaced with another amino acid residue from the same side chain family.
  • a "nonessential" amino acid residue is a residue that can be altered from the reference sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially alter one of these activities, for example, the activity is at least 60%, 70% or 80% of the reference sequence.
  • an "essential" amino acid residue is a residue that, when altered from the wild-type sequence of a reference polypeptide, results in abolition of an activity of the parent molecule such that less than 20% of the activity of the reference polypeptide is present.
  • essential amino acid residues include those that are conserved in polypeptides across different species.
  • propeptides of the present invention may be prepared by any suitable procedure known to those of skill in the art. Recombinant propeptides can be conveniently prepared using standard protocols as described for example in Sambrook, et ai, 1989 (supra), in particular Sections 13, 16 and 1 7; Ausubel et ai . Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994, in particular Chapters 10 and 16; and Coligan. et ai , 1995- 1997 (supra), in particular Chapters 1 , 5 and 6. Methods of purification include size exclusion, affinity or ion exchange chromatography/separation.
  • the identity and purity of peptides is determined for example by SDS-polyacrylamide electrophoresis or chromatographically such as by high performance liquid chromatography (HPLC).
  • the propeptides or parts of the propeptides may be synthesized by chemical synthesis, e.g. , using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard (supra) and in Roberge et ai, (1995, Science, 269: 202).
  • the propeptides are prepared by recombinant techniques.
  • the propeptides of the invention may be prepared by a procedure including the steps of: (a) preparing a construct comprising a nucleic acid sequence that encodes a propeptide and that is operably linked to a regulatory element; (b) introducing the construct into a host cell; (c) culturing the host cell to express the nucleic acid sequence to thereby produce the encoded peptide; and (d) isolating the propeptide from the host cell.
  • nucleic acid variants can be naturally-occurring (native), such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally- occurring.
  • Naturally-occurring nucleic acid variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as known in the art.
  • Non-naturally occurring polynucleotide variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms.
  • the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non- conservative amino acid substitutions (as compared in the encoded product) and glycosylation variants.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of a reference polypeptide.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a polypeptide.
  • Variants of a particular nucleic acid sequence will have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs known in the art using default parameters.
  • the immunoglobulin domain will possess at least about 80% sequence identity with a native or parent Fc domain or a hinge region thereof, preferably at least about 95% identity with a native or parent IgG2 Fc domain or a hinge region thereof, IgGl Fc or a hinge region thereof, or IgG4 Fc domain or a hinge region thereof.
  • Variants are generally made and selected in order to improve their binding affinity and/or stability and to reduce any unwanted effects. Cysteines in particular may be modified.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I, U) or the identical amino acid residue (e.g., Ala, Pro, Ser,, Thr, Gly, Val, Leu, He, Phe, l yr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i. e.
  • the identical nucleic acid base e.g., A, T, C, G, I, U
  • the identical amino acid residue e.g., Ala, Pro, Ser,, Thr, Gly, Val, Leu, He, Phe, l yr, Trp, Lys, Arg, His, Asp,
  • a comparison window may comprise additions or deletions (i.e. , gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. , resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • the invention provides a purified nucleic acid molecule that comprises a nucleotide sequence encoding the herein described propeptides including variants having substantial sequence identity or ability to cross hybridise under stringent hybridisation conditions, synonomous codon variants and codon optimised variants thereof.
  • stringency refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization and washing procedures. The higher the stringency, the higher will be the degree of complementarity between nucleic acid sequences that remain hybridized after washing.
  • high stringency etc refers to temperature and ionic conditions under which only nucleotide sequences having a high frequency of complementary bases will hybridize. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization.
  • nucleic acid constructs encoding a modified propeptide as described herein or a functional fragment thereof.
  • Illustrative nucleic acid sequences characterizing the subject nucleic acid molecules are set out in Table 2 and include SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30 and suitable combinations thereof.
  • the invention extends to vectors and other constructs comprising isolated nucleic acid molecules including those capable of expressing (producing) the subject propeptides and to isolated host cells comprising same.
  • vector is meant a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a nucleic acid molecule can be inserted or cloned.
  • a vector may contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e.
  • a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication e.g. , a linear or closed circular plasmid, an extra-chromosomal element, a mini- chromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self-replication.
