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WO2007011265A1 - Procede de blocage d'activite des cibles specifiques a noggin2 - Google Patents

Procede de blocage d'activite des cibles specifiques a noggin2 Download PDF

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Publication number
WO2007011265A1
WO2007011265A1 PCT/RU2006/000382 RU2006000382W WO2007011265A1 WO 2007011265 A1 WO2007011265 A1 WO 2007011265A1 RU 2006000382 W RU2006000382 W RU 2006000382W WO 2007011265 A1 WO2007011265 A1 WO 2007011265A1
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Prior art keywords
nuggin2
protein
noggin2
activity
proteins
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PCT/RU2006/000382
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English (en)
Russian (ru)
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WO2007011265A8 (fr
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Andrei Georgievich Zaraisky
Fedor Mikhailovich Eroshkin
Andrei Vyacheslavovich Bairamov
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Andrei Georgievich Zaraisky
Fedor Mikhailovich Eroshkin
Bairamov Andrei Vyacheslavovic
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Publication of WO2007011265A1 publication Critical patent/WO2007011265A1/fr
Publication of WO2007011265A8 publication Critical patent/WO2007011265A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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

Definitions

  • This invention relates to proteins that regulate cell differentiation by inhibiting proteins of the superfamily, TGF- ⁇ and the EGF-CFC family. This invention was made using grant funds from the Howard Hughes Medical Institute (Howard Scientific Medical Institute). Under the terms of the grant, Howard Hughes Medical Institute is not entitled to this invention.
  • TGF- ⁇ transforming growth factor ⁇
  • BMP bone morphogenesis proteins
  • BMP antagonists include, for example, chordin, ventroptin, noggin, cerberus, and follistatin proteins.
  • the proteins cerberus and vertebrate leggin induce the formation of head structures in the anterior ectoderm of vertebrate embryos.
  • the noggin protein is also able to change the differentiation of mesoderm primordia from ventral primordia, such as blood and mesenchyme, into dorsal primordia, such as muscle or chorda, or differentiation of epidermal primordia into neural primordia.
  • Chordin functions are similar to those of noggin, reflecting a similar mechanism of action of these proteins as antagonists of BMP.
  • TGF- ⁇ superfamily growth factor antagonists are important pharmaceutical, clinical, and laboratory tools for regulation of cell differentiation and therapeutic intervention.
  • Proteins secreted into the EGF-CFC family of epidermal growth factors are also characterized by common structural motifs. Proteins of this family were found only in vertebrate animals and to date, their involvement in a number of basic processes of ontogenesis has been shown (Salop et al., Bioresaus (1999) 21: 61-70; Salop et al., Endocr.Relat.Cancer (2000) 7 : 199-226; Shep and Sc Schier, Treps Gepet. (2000) 16: 303-309). In addition, it was found that violations and, in particular, an increase in the activity level of these proteins can lead to cell transformation and to the formation of cancer tumors ip vivo (M.
  • nuggin2 plays a role in the regulation of cell differentiation and is an antagonist of some members of the TGF- ⁇ superfamily and the EGF-CFC family. Moreover, the spectrum and nature of the action of nuggins2 are different from those of noggin (formerly “noggin”). In particular, unlike noggin, noggin2 does not induce the development of muscle tissue from embryonic ventral mesoderm. In addition, it may interfere with the well-known activity of noggin in relation to the induction of muscle development. At the same time, like noggin, noggin2 can induce the development of nervous tissue from the embryonic ectoderm. Thus, nuggin2 has a unique selective activity in relation to the induction of the development of nervous tissue.
  • nuggin2 has the ability to inhibit the activity of embryonic mesoderm inducers such as div / xpr (Xprs from Xeporis gave relat).
  • the mechanism of inhibition of half / Xprs activity by the nuggin factor 2 does not consist in direct binding of its molecules to half / Xprs molecules, but in their binding to the crypto factor, the presence of which is necessary for the interaction between half / Xprs and a specific receptor.
  • the present invention relates to methods and compositions that use nuggin2 or a nucleic acid encoding it.
  • the present invention relates to a method for altering the activity of a specific Nuggin2 target of the BMP family (e.g. BMP2, BMP4, BMP7), Sirto, or a combination thereof in the body, tissue, or cell, comprising administering an effective amount of Nuggin2, anti-Nuggin2 antibody, or a combination thereof the specified organism, tissue or cell, where these combinations alter the activity of nuggin2-specific targets (i.e., administration of nuggin2 inhibits the activity of these targets, while administration of an antibody leads to velicheniyu their activity).
