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WO2001009368A1 - Antagonistes de la follistatine - Google Patents

Antagonistes de la follistatine Download PDF

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Publication number
WO2001009368A1
WO2001009368A1 PCT/US2000/020444 US0020444W WO0109368A1 WO 2001009368 A1 WO2001009368 A1 WO 2001009368A1 US 0020444 W US0020444 W US 0020444W WO 0109368 A1 WO0109368 A1 WO 0109368A1
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Prior art keywords
activin
binding
follistatin
antagonist
domain
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PCT/US2000/020444
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English (en)
Inventor
Henry T. Keutmann
Alan Schneyer
Yisrael Sidis
Patrick Sluss
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The General Hospital Corporation
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Publication of WO2001009368A1 publication Critical patent/WO2001009368A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the field of the invention is biochemistry, cell biology, physiology, endocrinology and oncology.
  • Follistatin is a monomeric- glycoprotein whose only known function is to bind and neutralize growth factors, primarily activin. Activins are members of the TGF-jS superfamily of growth and differentiation factors.
  • FS Human FS exists naturally in several isoforms. Cloning and characterization of the human FS gene revealed that alternative splicing results in two mRNA transcripts encoding FS polypeptides of 288 (FS288) and 315 amino acids (FS315) , following signal cleavage (Shimasaki et al . , 1988, Proc. Natl . Acad. Sci . USA 85:4218-4222). Proteolytic processing yields a third FS polypeptide, which contains 303 amino acids (FS303) . Due to these differences in polypeptide length and differences in posttranslational glycosylation, the molecular weight of FS ranges from about 32 kD to about 44 kD.
  • mature FS After removal of a 29-amino acid signal sequence (leader) , mature FS contains 5 distinct structural domains, each encoded by a different exon: an N-Domain of 63 amino acids; FS Domains I, II, and III, which are three 73-75- amino acid repeats of the "follistatin module" (Hohenester et al., 1997, EMBO J. 16:3778-3786; Eib et al . , 1996, J. Neurochem. 67:1047-1055; Denzer et al . , 1998, EMBO J. 17:335-343); and, in FS315, a highly acidic C-Domain of 27 amino acids.
  • FS Domains I, II, and III which are three 73-75- amino acid repeats of the "follistatin module” (Hohenester et al., 1997, EMBO J. 16:3778-3786; Eib et al . , 1996, J. Neuro
  • follistatin A notable structural feature of follistatin is the large number of cysteine residues in the polypeptide.
  • FS is expressed in a variety of mammalian tissues, including ovary, testis, prostate, kidney, muscle, uterus, brain, pancreas and pituitary.
  • FS binds heparin sulfated proteoglycans exposed on cell membranes. This results in localized concentration of FS at the cell surface.
  • This cell surface-associated FS retains its activin-binding ability and forms a barrier, which impedes the access of extracellular activin to activin receptors in the cell membrane (Delbaere et al . , 1999, Endocrinology 140:2463- 2470)
  • Activins are dimeric proteins that function as potent, local regulators of cell growth (Vale et al . , "Reproductive and other roles of inhibins and activins," In: Knobil and Neill (Eds.) The Physiology of Reproduction, Raven Press, New York, pp 1861-78) .
  • Activin action is tissue-dependent. For example, activin stimulates cell growth in ovarian tissues. In contrast, activin inhibits basal and androgen-stimulated proliferation, and induce apoptosis, in human prostatic cancer cells (Wang et al . , 1996, Endocrinology 137:5476-5483) .
  • FS binds to (and neutralizes) activin in vivo and in vitro. This binding is essentially irreversible under physiological conditions, resulting in biologically inactive FS-Act complexes (Nakamura et al., 1990, Science 247:836- 838; Kogawa et al . , 1991, Endocrinology 128 : 1434-1440; Schneyer et al . , 1994, Endocrinology 135 : 667-674.