  • the vector can be one which, when introduced into the * host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector is preferably a viral or viral-derived vector, which is operably functional in animal and preferably mammalian cells.
  • Such vector may be derived from a poxvirus, an adenovirus or yeast.
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art and include the nptll gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene that confers resistance to the antibiotic hygromycin B.
  • Vectors useful for expressing the subject nucleotide sequence in subject host cells in vivo are known to those of skill in the art and are expressly contemplated. They include adenoviral vectors and adeno-associates virus vectors. Illustrative vectors include AAV8 or AAV6 described for example in Qiao el ai, Human Gene Therapy, /9. 00-000 (March 2008).
  • host cells comprising a nucleic acid construct encoding a modified propeptide as described herein, wherein the host cell expresses the propeptide.
  • Host cells are conveniently eukaryotic cells include mammalian, plant, yeast and insect cells as known in the art. Recombinant proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the modified propeptide in the host cells or, more preferably, secretion of the protein into the culture medium in which the host cells are grown.
  • Suitable mammalian cell lines include, but are not limited to, BHK, VERO, HT1080, HEK-293, -293T, -293F, 293T-Rex, RD, COS-7, CHO, Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2, MT-4.
  • H9, PM 1 , CEM, myeloma cells (e.g., SB20 cells) and CEM 174 are available, for example, from the ATCC.
  • Other host cells include without limitation yeast, e.g. Pichia pastoris, or insect cells such as Sf9 cells although such molecules would not ordinarily be glycoslyated.
  • the present invention provides a method of treating or preventing activin-induced conditions, such as muscle wasting, fibrosis, inflammation in a subject, the method comprising administering to the subject a compound comprising a modified propeptide as described herein or a nucleic acid construct encoding same which provides the modified propeptide to the subject.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, or a functional fragment thereof, the propeptide is linked to an immunoglobulin Fc domain.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, or a functional fragment thereof, the propeptide is linked to an immunoglobulin Fc domain.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29, or a functional fragment thereof, the propeptide is linked to an immunoglobulin Fc domain.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 1 , or a functional fragment thereof, with the proviso that the amino acid residues include SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variants thereof.
  • residue 64 of the propeptide comprising SEQ ID NO: 1 1 is Ser or a conservative amino acid substitution thereof and residue 65 Lys or a conservative amino acid substitution thereof.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, or a functional fragment thereof, with the proviso that the amino acid residues include SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variants thereof.
  • residue 64 is Ser or a conservative amino acid substitution thereof and residue 65 is Lys or a conservative amino acid substitution thereof.
  • the propeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25 or SEQ ID NO: 29, or a functional fragment thereof, with the proviso that the amino acid residues include SEQ ID NO: 8 or SEQ ID NO: 9 or conservative variants thereof.
  • Subjects contemplated in the present invention are humans or animals including laboratory or art accepted test or vehicle animals. “Subjects” include human subjects in need of treatment or prophylaxis.
  • compositions comprising a propeptide or nucleic acid as herein described.
  • the term “compound” includes “medicament”, “composition” and “phamacologically acceptable compound” or “pharmaceutical composition” and the like.
  • the composition comprises a pharmaceutically or physiologically acceptable carrier or diluent.
  • the polypeptides are for use in the treatment or prevention of conditions or symptoms of conditions promoted or exacerbated by TGF- ⁇ family ligand signalling.
  • compositions are conveniently prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Company, 1990. These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral or parenteral.
  • an effective amount includes “therapeutically effective amount” and “prophylactically effective amount” and mean a sufficient amount of a compound either in a single dose or as part of a series or slow release system which provides the desired therapeutic, preventative, or physiological effect in some subjects.
  • Undesirable effects e.g. side effects, may sometimes manifest along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining an appropriate "effective amount”.
  • the exact amount of composition required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact 'effective amount'.
  • treatment refers to any measurable or statistically significant amelioration in at least some subjects in one or more symptoms of a condition associated with disregulated or overactive TGFP-ligand signalling in a subject.
  • Prophylactic administration of the compound serves to prevent or attenuate onset of symptoms of a condition associated with disregulated or overactive TGFp-ligand signalling in a subject.
  • a "pharmacologically acceptable” composition is one tolerated by a recipient patient.