  • the invention relates to a method for blocking the activity of specific Noggin2 targets, comprising introducing Nuggin2 into a specified organism, tissue or cell in an amount effective to inhibit or reduce the activity of specific Noggin2 targets by inhibiting the binding of these targets or their fragments to specific protein receptors.
  • the present invention relates to a method for increasing the activity of noggin2-specific targets, comprising administering antibodies to nuggin2 into the specified organism, tissue or cell in an amount effective for increasing the activity of nuggin2-specific targets by binding of nuggins2.
  • the present invention relates to a composition for altering the activity of noggin2-specific targets, comprising an effective amount of nuggin2, anti-nuggin2 antibodies, or a combination thereof.
  • the invention relates to a composition for blocking the activity of noggin2 specific targets, containing nuggin2 in an amount effective to inhibit or reduce the activity of noggin2 specific targets by inhibiting the binding of said targets or fragments thereof to specific protein receptors.
  • the present invention relates to a composition for increasing the activity of specific nuggins2 targets, comprising antibodies against nuggins2 in an amount effective to increase the activity of specifics of nuggins2 by binding of nuggins2.
  • these methods and compositions are used to modify cell differentiation, where the specified method involves bringing the cell or the medium surrounding the cell into contact with the nuggin2 protein under conditions where nuggin2 specifically interacts with its target components of the medium and / or cell surface, for modification cell differentiation.
  • the exogenous protein nuggin2 is used; in another preferred embodiment, an expression construct is used comprising the nuggin2 encoding nucleic acid under the control of a suitable promoter.
  • a method for altering cell differentiation involves the creation of an expression construct comprising a nuggin2 encoding nucleic acid under the control of a suitable promoter; introducing said expression construct into a cell for expression of nuggin2 or a functional chimeric protein containing nuggin2, where the expression of nuggin2 or said chimeric protein leads to a change in cell differentiation.
  • Figure 1 shows the difference between the effects of nuggins and nuggins2 on the differentiation of nervous and muscle tissues in Heporys lavis embryos and their tissue explants.
  • A, B. Ventral microinjections of 40 pg of synthetic noggin gene mRNA in the X. lavis embryos at the early stages of development lead to the formation of an additional axis of the body (the black arrow indicates the main axis of the body, the gray arrow indicates the additional axis of the body), while microinjections of such the same amounts of synthetic nuggin mRNA lead to the formation of a mushroom-like phenotype (A: 80%, 68 out of 85 embryos in total; B: 90%, 68 out of 75 embryos in total).
  • Figure 2 shows the scheme (A) and the results (B) of experiments to study the effects of nuggins and nuggins2 in the explants of embryonic tissues.
  • A Scheme of experiments with ectoderm explants of the animal region of the embryo (animal caps - AC) and explants of the ventral marginal zone of the embryo (VMZ).
  • B Scheme of experiments with ectoderm explants of the animal region of the embryo (animal caps - AC) and explants of the ventral marginal zone of the embryo (VMZ).
  • noggin and noggin2 activate the expression of neural markers (NCAM, Xapp-1) and inhibit the expression of epidermal (kerati), while only moggin mRNA detects the ability to activate the expression of the muscle molecular marker ⁇ -actin in explants of the ventral marginal zone.
  • Fig. 3 shows the difference between the effects of nuggins and nuggins2 on the expression of genes broxuir, BF-I and Pax-6.
  • a 5 B, C, D, D, E - results of hybridization of ip sit and total preparations of Heporus lakarvis embryos with probes to the corresponding genes (the hybridization signal ip sit is shown in black);
  • G ', D', E ') - the same embryos are shown under ultraviolet light, which allows visualization of microinjected cells containing, in addition to the mRNA label - fluorescein-lysine-dextran (FLD).
  • A, A '. Suppression of the expression of a mesoderm molecular marker (braxuyr) in embryos microinjected with ngingin mRNA2.
  • a - green fluorescent dye shows the distribution of microinjected material.
  • B, B '. Expression of the mesoderm molecular marker ⁇ braskhuyr) in the embryos microinjected with moggin mRNA does not show changes compared with the control embryo (arrow).
  • B, B ', D, D. Formation of the additional axis of the body in embryos microinjected with 2-5 ng ngingin2 mRNA into one of the ventral blastomeres.