  • FS limits the activity of activin in a given tissue, or limits the range of tissues in which activin is active, or both. All known isoforms of FS display similar activin binding characteristics (de Winter et al., 1996, Mol . Cell . Endocrinol . 116:105-114; Inouye et al., 1991, Endocrinology 129:815-822) .
  • the FS antagonist includes amino acids 3- 63 of a non-activin-binding FS N-Domain (amino acids 3-63 of SEQ ID NO:l), or amino acids 3-61 of a non-activin-binding FLRG N-Domain (amino acids 3-61 of SEQ ID NO: 2) ; and a heparin sulfate-binding moiety; wherein the tryptophan residue (W) at position 4 or 36 of the non-activin-binding N-Domain (from FS or FLRG) is substituted by A, G, L, I, V, P, S, T, M, C, N, Q, D, E, K, R, Y or H.
  • the non-activin-binding N-Domain is located N-terminally, relative to the heparin sulfate-binding moiety.
  • the heparin sulfate-binding moiety contains the "heparin sulfate site" of native FS (amino acids 74-87 of SEQ ID NO:l) .
  • the heparin sulfate-binding moiety can include amino acids 64-136 of SEQ ID NO:l, 64-211 of SEQ ID NO:l, or amino acids 64-288 of SEQ ID NO:l.
  • the tryptophan residue (W) at position 4 or 36 of the non-activin-binding N-Domain from FS or FLRG is substituted by A, G, S, T, N, Q, R, D or E. More preferably, it is substituted by A or G, and most preferably, A.
  • the invention also features a nucleic acid encoding the above-described FS antagonist, and a vector containing that nucleic acid.
  • the vector can include expression control sequences operatively linked to the nucleic acid encoding the FS antagonist.
  • the invention also includes a host cell transformed with the vector.
  • the invention also includes a method of increasing activin activity in an activin-sensitive tissue in a mammal.
  • the method includes contacting the cells of the activin- sensitive tissue with the FS antagonist.
  • contacting the cells of the activin-sensitive tissue with the FS antagonist is accomplished by expressing the FS antagonist in vivo from a vector containing a nucleic acid encoding the FS antagonist polypeptide. This method can be used where the activin-sensitive tissue is prostate tissue.
  • Fig. 1 is the amino acid sequence of FS288 (SEQ ID NO:l) .
  • the signal (leader) sequence, the N-Domain, Domains I-III, and the C-Domain are indicated. Domains I-III are shown with the 10 cysteine residues in each domain aligned.
  • the heparin sulfate site is indicated in Domain I, and the epitopes recognized by three different monoclonal antibodies are indicated.
  • Figs. 2A-2C are a histograms summarizing data on activin binding, heparin binding, and 7FS30 binding, respectively, by synthetic peptides corresponding to different segments of human FS.
  • Fig. 3 is a histogram summarizing data on activin binding by synthetic peptides FS1-26 and FS44-59 each alone, and each in competition with native follistatin FS288, or native follistatin FS315.
  • Fig. 4A is a line graph showing dose-response curves for activin binding by synthetic peptides FS1-26, FS3-26, FS6-26, FS14-26 and FS10-26.
  • Fig. 4B is a line graph showing done-response curves for activin binding by a series of synthetic peptides corresponding to FS amino acids 44-59, 47-59, 53-59, 49-59, 51-59 and 44-59 scrambled (negative control) .
  • Fig. 5 is a histogram summarizing data on activin binding by a series of mutant FS288 proteins containing specific, site-directed mutations. Activin binding is expressed as % of control, with native FS288 being the positive control. Non-conditioned medium is the negative control.
  • W4A tryptophan 4 changed to alanine
  • R6A arginine 6 changed to alanine
  • K9A lysine 9 changed to alanine
  • KW(48,49)AA lysine 48 and tryptophan 49 both changed to alanine
  • M50E methionine 50 changed to glutamate .