  • a "pharmaceutically acceptable carrier and/or a diluent” is a pharmaceutical vehicle comprised of a material that is not otherwise undesirable i.e., it is unlikely to cause a substantia] adverse reaction by itself or with the active composition.
  • Carriers may include all solvents, dispersion media, coatings, antibacterial and antifungal agents, agents for adjusting tonicity, increasing or decreasing absorption or clearance rates, buffers for maintaining pH, chelating agents, membrane or barrier crossing agents.
  • a pharmaceutically acceptable salt is a salt that is not otherwise undesirable.
  • the agent or composition comprising the agent may be administered in the form of pharmaceutically acceptable non-toxic salts, such as acid addition salts or metal complexes.
  • compositions can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed.
  • Tablets may contain a binder such as tragacanth, com starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active composition can be encapsulated to make it stable to passage through the gastrointestinal tract. See for example, International Patent Publication No. WO 96/1 1698.
  • the composition may be dissolved in a carrier and administered as a solution or a suspension.
  • appropriate penetrants known in the art are used for delivering the composition.
  • delivery uses any convenient system such as dry powder aerosol, liquid delivery systems, air jet nebulizers, propellant systems.
  • the formulation can be administered in the form of an aerosol or mist.
  • the compositions may also be delivered in a sustained delivery or sustained release format.
  • biodegradable microspheres or capsules or other polymer configurations capable of sustained delivery can be included in the formulation.
  • Formulations can be modified to alter pharmacokinetics and biodistribution.
  • the formulations may be incorporated in lipid monolayers or bilayers such as liposomes or micelles.
  • Targeting therapies known in the art may be used to deliver the agents more specifically to certain types of cells or tissues.
  • the present therapeutic or prophylactic methods are suitable for treating or preventing cachexia-anorexia syndrome. In some another embodiments the present therapeutic or prophylactic methods are suitable for treating or preventing activin-induced inflammatory conditions. In some embodiments the activity of activin A is reduced. In other embodiments the activity of activin A and B is reduced.
  • the invention provides a method for down regulating the activity of endogenous activin A in a subject, the method comprising administering a modified activin A propeptide or a modified activin B propeptide as described herein or a nucleic acid construct encoding same.
  • the invention provides a method of down regulating the activity of endogenous activin A and B in a subject, the method comprising administering a modified activin B propeptide as described herein or a nucleic acid construct encoding same.
  • the invention provides a method of down regulating the activity of endogenous B in a subject, the method comprising administering a modified activin B propeptide as described herein or a nucleic acid construct encoding same.
  • modified activin propeptides and nucleic acid constructs including expression vectors comprising same, for use in therapy or in the manufacture of a medicament for use in therapy.
  • the specification describes a process for producing a modified TGF- ⁇ family propeptide, the method comprising co-expressing the propeptide together with an immunoglobulin Fc domain directly or indirectly covalently linked to the N- terminus or C-terminus of the propeptide, wherein the presence of the Fc domain enhances the affinity of the modified propeptide for its mature domain.
  • Recombinant propeptides for example, are conveniently produced by transfecting host cells with a nucleic acid molecules encoding the modified propeptide, culturing the host cell to express the recombinant propeptide and isolating or purifying the propeptide.
  • the modified TGF- ⁇ propeptides of the present invention preferably have an enhanced affinity for their mature domains compared to the affinity of the propeptide before modification.
  • the TGF- ⁇ propeptides selected for modification are those propeptides which, in their native form, have a lower affinity for their mature domain than the affinity between the mature domain and a signalling receptor, that is, the mature wild type ligand is typically expressed in active form.
  • most TGF- ⁇ propeptides can be thus characterised.
  • Such propeptides do not include, in some embodiments, TGF- ⁇ , ⁇ - ⁇ 2, ⁇ - ⁇ 3, myostatin and GDF 1 1.
  • the specification describes a process for producing a modified TGF- ⁇ family propeptide, wherein the pre-modified form of the propeptide has a lower affinity for its mature domain than the affinity between the mature domain and a signalling receptor, the method comprising co-expressing as a fusion protein the propeptide together with an immunoglobulin Fc domain directly or indirectly covalently linked to the N-terminus or C-terminus of the propeptide, wherein the presence of the Fe domain enhances the affinity of the modified propeptide for its mature domain.