  • Pax-6 indicates the presence in the additional axes of the forehead and eye structures.
  • Figure 4 shows the results of the analysis of the ability of the nuggin2 protein to bind to potential partner proteins.
  • the Nuggin2 protein exhibits the ability to bind bone morphogenetic protein (BMP) molecules and the Cirto protein molecule and does not show the ability to bind activin and Xpr molecules.
  • BMP bone morphogenetic protein
  • ortholog refers to a polypeptide or protein derived from one species that is a functional copy of a polypeptide or protein of another species. Differences in ortholog sequences are the result of speciation.
  • the term “homologue” or “homology” represents is a term used in this field to describe the affinity of the nucleotide or protein sequence of another nucleotide or protein sequence, determined by the degree of identity and / or similarity between these compared sequences.
  • methods for changing the activity of certain members of the TGF- ⁇ superfamily and the EGF-CFC family are disclosed, as well as compositions containing the nuggin2 protein encoding its nucleic acid or anti-nuggin2 antibodies for use in them.
  • compositions for their practical use can be used in a number of research, diagnostic or therapeutic applications, including, but not limited to, inhibition or reduction of inflammation; regulation of bone reconstruction in the adult skeleton; the prevention and treatment of BMP-related disorders in animals, in particular humans; research and treatment of heart diseases and neurological disorders.
  • the present invention relates to the use of the specific function of nuggin2.
  • nuggin2 plays a role in the regulation of cell differentiation and differs from nuggins and other well-known growth factor antagonists belonging to superfamily TGF- ⁇ .
  • the authors also found that the nuggin2 proteins from Gallis gallis, having the sequences of SEQ ID NOs: 6 and 8, have the same biological properties as the nuggin2 from Heporys lavis.
  • noggin2 suppresses the general differentiation of the embryonic mesoderm, inhibiting the action of the mesoderm inducers by distance / hpg.
  • nuggin2 induces the development of nervous tissue from the embryonic ectoderm.
  • nuggin2 has a unique selective activity in relation to the induction of the development of nervous tissue.
  • the revealed ability of nuggins2 to inhibit dorsalizing activity of nuggins is the result of ⁇
  • an additional factor that binds to nuggin2 is the representative of the Epidemic Growth Factor Family (EGF-CFC) factor Sirto and related proteins, the activity of which is necessary for the induction of muscles from the embryonic mesoderm.
  • EGF-CFC Epidemic Growth Factor Family
  • the term “nuggin2” means a protein having a single and selective activity for inducing the development of neural tissue, binding to specific nuggin2 binding targets selected from the group consisting of BMP2, BMP4, BMP7, Sirto or a combination thereof, and being a homolog and an ortholog of the nuggin2 proteins selected from the group consisting of the nuggins2 of Xeporis lavis; nuggin2 Heporys trisalis; nuggin2 Figures and ribres; noggin2 gallis gallis (SEQ ID NO: 2, 4, 6, 8; corresponding Assessiop Nos in the Gepe Vapk database: AAX07470, AAX07471, AAX07474, AAX07476).
  • Noggin2-specific activity or function can be determined using known cell biology approaches in ip vitro or ip vivo systems, for example, ip vitro binding assays, analyzes in cell culture, in animals (e.g. immune response, gene therapy, transgenic animals, etc. .) etc.
  • Binding assays include any assay that evaluates the specific molecular interaction of nuggin2 with a binding target.
  • the target for binding may be a natural target for binding or non-natural target for binding, such as a specific immune protein, for example, an antibody.
  • binding specificity is assessed by biological analysis (for example, the ability to induce nerve tissue from an injected embryonic ectoderm) or by determining the binding constant of nuggin2 to its targets from the TGF- ⁇ superfamily, etc.
  • protein homologue a protein whose amino acid sequence is at least about 68%, typically at least about 75% and most often at least about 85% is identical to the known amino acid the nuggin2 sequences given in SEQ ID NO: 2, 4, 6, 8, as determined using the MegAligp algorithm, DNAstar clustal algorithm, as described in DG Niggps app R.M. Sharr, "Fast apd Sepsitive multirle Sequepse Aligpmepts on a Misrosomruter", CABIOS 5 May pp. 151-3 (1989) (using the multiplicity 1 parameters, the penalty for skipping 3, windows 5 and saved diagonals 5).