  • Fig. 6 is a sequence comparison showing the amino acid sequence of the N-Domain of FS (amino acids 1-63 of SEQ ID NO:l) and the N-Domain of FLRG (SEQ ID NO: 2) .
  • the mature FS antagonist is FS288 (amino acids 1-288 of SEQ ID NO:l) in which the critical tryptophan residues at positions 4 and 36 are replaced by alanine residues. Replacement of either tryptophan residue reduces activin binding by more than 90%, and results in a useful antagonist. Replacement of both further reduces activin binding.
  • the FS antagonist efficiently binds heparin sulfate by virtue of a basic (positively charged) region of the FS Domain I called the "heparin sulfate site" (amino acids 74-87 of SEQ ID NO:l) .
  • the acidic (negatively charged) FS C-Domain (amino acids 289-315 of SEQ ID NO:l) is not included in the FS antagonist polypeptide, because if present, it would undergo intramolecular binding to the heparin sulfate site. This would prevent the FS antagonist from binding to heparin sulfate exposed on cell membranes, thereby impairing the in vivo effectiveness of the FS antagonist.
  • the above-described FS antagonist can be isolated from a recombinant expression system, or chemically synthesized, and then delivered, as an exogenous polypeptide, to cells in a target tissue.
  • the FS antagonist is expressed in vivo from a suitable vector introduced into the cells of a target tissue.
  • in vivo expression provides a continuous supply of the FS antagonist in intimate contact with the target cells (cells of the target tissue) , for a period of several weeks, or indefinitely, depending on the type of vector employed. This is important because optimal effectiveness of the FS antagonist occurs when it has displaced endogenous FS from the heparin sulfate sites on the target cell surface.
  • the above-described FS antagonist is expressed initially with a signal sequence (amino acids (-)29-(-)l of SEQ ID NO:l) at its N-terminus.
  • the signal sequence causes the FS antagonist polypeptide to be secreted from the cell.
  • the cell cleaves the signal sequence during the secretion process, thereby yielding the mature form of the FS antagonist.
  • the mature FS antagonist As the mature FS antagonist emerges at the cell surface, it is in proximity to the heparin sulfate groups on the cell surface, and binding of the FS antagonist to the heparin sulfate groups occurs readily (if binding has not already occurred) .
  • the native FS can be displaced by the FS antagonist through competitive binding. The extent to which such displacement occurs depends on the relative amounts of native FS and FS antagonist.
  • the local concentration of FS antagonist is increased, e.g., by use of strong, constitutive promoter to drive in vivo expression of the FS antagonist from an FS antagonist transgene.
  • the FS antagonist displays a structure extremely similar to natural, native FS. Because of such similarity, the cellular machinery treats the FS antagonist as if it were native FS, a molecule naturally present in the cell . Consequently, the FS antagonist causes minimal disruption to normal cellular processes, i.e., the FS antagonist's side effects are minimized. In addition, because the cell treats the FS antagonist as if it were native FS, the antagonist undergoes any FS-specific transport or localization that takes place in the target cell or target tissue. This is advantageous, because to the extent that any localization of native FS takes place, the FS antagonist is similarly localized so as to maximize its competition with the native FS.
  • a critical tryptophan residue position 4 or 36 of FS Domain I
  • the substituted amino acid is not phenylalanine.
  • Replacement of a critical tryptophan residue by phenylalanine does not substantially diminish activin binding.
  • Substitute amino acids with small, non-hydrophobic chains are preferred, e.g., alanine and glycine.
  • FS antagonist when expressed initially with a signal sequence, it is not necessary to use the signal sequence of natural, human FS (amino acids (-)29-(-)l of SEQ ID NO:l).
  • suitable, eukaryotic signal sequences are known in the art and can be employed in the FS antagonist.
  • the FS antagonist incorporate a mutated N-Domain of FLRG instead of a mutated N-Domain of FS.
  • the tryptophan at position 4 or position 36 (or both) is mutated to a non- aromatic amino acid, e.g., alanine, just as in a mutated FS N-Domain.