  • the specification describes a process for producing an activin antagonist, the method comprising providing a nucleic acid encoding an activin propeptide, such as SEQ ID NO: 10 or SEQ ID NO: 24 or a functional fragment thereof and introducing a sequence of bases encoding the peptide of SEQ ID NO: 8 or SEQ ID NO: 9 of a conservative variant thereof in the region of activin propeptide corresponding to the fastener region of ⁇ , wherein the presence of SEQ ID NO: 8 or SEQ ID NO: 9 of a conservative variant thereof enhances the affinity of the modified propeptide for its mature domain.
  • an activin propeptide such as SEQ ID NO: 10 or SEQ ID NO: 24 or a functional fragment thereof
  • the encoding nucleic acid such as SEQ ID NO: 10 is further modified if required to encode Ser at the residue corresponding to Ser at residue 55 of ⁇ or a conservative amino acid substitution thereof, and Lys at the residue corresponding to Lys at residue 56 of ⁇ (see Figure 4) or a conservative amino acid substitution thereof.
  • the region of activin propeptide corresponding to the fastener region of TGF i is the region corresponding to 102-130 of INHBA or 122 to 150 of ⁇ as described in Example 1.
  • the present invention is further directed to the use of the herein described polypeptides in, or in the manufacture of a medicament for, the treatment or prevention of a condition or one or more symptoms of a condition promoted or exacerbated by a TGF- ⁇ family ligand signalling.
  • anatagonists are proposed for use inter alia in treating the pathological consequences of over expression by one or more TGF- ⁇ family ligands such as of activin B, activin A, activin C, activin E, active BMP7, BMP5, BMP6, BMP8A, BMP8B, BMP2, BMP4, BMP10, GDF2, GDF5, GDF6, GDF7, BMP3, BMP3B, lefty 1 , lefty2, GDF1 , GDF3, NODAL, BMP1 5, GDF9, GDF 15, MIS and inhibin.
  • TGF- ⁇ family ligands such as of activin B, activin A, activin C, activin E, active BMP7, BMP5, BMP6, BMP8A, BMP8B, BMP2, BMP4, BMP10, GDF2, GDF5, GDF6, GDF7, BMP3, BMP3B, lefty 1 , lefty2, GDF1 , GDF3, NODAL, BMP1 5, GDF9, GDF
  • the present invention provides a method for treatment or prevention of a condition or one or more symptoms of a condition promoted or exacerbated by activin signalling in a subject said method comprising administering to a subject in need an effective amount of a composition comprising an activin antagonist as described herein.
  • Activin antagonists are proposed for use inter alia in restoring homeostasis, enhancing liver growth and repair, treating inflammatory conditions, such as sepsis, the . prevention of cachexia-anorexia syndrome.
  • BMP-2 antagonists are proposed for use inter alia in promoting hair growth and to block decidualization required for pregnancy establishment.
  • BMP-4 antagonists are proposed for use inter alia in promoting hair growth and to treat diseases associated with heterotopic bone formation, such as fibrodysplasia ossificans progressiva.
  • GDF-3 antagonists are proposed for use inter alia in control of diet-induced obesity and fat deposition.
  • BMP-3 antagonists are proposed for use inter alia in maintaining and enhancing bone formation.
  • BMP-7 antagonists are proposed for use inter alia in treating kidney and skeletal abnormalities.
  • BMP-5 antagonists are proposed for use inter alia in treating axial skeletal abnormalities.
  • BMP-6 antagonists are proposed for use inter alia in treating anaemia of inflammation due to hepcidin excess.
  • BMP-8A and BMP-8B antagonists are proposed for use inter alia in the regulation of spermatogenesis.
  • BMP- 10 antagonists are proposed for use inter alia as anti-angiogenic agents capable of blocking vascularization.
  • GDF-2 antagonists are proposed for use inter alia as anti-angiogenic agents capable of blocking vascularization.
  • GDF-5 antagonists are proposed for use inter alia to treat diseases associated with skeletal abnormalities, such as symphalangism proximal syndrome and multiple synostoses syndrome type 2.
  • GDF-6 antagonists are proposed for use inter alia in the treatment of ocular and skeletal anomalies.