  • the homologues of interest have a much greater sequence identity, e.g., 90% (e.g., 92%, 93%, 94%) or higher, e.g., 95%, 96%, 97%, 98%, 99%, 99, 5%, especially for the amino acid sequence that forms the functional regions of the protein.
  • Tayuka are of interest to proteins that are substantially identical to nuggin2 proteins with the amino acid sequences of SEQ ID NO 2, 4, 6, 8, where significant identity means that the amino acid sequence of the protein is at least about 68% identical to that of the protein, as typically at least about 75% and most often at least about 80%, where in some cases the identity may be more than, for example, 85%, 90%, 95% or more.
  • Proteins of interest include naturally occurring proteins and recombinant proteins containing the amino acid sequence of nuggin2 or its functional protein fragments having definable analysis of the specific activity of nuggin2.
  • proteins can be deletion mutants of the natural nuggin2 proteins, and they can be obtained in the form of hybrid products containing the nuggin2 polypeptides or its functional protein fragments.
  • polypeptides typically at least about 30 amino acids long, typically at least about 50 amino acids long, preferably at least about 75 to 100 amino acids long, and can be up to 300 amino acids or more in length , but, as a rule, do not exceed the length of 250 amino acids, where the fragment contains a portion of the amino acid sequence identical to the protein in question, at least about 25 amino acids in length, and usually, at least 45 amino acids, and in many embodiments, at least 50 amino acids.
  • the subject polypeptides are approximately 25 amino acids, approximately 50, approximately 75, approximately 100, approximately 125, approximately 150, approximately 200, or approximately 250 amino acids in length, up to a full length protein.
  • the fragments of interest retain all or substantially all of the specific functions of the wild-type protein.
  • proteins that are mutants or derivatives of naturally occurring proteins with specific nuggin2 activity. Mutants are understood to be proteins obtained either as a result of a limited substitution of amino acid residues in noggin2 proteins (as a rule, such a substitution can affect no more than 20% of all amino acid residues of the original protein), or as a result of deletions of certain fragments of noggin2 proteins. Derivatives of proteins are understood as recombinant proteins obtained on the basis of noggin2 proteins or their fragments, to which fragments of other proteins are added. Mutants and derivatives may retain the biological properties of wild-type proteins (eg, those found in nature) or may possess biological properties different from those of wild-type proteins.
  • biochemical property of the proteins of the present invention refers to biochemical properties, such as the stability of ip vivo and / or ip vitro (for example, half-life); ripening speed, ability to aggregate or to form oligomers and other such properties, but is not limited to them. Mutations include substitutions of individual amino acids, deletions or insertions of one or more amino acids, shortening or lengthening at the N-terminus, shortening or lengthening at the C-terminus, and the like.
  • nuggin2 nucleic acid containing a nucleic acid selected from the group consisting of SEQ ID NO: 1, 3, 5, 7 (corresponding Assessiop Nos in the Gepe Vapk base: AY779052, AY779053, AY779056, AY779058) nucleic acids, in substantially identical to these nucleic acids, and their mutants, derivatives and homologs.
  • nucleic acid homologue is meant a nucleic acid identical to the corresponding nucleotide sequence in at least about 30%, and preferably about 40%, 50%, 55%, 60%, 65%, 70% or higher, including 75%, 80%, 85%, 90% and 95% or higher.
  • the source of homologous nucleic acids and protein can be any species of plants or animals, or the sequence can be fully or partially synthesized, including nucleic acid analogs.
  • Mutants or derivatives of nuggin2 proteins and nucleic acids can be formed using standard molecular biology methods to the matrix nucleic acid encoding the nuggin2 protein by altering, deleting, or adding one or more nucleotides of the template sequence or a combination thereof to form a variant of the template nucleic acid. Substitutions, additions or deletions can be made by any method known in the art (see, for example, Gustip et al., Biotheshques (1993) 14: 22; Varapu, Gepe (1985) 37: 111-123 and Colicelli et al., MoI Gep. Hepet.
  • Substitutions, additions or deletions can also be introduced by a method involving recombination, recursive recombination of sequences, mutagenesis using modified phosphotioat DNA, mutagenesis using a uracil containing matrix, mutagenesis using a duplex rupture, mutagenesis using the repair of point inconsistencies, mutagenesis using a host strain with a deficiency in the recovery system, chemical mutagenesis, radiogenic mutagenesis, mutagenesis through deletions, mutagenesis using restriction-selection, mutagenesis using restriction-purification, synthetic synthesis genes, group mutagenesis, the creation of multimers of chimeric nucleic acids and their combination.