  • the FS antagonist can include one or more amino acid substitutions in the non-activin-binding FS N-Domain (SEQ ID NO:l), or the non-activin-binding FLRG N- Domain. Preferably, such substitutions are conservative amino acid substitutions.
  • those domains can include one or more amino acid substitutions.
  • substitutions (1) do not involve the heparin sulfate site; (2) do not involve cysteine residues; (3) do not create highly acidic regions likely to interfere with the heparin sulfate-binding function of the heparin sulfate site; and (4) are conservative amino substitutions.
  • the complete nucleotide sequence of human FS gene exons 1-6 is found in Shimasaki et al . , 1988, Proc. Natl . Acad. Sci . USA 85:4218-4222; and in GenBank (Accession Nos. M19480 and M19481) . Using this sequence information with commercially available PCR technology, DNA encoding human FS288, including the FS signal sequence can be prepared routinely and reliably, without undue experimentation.
  • RNA is isolated from a sample of a human tissue type that expresses FS, e.g., ovarian tissue.
  • the RNA is then used for synthesis of cDNA.
  • the desired coding sequence is amplified by standard PCR techniques.
  • Well-known, PCR-based, site-directed mutagenesis techniques are applied to the FS-encoding DNA to obtain a DNA encoding FS288 in which tryptophan residue 4 and/or tryptophan residue 36 are replaced with a suitable amino acid, e.g., alanine. This yields a complete FS antagonist coding sequence.
  • the FS antagonist coding sequence is then inserted into an expression vector suitable for use in mammalian cells.
  • Suitable expression vectors are commercially available.
  • alternative methods of reliably obtaining the DNA of the invention are known and can be used. For recombinant DNA techniques, see generally,
  • Vectors suitable for delivery of DNA encoding an FS antagonist to a target tissue are known in the art.
  • a viral or nonviral vector can be used.
  • At least one nonviral vector has been used successfully for intraprostatic gene therapy.
  • Preliminary studies with this vector have demonstrated the safety and efficacy of intratumoral injection of liposome-DNA complexes. This is described in Naitoh et al . , 1998, "Intraprostatic interleukin-2 (IL-2) gene therapy:
  • the therapeutic gene product is secreted by transformed cells. Consequently, it is not necessary to achieve genetic transformation of all cells in the target tissue to achieve beneficial results. For example, in the treatment of benign prostate hyperplasia or prostate cancer, transformation of a substantial number of prostate cells will result in secretion of the FS anagonist.
  • the secreted FS antagonist then can be utilized by non-transformed prostate cells neighboring the transformed cells.
  • the anatomic location of the human prostate facilitates intraprostatic delivery of DNA vectors to treat local disease.
  • routes are transperineal and transrectal.
  • a transrectal ultrasound probe is used to guide the placement of needles through the perineum into the prostate .
  • An advantage of the transperineal route is decreased risk of infection, as compared to the transrectal route.
  • Transrectal delivery typically is performed in a manner similar to an ultrasound-guided biopsy of the prostate.
  • Fig. 1 shows the peptides synthesized and the position of the follistatin sequences they represent within the native FS protein.
  • Synthetic peptides were designed to: (1) represent the entire FS N-Domain for mapping the activin binding site; (2) represent the heparin sulfate binding site; (3) represent the human-specific antigenic epitope of human FS; (4) represent intra-cysteine sequences from each of the "follistatin module" domains of the FS288 core protein.
  • Peptides were also used to generate site-directed monoclonal antibodies for use as structural probes of the C-termini of FS288 and FS315. Progressive N-terminal truncations were introduced in the two peptides identified as containing potential activin-binding epitopes.