  • BMP-3 antagonists are proposed for use inter alia in maintaining and enhancing bone formation.
  • Nodal antagonists are proposed for use inter alia in regulating endometrial remodelling and in reducing tumour cell aggressiveness.
  • BMP- 15 antagonists are proposed for use inter alia in the regulation of folliculogenesis.
  • GDF-9 antagonists are proposed for use inter alia in the regulation of folliculogenesis.
  • GDF- 15 antagonists are proposed for use inter alia for the treatment of cancer anorexia and weight loss, as well as of obesity.
  • TGF- ⁇ ligands including GDF-7, BMP-3B, GDF-1 and MIS
  • further characterisation of their physiological roles in the adult are required before therapeutic applications for their antagonism become apparent.
  • Still other TGF- ⁇ ligands including inhibin A, inhibin B, Leftyl and Lefty2, act as antagonists of other family members.
  • Antagonising the actions of inhibin A would promote activin A signalling, which may be desirable in certain settings.
  • the present invention provides propeptides, and their encoding nucleic acid molecules, which are suitably in isolated, recombinant or purified form.
  • isolated and purified means material that is substantially or essentially free from components that normally accompany it in its native state.
  • an isolated nucleic acid molecule refers to a nucleic acid or polynucleotide, isolated from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • an isolated propeptide includes in vitro isolation and/or purification of a propeptide from association with other components of the cell.
  • an isolated nucleic acid, polynucleotide, propeptide, peptide, or polypeptide can refer to a sequence that is isolated by purification and to a sequence that is produced by recombinant or synthetic means.
  • recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e. , through the expression of a recombinant polynucleotide.
  • Polypeptide “peptide,” “protein” and “propeptide” are used interchangeably herein to refer to molecules comprising or consisting of a polymer of amino acid residues and to variants and synthetic analogues of the same.
  • Lumi-light Western blotting substrate was from Roche (Basel, Switzerland), BioXact short DNA polymerase was from Bioline (Taunton, MA), PCR/plasmid purification kits were from Promega (Madison, Wl), and DMEM, Opti-MEM and SeeBlue Plus2 were from Invitrogen (San Diego, CA).
  • 125 I-Activin A was prepared using the chloramine T method as previously described (Harrison et al. Endocrinology 147: 2744-2753, 2006).
  • LbetaTl cell bioassay The in vitro bioactivities of the AT propeptide, follistatin, soluble ActRIIA and soluble ActRIIB were determined based on their ability to suppress the release of FSH by a mouse pituitary gonadotrope cell line ( ⁇ 2). ⁇ 2 cells were plated in 48-well plates at a density of 2.5 x 10 3 cells/well. The cells were allowed to recover for 24 h in DMEM supplemented with 10% fetal calf serum.
  • FSH levels were determined by a specific rat FSH immunofluorometric assay as previously described (Makanji et ai. J Biol Chem 283: 16743- 16751 , 2008) employing reagents kindly provided by A. Grootenhuis and J. Verhagen of (N.V. Organon).
  • the sensitivity of the assay was 12.5 pg/well (human inhibin A immunoreactive preparation).
  • the mean index of precision ( ⁇ ) was 0.078.
  • NIH3T3 cell bioassay - To assess the effect of the an AT propeptide on TGF- ⁇ activity, NIH-3T3 cells stably expressing the TGF-p-responsive pCAGA reporter were used (kindly provided by Dr Hong-Jian Zhu, University of Melbourne). Stable ⁇ -3 ⁇ 3 cells were plated at 15,000 cells/well in DMEM/10%FCS and treated the following day with 80 pM TGF- ⁇ and increasing doses of the AT propeptide or the TGF- ⁇ ⁇ propeptide as a positive control.
  • solubilization buffer 1 % Triton X-100, 25 mM glycylglycine (pH 7.8), 15mM MgS04, 4 mM EGTA, and 1 mM dithiothreitol
  • reporter activity was measured.