  • degenerate variants of the nuggin2 nucleic acid encoding proteins can be obtained.
  • Degenerate nucleic acid variants contain substitutions of nucleic acid codons for other codons encoding the same amino acids.
  • degenerate nucleic acid variants are prepared to enhance their expression in the host cell.
  • nucleic acid codons that are not preferred or less preferred in the host cell genes are replaced by the codons most represented in the host cell coding sequences, wherein said substituted codons encode the same amino acid.
  • Derivatives can also be obtained using standard methods, including RNA modification, chemical modifications, post-translational and post-transcriptional modifications, etc.
  • derivatives can be obtained using methods such as changing the profile of phosphorylation or glycosylation or acetylation or lipidization or using various types of cleavage of mature forms and the like.
  • Proteins and fragments of interest are isolated, i.e. exist outside the natural environment.
  • the considered proteins and protein domains can be synthesized or obtained by recombinant technologies or isolated from their natural environment.
  • the proteins of the present invention are recombinant proteins obtained using standard approaches using the Ngingin encoding nucleic acid, for example, a nucleic acid selected from the group consisting of SEQ ID NO: 1, 3, 5 or 7.
  • the nucleic acid molecule referred to here is a DNA molecule, such as genomic DNA molecules or cDNA molecules or RNA molecules, such as mRNA molecules.
  • said nucleic acid molecules are open reading frame cDNA molecules encoding the nuggin protein of the present invention or a functional fragment thereof and are capable of being expressed under the appropriate conditions as the active nuggin2 or functional domain of the nuggin of the present invention.
  • the genomic sequence of interest may contain a nucleic acid located between the initiating codon and the stop codon, as defined in the listed sequences, including all introns normally present in the original chromosome.
  • the genomic sequence of interest can include 5'- and 3'-untranslated regions located in mature mRNA, as well as specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 t. bp, but possibly more, flanking genomic DNA at either the 5 ′ or 3 ′ end of the transcribed region.
  • nucleic acids of interest can be isolated and obtained in substantially purified form.
  • substantially purified form means that the nucleic acids are at least about 50% pure, typically at least 90% pure and, as a rule, are "recombinant", i.e. flanked by one or more nucleotides with which they are normally not associated in the natural chromosome in their natural host organism.
  • Nucleic acids of the present invention, corresponding cDNAs, full-length genes and constructs can be artificially produced using various protocols known to those skilled in the art. Appropriate nucleic acid constructs are purified using standard recombinant DNA methods, for example, as described in SAMBROOK et al.
  • Fusion proteins may contain, for example, the nuggin2 protein and the second polypeptide ("fusion partner"), combined into a single reading frame at the N-terminus and / or C-terminus of the nuggin2 polypeptide.
  • Fusion partners include, but are not limited to, polypeptides capable of binding antibodies specific to the fusion partner (e.g., epitope tags), antibodies or their binding domains, polypeptides providing catalytic function or induction of a cellular response, ligands or receptors, or mimetics thereof etc.
  • the fusion partner is typically associated with a portion of the fusion protein, which is the nuggin2 protein, a non-natural method and, as a rule, is not the nuggin2 protein of the present invention or its derivative / fragment.
  • Nucleic acids encoding the fusion proteins described above are also of interest.
  • a vector and other nucleic acid constructs comprising a nuggin2 encoding nucleic acid.
  • Applicable vectors include viral and non-viral vectors, plasmids, cosmids, phages, etc., preferably plasmids, and are used for cloning, amplification, expression, transfer, etc. the nucleic acid sequences of the present invention in a suitable host.
  • the selection of a suitable vector is well known to the person skilled in the art, and many such vectors are commercially available.
  • a partial or full-length nucleic acid is inserted into a vector, usually by attaching a DNA ligase to a restriction enzyme digested site in a vector.
  • the desired nucleotide sequence can be inserted by homologous recombination of ip vivo, typically by attaching to the vector on the sides of the desired nucleotide sequence of homology regions.
  • homology regions are added by ligation of nucleotides or by polymerase chain reaction using primers containing a homology region and part of a desired nucleotide sequence.
  • the expression cassette can exist as an extrachromosomal element, or it can be integrated into the genome of a cell as a result of introducing said cassette into the cell.