  • the monoclonal antibody recognizing FS Domain II was generated in mice immunized with recombinant human FS 288 essentially as previously described (MacDonald et al . , Biol . Reprod. 52/Supplement, 77; McConnell et al . , 1998, J. Clin . Endocrinol . Metabol . 83:851-858). Monoclonal antibodies against FS Domain III and the C-Domain were generated against synthetic peptides representing amino acid residues 274-287 and 300-315, respectively. Mice were immunized with protein or synthetic peptide-BSA conjugates emulsified in Freund's Complete Adjuvant (Sigma Chemical Co., St. Louis, MO) .
  • Booster immunizations were composed of protein or peptide conjugated to BSA in Freund's Incomplete Adjuvant emulsifants. Both primary and booster immunizations were administered subcutaneously. After achieving suitable serum titers, spleen cells were harvested and fused with SP2/0 myeloma cells obtained from the ATCC (Bethesda, MD) . Hybridomas were selected by standard dilution cloning and enzyme immunoassay methods. Monoclonal antibodies were purified using protein G affinity chromatography from ascites fluid generated from pristane-primed mice inoculated (ip) with 5 x 10 s hybridoma cells. Harlan Laboratories Inc.
  • mice (Indianapolis, ID) generated and harvested the ascites fluids. The use of mice in these studies was performed in compliance with NIH guidelines. Experimental procedures were reviewed and approved by an internal review board for the use of animal resources. Animals were housed and cared for in AAALAC accredited facilities operated by the Massachusetts General Hospital or Harlan Laboratories.
  • FS peptides or full-length FS were coated onto Immulon-4 ELISA plates (Dynex Technologies, Chantilly, VA) by incubating 100 uL of the appropriate solution at 4° C overnight.
  • FS peptides were initially dissolved in 0.1 M acetic acid to 10 mg/mL and then diluted with 0.1 M sodium bicarbonate to 0.3, 1, 3, 10, 30 or 100 ⁇ g/mL.
  • Native FS was coated in a similar fashion using a 0.2 ⁇ g/mL solution.
  • Non-specific binding sites on ligand-coated (peptide or full-length FS) plates were blocked by incubation with 200 ⁇ L of a blocking solution (PBS containing 0.05% gelatin and 0.05% Tween-20) at room temperature for 2 hrs . Binding of [ 125 I] -activin, [ 3 H] -heparin, or monoclonal anti-follistatin IgG was determined by incubating these probes in a total volume of 200 uL in ligand-coated or control wells at room temperature for 4 hrs with gentle shaking. After incubation, unbound probes were decanted and the plates were washed 3 times with PBS-0.05% Tween-20 to reduce non-specific binding.
  • PBS containing 0.05% gelatin and 0.05% Tween-20
  • Bound [ 125 I] -activin was measured by direct counting of the wells (inserted into 12 x 75 mm plastic tubes) in a gamma counter. Tritiated heparin bound to peptide or follistatin was measured by submerging washed wells in 10 mL of liquid scintillation fluid in a 20 mL glass scintillation vial and counted in a beta counter (Beckman, Los Angeles, CA) .
  • Bound antibody was measured with an ELISA reader (405 nM absorbance) after sequentially incubating with an anti- mouse IgG-alkaline phosphatase conjugate (Pierce Chemical Co., Chicago, IL; 1/4000 dilution of conjugated antibody) then a PNPP substrate (Pierce Chemical Co.; 2.7 mM solution) .
  • Iodine-labeled activin was prepared using lactoperoxidase and purified by gel eletrophoresis as previously described (Schneyer et al . , 1994, Enodocrinology 135:667-674). The specific activity of the [ 125 I] -activin was approximately 30 ⁇ Ci/ ⁇ g. Tritiated heparin was obtained from New England Nuclear (Boston, MA) .
  • FS288 in the absence or presence (5 and 50 ng/ml) of activin was allowed to bind to monoclonal antibody 7FS30 coated on the bottom of ELISA wells by incubating at room temperature for 12 h. After removal of unbound FS by washing, tritiated heparin was added and incubated at room temperature for an additional 3 hr to allow its binding to follistatin (free or activin-bound) captured by the solid- phase antibody.