  • Receptor binding assays - HEK293T cells were plated at 10 5 cells/well in 24 well plates coated with poly-D-lysine. The following day, the cells were transfected with 15 ng of vector (pcDNA3.1 ) control, ActRIIA or ActRIIB cDNA using the Lipofectamine transfection reagent and incubated for 48 h at 37 °C. The cells were washed in binding buffer (DMEM/0.1 % bovine serum albumin) and incubated for 4h at room temperature
  • HE -293T cells are transfected with a Smad2/3-responsive reporter (A3-luc) and FAST-2 (a transcription factor).
  • Smad2/3-responsive reporter A3-luc
  • FAST-2 a transcription factor
  • BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP-15 modified propeptides as antagonists
  • HEK-293T cells are transfected with a Smadl/5/8-responsive reporter (BRE-Iuc).
  • BRE-Iuc Smadl/5/8-responsive reporter
  • transfected cells are treated with a fixed dose of a TGF- ⁇ family ligand together with increasing doses of the inhibitory propeptide.
  • modified propeptide is contacted with 2 or 3 ligands that should not be inhibited.
  • an immunoglobulin Fc domain was incorporated into the AT propeptide.
  • the Fc domain was also added to the wild type activin A propeptide.
  • the presence of the Fc domain on the wild type activin A propeptide increased its affinity for activin A, such that it was at least as good an activin antagonist as the AT propeptide.
  • a FLAG tag (see construct 2A, Figure 5) was added to the C-terminus of the activin A prodomain, followed by the Fc domain of murine IgG2A (see construct 3 A, Figure 5).
  • the Fc region comprises the CH2 and CH3 domains of the IgG heavy chain and the hinge region.
  • the hinge serves as a flexible spacer between the two parts of the Fc- Fusion protein, allowing each part of the molecule to function independently.
  • activin A prodomain lacks the key 'fastener' residues (Tyr, Tyr/His and Ala; see Figure 4); it retains relatively high sequence homology with myostatin and GDF-1 1 in the surrounding regions (see below).
  • a FLAG tag (see construct 2B, Figure 5) was added to the C-terminus of the activin B prodomain, followed by the Fc domain of murine lgG2A (see construct 3B, Figure 5).
  • this fusion protein was expressed in HE 293f cells, it was found to be proteolytically cleaved (see Figure 6). However this did not occur in the other cells such as COS7 cells as shown in Figure 6.
  • the activin B prodomain lacks the key 'fastener' residues (Tyr, Tyr/His and Ala; see Figure 4); it retains relatively high sequence homology with myostatin and GDF-1 1 in the surrounding regions (see above).
  • the non-conserved region of the activin B prodomain 123 IPHLDGHASPGADGQERVSE 142 ; shaded grey, above) were substituted with the 'fastener' region of the myostatin prodomain ( 107 EDDDYHATTET' 17 ) (SEQ ID NO:9) (see construct 4B, Figure 5).
  • HE 293F cells were transfected with Pro-Activin A-Fc (construct 5A, Figure 5) or Pro-Activin B-Fc (construct 4B, Figure 5). After 48h, conditioned medium was collected and analysed by SDS-PAGE and Western blot. Pro-Activin A-Fc was secreted as a covalent dimer of 120 kDa ( Figure 6A).
  • Pro-activin B-Fc was secreted in various forms: (i) a 120 kDa covalent dimer; (ii) a 105 kDa form (presumed to be generated by proteolysis of one prodomain chain); and (iii) a 90 kDa form (presumed to be generated by proteolysis of both prodomain chains).
  • the 90 and 105 kDa forms of Pro-Activin B-Fc are not expected to have any inhibitory activity as they lack the prodomain regions ( l and a2 helicies) that contact the mature domain.
  • COS7 cells were transfected with Pro-Activin B- Fc (construct 4B, Figure 5).
  • HEK293T cells were transfected with a Smad2/3 -responsive luciferase reporter and 24 h later were stimulated with 200 pM activin A ( ⁇ ), activin B ( ⁇ ), myostatin ( A ) or GDF 1 1 ( ⁇ ), in the absence or presence of increasing concentrations of Pro-Activin A-Fc (construct 5 A, Figure 5).
  • HEK293T cells were transfected with a Smad2/3-responsive luciferase reporter and 24 h later were stimulated with 200 pM activin A ( ⁇ ), activin B ( ⁇ ), myostatin ( A ) or GDF 1 1 ( ⁇ ), in the absence or presence of increasing concentrations of Pro-Activin B-Fc (construct 3B, Figure 5). After 24 h, cells were harvested in solubilization buffer [1% Triton X- 100, 25 mM glycylglycine (pH 7.8), 15 mM MgS04, 4 mM EGTA, and 1 mM dithiothreitol], and the luciferase activity was measured.