  • the product of the gene encoded by the nucleic acid of the invention is expressed in any suitable expression system, including, for example, bacterial, yeast, insect, amphibian or mammalian systems.
  • the nucleic acid of the invention is operably linked to a regulatory sequence, which may contain promoters, enhancers, terminators, operators, repressors and inducers.
  • Cell lines stably expressing nuggin2 can be selected using methods well known in the art (for example, co-transfection with a selective marker such as dhfr, gpt, neomycin, hygromycin, which allows identification and isolation of transfected cells containing the gene integrated into the gene).
  • the expression systems described above can be used in prokaryotic or eukaryotic hosts.
  • host cells as E. coli, B. sibtilis, S. servisiae, insect cells in combination with baculovirus vectors, or cells of higher organisms such as vertebrates, for example, C0S7, HEK293, CHO cells, Xepopus ⁇ can be used ⁇ etc.
  • an expressed protein or polypeptide fall within the scope of the invention as a product of a host cell or host organism.
  • the product can be isolated by any suitable methods known in the art.
  • Antibodies specifically binding to the nuggin2 of the present invention are also disclosed. Applicable antibodies can be prepared using methods known in the art.
  • polyclonal antibodies can be obtained as described in Narrow Apd Lap Optibodies: A Labor Mapul, (1988) CoId Sprig Paror Lab, Parallel, as described below, it is possible ⁇ radrontise: ⁇ rodustiop apd Arlistiop THERf Monoclonal Certification ip CeIl Biologu, ⁇ i demanderhemistr apd Immopolisu; Zrd editiop, (1996) Academis Press.
  • Hybrid antibodies are also of interest, including humanized antibodies, as well as single chain antibodies and antibody fragments such as Fv, F (ab ') 2 and Fab.
  • Hoggin2 as well as the other components of the present invention described above, find application in many different applications, including the use as immunogens, targets in screening assays, biologically active substances for modulating cell growth, differentiation and / or functioning, etc.
  • the present invention relates to a method for changing the activity of noggin2-specific targets (for example, BMP2, BMP4, BMP7, Sirto, or a combination thereof), comprising administering a sufficient amount of noggin2, anti-noggin2 antibodies, or a combination thereof, to the specified organism, tissue or cell, where these combinations alter the activity of nuggin2-specific targets (i.e., the administration of nuggin2 inhibits the activity of these targets, while the administration of an antibody increases their activity).
  • noggin2-specific targets for example, BMP2, BMP4, BMP7, Sirto, or a combination thereof
  • the present invention relates to a method for blocking the activity of noggin2-specific targets, the method comprising administering nuggin2 into a specified organism, tissue or cell in an amount sufficient to inhibit or reduce the activity of specific noggin2 targets by preventing binding these targets or their fragments with specific protein receptors.
  • the present invention relates to a method for increasing the activity of noggin2 specific targets, comprising administering antibodies to nuggin2 into said organism, tissue or cell in an amount sufficient to increase the activity of specific noggin2 targets by binding to nuggin2.
  • the methods and compositions provided are used to modify cell differentiation.
  • a method for modifying cell differentiation involves bringing the cell or the environment surrounding the cell into contact with the nuggin protein under conditions where nuggin specifically interacts with its targets, the components of the medium and / or extracellular surface, to effect a change in cell differentiation.
  • the exogenous protein nuggin2 is used; in another preferred embodiment, an expression construct is used comprising the nuggin2 encoding nucleic acid under the control of a suitable promoter.
  • Nuggin2 protein can be added to the vitro culture medium and physiological fluids such as blood, synovial fluid, etc.
  • the protein can also be introduced or expressed in specific cell populations by any suitable method, such as microinjection, promoter-dependent expression of the recombinant enzyme, targeted delivery of lipid particles, etc. These methods can be used to reduce unwanted osteogenesis, inhibit cell growth requiring morphogenetic protein (e.g. BMP-dependent neuroblastomas and gliomas), regulate cartilage formation and growth, change morphogen-dependent cell development / differentiation directions in culture, such as cells for transplantation or infusion, etc.
  • morphogenetic protein e.g. BMP-dependent neuroblastomas and gliomas
  • nuggin2 Since nuggin2 causes activity in relation to the induction of nerve tissue, it can be used to treat peripheral neuropathies with the growth of the spinal cord and processes of sensitive neurons, for example, it can be included in therapeutic treatment for the regeneration of processes of neurons after strokes, brain damage caused by head injuries , and paralysis caused by spinal cord injuries.