  • the stable pyridylethylcysteine residues formed were quantitated by amino acid analysis (Beckman model 6300 analyzer) after total acid hydrolysis (6N HCI, 110 C, 24 hr) . N-terminal sequence analysis to characterize Cys at position 3 was performed on the Applied Biosystems Model 477A microsequencer.
  • Fig. 2A Data on binding of recombinant human activin-A to to peptides representing linear sequences from human FS are summarized in Fig. 2A.
  • Activin bound to only two synthetic fragments, and FS 44-59 , of the N-Domain (amino acids 1- 63) .
  • no activin binding was observed to synthetic peptides- representing epitopes with the FS Domains I, II, or III.
  • Fig. 2 Also shown in Fig. 2 is the mapping of tritiated heparin (Figure 2B) and monoclonal antibody 7FS30 ( Figure 2C) using this series of synthetic peptides. Heparin binding mapped to separate epitopes within the N-Domain and FS Domain I, respectively. Heparin binding to the 74-86 fragment confirmed the previously identified heparin binding site.
  • the monoclonal antibody 7FS30 bound only to the 168-178 fragment within the FS Domain II.
  • Figs. 2 and 3 Specific activin binding to two non-adjacent, linear sequences (Figs. 2 and 3) indicated that there may be at least two distinct activin binding epitopes (1-26) and (44- 59) located with the FS N-Domain.
  • a series of N-terminal truncated peptides was synthesized. Each series of peptides successively truncated at the N-terminus was directly tested as solid-phase ligands for ability to bind activin.
  • Fig. 4A summarizes the results of mapping the binding of activin to peptide FS 1-26 .
  • the equipotent activin binding displayed by peptide and peptide FS 3-26 showed amino acid residues 1 and 2 of FS not to be required for activin binding.
  • Fig. 4B shows data summarizing the mapping of the
  • FS 3 . 26 and FS 47-59 bind activin in linearized native FS or constitute a conformation-dependent binding site(s) within the N-Domain.
  • Ligand blotting [ 125 I] -activin
  • SDS gel-electrophoresis of reduced or unreduced FS 288 was used to assess the ability of activin to bind epitopes within the unfolded protein.
  • Activin binding to FS288 depended on the disulfide bonding of the protein (data not shown) .
  • the lower limit of detection by ligand blotting was approximately 1 ⁇ g per lane. No activin binding was detected when 20 ⁇ g of reduced FS was loaded.
  • the cysteine residue at position 3 is part of an activin binding mimotope (Figs. 4A and 4B) . Its importance was indicated by loss-of-function mutants created by amino acid insertions between residues 2 and 3 of follistatin. This cysteine could be part of a disulfide bond in the native FS protein, or it could be present in an unbonded, reduced state. If Cys 3 is reduced in the native protein, its free sulfhydryl group might participate in post-binding reactions between activin and follistatin, explaining the essentially irreversible behavior of the follistatin-activin complex. N-terminal composition and sequence analysis was performed after reacting FS288 with
  • compositional analysis revealed the pyridylethyl- cysteine residue eluting at 51 minutes .in the reduced vinylpyridine-treated control preparation to be absent from the derivatized, native FS (data not shown) .
  • each preparation was subjected to Edman microsequence analysis through 15 cycles. Phenylthiohydantoins representing pyridylethylcysteine, eluting between Tyr and Pro by on-line HPLC, were observed at positions 3 and 13 of the reduced, pyridylethylated control sample.
  • Tritiated heparin was used as a probe for the heparan sulfate binding site located in Domain I (Fig. 2B) .
  • Domain II was probed using a monoclonal antibody designated 7FS30, specific to the 168-178 sequence (Fig. 2C) .
  • Activin induced changes in FS Domains I or II were probed simultaneously using a "sandwich" type immunoassay composed of solid-phase antibody bound to plastic plates to capture follistatin and the tritiated heparin to detect the antibody-bound protein.
  • FS response curves (which require binding of both probes) were superimposable . Thus, activin binding did not impair recognition by specific probes to either Domain I or II.