  • solubilization buffer 1% Triton X- 100, 25 mM glycylglycine (pH 7.8), 15 mM MgS04, 4 mM EGTA, and 1 mM dithiothreitol
  • Pro-Activin B-Fc antagonised both activin A (IC50 0.4nM) and activin B (IC50 0.65nM) activity, but had no effect on myostatin or GDF l 1 signalling (data not shown). As such, Pro-Activin B-Fc is a more potent activin antagonist than Pro-Activin A-Fc, however, it is less specific as it blocks the activity of both activin isoforms.
  • C The potency of Pro-Activin B-Fc to inhibit activin B activity (IC50 0.65nM) was 3-fold lower than the common activin antagonist, follistatin (IC50 0.22nM), but was greater than the soluble activin type II receptors (ActRIIA and ActRIIB). Importantly, follistatin, sActRIIA and sActRIIB antagonise the actions of multiple TGF- ⁇ proteins, including activin A, activin B, myostatin and GDF-1 1.
  • HE 293T cells were transfected with a Smad2/3-responsive luciferase reporter and 24 h later were stimulated with 200 pM activin A ( ⁇ ), activin B ( ⁇ ), myostatin ( A ) or GDF 1 1 ( ⁇ ), in the absence or presence of increasing concentrations of Pro-Activin A-Fc (construct 5A, Figure 5).
  • solubilization buffer [1 % Triton X- 100, 25 mM glycylglycine (pH 7.8), 15 mM MgS04, 4 mM EGTA, and 1 mM dithiothreitol]
  • Pro-Activin A-Fc specifically antagonised activin A activity (IC50 5nM) (see Figure 7A). Even at high doses of Pro- Activin A-Fc (40nM), activin B, myostatin and GDF 1 1 signalling was not inhibited.
  • HE 293T cells were transfected with a Smad2/3 -responsive luciferase reporter and 24 h later were stimulated with 200 pM activin A ( ⁇ ), activin B ( ⁇ ), myostatin ( A ) or GDF l 1 ( ⁇ ), in the absence or presence of increasing concentrations of Pro-Activin B-Fc (construct 4B, Figure 5).
  • Pro-Activin B-Fc is a more potent activin antagonist than Pro-Activin A-Fc, however, it is less specific as it blocks the activity of both activin isoforms.
  • the potency of Pro-Activin B-Fc to inhibit activin B activity (IC50 0.65nM) was 3-fold lower than the common activin antagonist, follistatin (IC50 0.22nM), but was greater than the soluble activin type II receptors (ActRIIA and ActRIIB) (see Figure 7C).
  • follistatin, sActRIlA and sActRIIB antagonise the actions of multiple TGF- ⁇ proteins, including activin A, activin B, myostatin and GDF-1 1.
  • AAV6-activin A vector 10 viral genomes in both the left and right tibialis anterior
  • AAV6-modified activin A prodomain comprising construct 5A; 10 10 or
  • AAV6 vector 10 1 1 viral genomes was co-delivered to the right TA, whereas empty AAV6 vector (10 10 or 10 1 1 viral genomes) was co-delivered to the left TA (See Figure 8).
  • a group of control 10 1 1 viral genomes was co-delivered to the right TA, whereas empty AAV6 vector (10 10 or 10 1 1 viral genomes) was co-delivered to the left TA (See Figure 8).
  • mice received empty AAV6 vector alone (10 viral genomes) in their left TA. Mice were culled after 4 weeks and the mass of the left and right TA muscles were measured. Results are presented graphically in Figure 8.
  • Activin A caused the anticipated 30% decrease in muscle mass in the left TA, but the activin effect in the right TA was inhibited by co- expression of the modified activin A prodomain (at the 10 1 ° viral dose).
  • the modified activin A prodomain At the higher viral titre (lo" ), the modified activin A prodomain not only inhibited the activin response in the injected right TA, but also reduced activin-induced muscle wasting in the left TA. This strongly suggests that the modified activin A prodomain can get into the circulation and act at distant sites.