  • the use may also be in the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and multiple sclerosis, by stimulating the processes of neurons. Additional applications may include restoration of dissected axons, which often occurs in foci of damage in multiple sclerosis.
  • nuggin2 or a fragment thereof can be used as a suppressor of the activity of these factors, for example, morphogenetic bone protein (BMP), and epidermal growth factor Sirto or their homologues.
  • BMP morphogenetic bone protein
  • the invention relates to a method of counteracting the functioning of said specific target, comprising bringing said target into contact with the nuggin2 protein or fragment thereof.
  • the method according to the invention is carried out under conditions when nuggin2 or a fragment thereof binds to a specific target.
  • nuggin2 may be useful for the prophylaxis or treatment of BMP-related disorders in animals, in particular humans.
  • nuggin2 can be used to treat diseases or disorders that include, but are not limited to, progressive ossifying fibrodysplasia (FOP), and also to treat abnormal bone growth such as abnormal bone growth after hip replacement surgery, trauma, burns, or spinal cord injuries.
  • FOP progressive ossifying fibrodysplasia
  • nuggin2 can be used to treat or prevent the undesirable effects of BMP associated with abnormal bone growth observed in association with metastatic prostate cancer or osteosarcoma. Since it is known that the content of the ligand BMP2 and BMP4 significantly increases in asthma (Rosephal et Al, Am J Respir CeIl MoI Biol.
  • nuggin2 can also be used to bind BMP and, thus, to treat inflammatory syndromes.
  • nuggin2 can bind and thereby inhibit the activity of the protein, it also finds use in the prevention and treatment of cancer tumors, the formation of which in vertebrates is associated with an abnormal increase in the activity of this epidermal growth factor.
  • nuggin2 instead of the monoclonal antibodies and antisense RNA used to date for the treatment of a wide range of cancers, such as tumors of the breast, uterus, stomach, pancreas, lungs, ovaries and others.
  • nucleic acids, proteins, fragments and antibodies to nuggin2 of the invention can be used to alter the activity of specific nuggin2 targets in animals, in particular humans.
  • Compositions of interest may include a nuggin2 protein, for example a recombinant purified protein.
  • composition may also contain a suitable pharmaceutical carrier for systemic or local administration of ip vivo in any suitable manner, including, but not limited to, injection (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, endoneural, perineural, intraspinal, intraventricular, intravitreal, and intrathecal etc.) by absorption through the epithelial or mucocutaneous lining (e.g., through the oral mucosa, rectal or intestinal mucosa, and .d.) or a sustained release implant, including a cellular or tissue implant.
  • injection e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, endoneural, perineural, intraspinal, intraventricular, intravitreal, and intrathecal etc.
  • absorption e.g., through the epithelial or mucocutaneous lining (e.g., through the oral mucosa, rectal or intestinal mucosa, and .d.) or a sustained release implant, including
  • the composition may contain a liquid carrier, such as saline, be enclosed in liposomes, microcapsules, polymer or paraffin-based controlled-release formulations, or be formulated in the form of a tablet, pill or capsule.
  • a liquid carrier such as saline
  • the concentration of the active ingredient used (s) depends on the type of disease or signs that are affected, the method of administration used, as well as the characteristics of the biological system. Effective doses can be obtained by extrapolating the dose-response curves obtained in ip vitro test systems or in animal models.
  • the term "effective amount” or "effective dose” refers to the amount of protein necessary to achieve a biological effect, i.e.
  • an effective dose is in the range of 0.1-100 mg / kg of the subject.
  • nuggin2 may result in the distribution of nuggin2 in the body or in a localized area.
  • intravenous or intrathecal administration of nuggin2 can be used.
  • local administration may be used.
  • an implant containing a nuggin2 can be placed in or near a specific area. Suitable implants include, but are not limited to, a gelatin sponge, wax, or microparticle based implants.
  • Example 1 Cloning of nuggin2 cDNA coding of nuggin2 cDNA was cloned during the search for the Xeporis lavis genes involved in the regulatory network of the homeobox-containing tepaXapf-1. 1999, v. 126, pp. 4513-4523).
  • nuggin2 The nucleotide and amino acid sequences of nuggin2 are shown in SEQ ID NO: 1 and 2.