  • activin binding to FS resulted in a marked decrease in recognition by the monoclonal antibody directed against the Domain III epitope (data not shown) .
  • Activin binding also effected changes in the C- Domain, found only in FS315.
  • activin binding enhanced antibody binding to the C-Domain epitope (data not shown) .
  • This observation suggested conformational changes associated with activin binding, resulting in a more favorable C-Domain antigenic/epitope, either through higher affinity recognition or an enriched ensemble of favorable conformers among the population of FS315 molecules.

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Abstract

L'invention concerne des antagonistes de la follistatine. Ces antagonistes sont des polypeptides comprenant un domaine N de FS de liaison non activine ou un domaine N de FLRG de liaison non activine; et une fraction de liaison sulfate-héparine; les residues de tryptophane (W) se trouvant à la position 4 ou 36 du domaine N de liaison non activine étant substitué par un acide aminé non aromatique tel que l'alamine. An example of a heparin sulfate-binding moiety is Domains I-III of human follistatin.
PCT/US2000/020444 1999-07-30 2000-07-27 Antagonistes de la follistatine WO2001009368A1 (fr)

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Cited By (7)

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US9399669B2 (en) 2007-02-02 2016-07-26 Acceleron Pharma Inc. Variants derived from ActRIIB
US9439945B2 (en) 2008-08-14 2016-09-13 Acceleron Pharma Inc. Isolated nucleotide sequences encoding GDF traps
US9480742B2 (en) 2005-11-23 2016-11-01 Acceleron Pharma Inc. Method of promoting bone growth by an anti-actriia antibody
US9919030B2 (en) 2008-06-26 2018-03-20 Acceleron Pharma Inc. Follistatin fusion proteins and uses thereof
US9975934B2 (en) 2015-03-26 2018-05-22 Acceleron Pharma Inc. Follistatin-related fusion proteins
US10010498B2 (en) 2014-06-04 2018-07-03 Acceleron Pharma Inc. Methods for treatment of amyotrophic lateral sclerosis with follistatin fusion proteins
US10023621B2 (en) 2014-06-04 2018-07-17 Acceleron Pharma Inc. Follistatin-related fusion proteins

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US6004937A (en) * 1998-03-09 1999-12-21 Genetics Institute, Inc. Use of follistatin to modulate growth and differentiation factor 8 [GDF-8] and bone morphogenic protein 11 [BMP-11]

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WO1989001945A1 (fr) * 1987-08-28 1989-03-09 The Salk Institute For Biological Studies Follistatine et procede de purification
US5366964A (en) * 1988-12-15 1994-11-22 Lindstrom Richard L Viscoelastic solution
US5703048A (en) * 1992-09-16 1997-12-30 Genentech, Inc, Protection against liver damage by HGF
US5942420A (en) * 1997-11-17 1999-08-24 Millennium Biotherapeutics, Inc. Molecules of the follistatin-related protein family and uses therefor
US6004937A (en) * 1998-03-09 1999-12-21 Genetics Institute, Inc. Use of follistatin to modulate growth and differentiation factor 8 [GDF-8] and bone morphogenic protein 11 [BMP-11]

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9480742B2 (en) 2005-11-23 2016-11-01 Acceleron Pharma Inc. Method of promoting bone growth by an anti-actriia antibody
US10239940B2 (en) 2005-11-23 2019-03-26 Acceleron Pharma Inc. Method of promoting bone growth by an anti-actriia antibody
US9399669B2 (en) 2007-02-02 2016-07-26 Acceleron Pharma Inc. Variants derived from ActRIIB
US9919030B2 (en) 2008-06-26 2018-03-20 Acceleron Pharma Inc. Follistatin fusion proteins and uses thereof
US9439945B2 (en) 2008-08-14 2016-09-13 Acceleron Pharma Inc. Isolated nucleotide sequences encoding GDF traps
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