  • the subject propeptide to specifically antagonise activin signalling suggests they will be useful for preventing the development of cachexia- anorexia syndrome in response to activin-secreting tumours (Zhou et al, Cell 142(4): 53 1 - 543, 2010).
  • upregulated activin A modulates the release of key proinflammatory cytokines in response to endotoxemia and is negatively correlated with survival
  • the subject activin propeptides may have therapeutic potential for treating inflammatory disorders, such as sepsis.
  • propeptide comprising fastener encoding residues from myostatin and GDF 1 1 ( Figure 10)
  • modified Activin B comprising fastener residues from TGF family prodomain e.g. myostatin, ( Figure 12)

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Abstract

L'invention concerne un propeptide modifié comprenant une séquence d'acides aminés qui est identique à au moins 90 % à la SEQ ID NO: 11 ou SEQ ID NO: 19 ou un fragment fonctionnel correspondant, ledit propeptide étant lié par covalence à un domaine de dimérisation ou à un acide nucléique codant celui-ci. Cette invention concerne également un propeptide modifié comprenant une séquence d'acides aminés qui est identique à au moins 90 % à la SEQ ID NO: 25, ou un fragment fonctionnel correspondant, ledit propeptide étant lié par covalence à un domaine de dimérisation ou à un acide nucléique codant celui-ci. Cette invention concerne aussi des compositions pharmaceutiques renfermant ces composés. L'invention concerne en outre une méthode pour traiter ou prévenir une atrophie musculaire induite par l'activine. Cette invention se rapporte aussi à une méthode pour traiter ou prévenir un syndrome d'anorexie-cachexie. L'invention concerne par ailleurs une méthode pour réguler à la baisse l'activité d'une activine A et/ou d'une activie B endogène.
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US10869909B2 (en) 2012-03-27 2020-12-22 Ngm Biopharmaceuticals, Inc. Compositions and methods of use for treating metabolic disorders
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US9828415B2 (en) 2013-01-30 2017-11-28 Ngm Biopharmaceuticals, Inc. Compositions and methods of use for treating metabolic disorders
US9827291B2 (en) 2013-01-30 2017-11-28 Ngm Biopharmaceuticals, Inc. Compositions and methods of use for treating metabolic disorders
US9161966B2 (en) 2013-01-30 2015-10-20 Ngm Biopharmaceuticals, Inc. GDF15 mutein polypeptides
US10323075B2 (en) 2013-01-30 2019-06-18 Ngm Biopharmaceuticals, Inc. Compositions and methods of use for treating metabolic disorders
US11358995B2 (en) 2014-07-30 2022-06-14 Ngm Biopharmaceuticals, Inc. Compositions and methods of use for treating metabolic disorders
US9834586B2 (en) 2014-07-30 2017-12-05 Ngm Biopharmaceuticals, Inc. Compositions and methods of use for treating metabolic disorders
US9920118B2 (en) 2014-10-31 2018-03-20 Ngm Biopharmaceuticals, Inc. Compositions and methods of use for treating metabolic disorders
US10562965B2 (en) 2014-10-31 2020-02-18 Ngm Biopharmaceuticals, Inc. Compositions and methods of use for treating metabolic disorders
US11530260B2 (en) 2014-10-31 2022-12-20 Ngm Biopharmaceuticals, Inc. Compositions and methods of use for treating metabolic disorders
WO2016120647A3 (fr) * 2015-01-30 2016-11-17 Debreceni Egyetem Régénération musculaire
WO2016120647A2 (fr) 2015-01-30 2016-08-04 Debreceni Egyetem Régénération musculaire
WO2016181177A2 (fr) 2015-05-14 2016-11-17 Alu-Öntö Kft. Procédé de coulage et moule en sable pourvu d'un système d'admission pour produire des pièces coulées en aluminium à parois au moins partiellement minces avec la technologie de moulage au sable au moyen d'un coulage par gravité
US10174119B2 (en) 2016-03-31 2019-01-08 Ngm Biopharmaceuticals, Inc. Binding proteins and methods of use thereof
US10975154B2 (en) 2016-03-31 2021-04-13 Ngm Biopharmaceuticals, Inc. Binding proteins and methods of use thereof
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

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