  • Example 2 The effect of nuggin2 on the direction of cell development
  • the pSP64T vector containing the complete coding part of the nuggin2 cDNA (pSP64T-nuggin2 vector) was obtained as described in Example 1.
  • the complete coding sequence was obtained by PCR nogginl amshshfikatsii Heporis laevis cDNA sample using nogginl - specific primers 5'-T ATCC ATGGC AAGAAATSGGG AGC A-3 'and 5'ATTSTSGAGTSTS AGC ATGAGC ATTTGC A-3 1 and cloned into vector pSP64T ( vector pSP64T-nogginl).
  • Synthetic muggins noggin2 and noggin were obtained using the mMESSAGE MASHINE Kit reagent kit (Ambiop) from the pSP64T-nuggin vectors and the pSP64T-noggin vector, respectively, linearized with the restriction endonuclease EcoRI (Fermeptas).
  • the effects of microinjections of nuggin mRNA on the development of Xeporis laevgs were compared with the previously described effects of noggin mRNA injections (Smith, Ap. Narlapd, CeIl 1992, v. 70, pp. 829-840; Lamb et al., Sciepse 1993, v. 262, pp.
  • nuggin2 despite its alleged ability to bind BMP, did not induce the secondary axis of the body when its mRNA was injected into both ventral blastomeres of 8-cell embryos. Instead, abnormal mushroom-shaped embryos arose.
  • VMZ ventral marginal zone
  • OT-PCR reverse transcription-PCR
  • VMZ ventral marginal zone
  • AC explants were removed from the embryos subjected to injection using a micro-knife and a glass rod, as previously described in Zarisk et al. Developed Biolog 1992, v. 152, pp. 373-382. All explants were incubated overnight in a 0.5xMMR solution. Out of ten explants of each type, total RNA was isolated and RT-PCR was performed as described in Zarisk et al. Developed Biolog 1992, v. 152, pp. 373-382.
  • ⁇ -actin gene 5'-GCTGACAGAATGCAGAAG and 5'-TTGSTTGGAGGAGTGTGT; brashuiru: 5'-GCTGGAAGTATGTGAATGGAG AND 5'-TTAAGTGSTGTAATSTSTTS; Cerberus: 5'-
  • RT-PCR based on total RNA from AC and VMZ explants of embryos injected with leggin or nuggin2 mRNA was performed with primers for markers of neuroectoderma (NCAM, Xanf-1), epidermis (keratin), muscle ( ⁇ -actin), late mesoderm (brushuiru) and entomesoderm (cerberus) and Ef-1- ⁇ as a control (Fig. 2B).
  • Nuggin and noggin2 have been shown to activate the expression of neural markers (NCAM, Xanf-1) and inhibit the epidermal marker (keratin), while only noggin activates the expression of a muscle marker ( ⁇ -actin) in VMZ explants.
  • nuggin2 showed a strong neuralizing activity similar to that of noggin (Smith appd Härläpd, CeIl 1992, v. 70, pp. 829-840; Lamb et al., Sciepse 1993, v. 262, pp. 713-718) inducing the expression of markers of the anterior part of the nervous system, but inhibiting the epidermal marker.
  • the ability of nuggin2 to neutralize the ectoderm, together with its ability to suppress dorsalizing activity of nuggins looks very unusual and means that nuggin2, in addition to BMP, can bind some other factors necessary for differentiation of muscles.
  • noggin2 mRNA microinjections on the differentiation of embryonic mesoderm was assessed by analyzing the expression of the universal marker of the early stages of mesoderm development - the baxuiru gene - at the stage of the early gastrula Heporis laevis.
  • either noggin or noggin2 mRNA, obtained as described in example 2 was injected into the equatorial region of one of the ventral blastomeres at the stage of 8 blastomeres in an amount of 40 Ig using the Errepdorff microinjector.
  • FIG. 3 The experimental results are shown in FIG. 3.
  • the noggin2 mRNA microinjection causes inhibition of the expression of the bashui gene in the microinjection region, which indicates the suppression of differentiation of embryonic mesoderm at this site.
  • similar microinjections of Noggin mRNA do not cause inhibition of expression of briskuir and, accordingly, inhibition of the development of mesoderm (Fig. 3 B and B ').
  • nuggin2 has a unique ability to suppress the differentiation of embryonic mesoderm.
  • nuggin2 The ability of nuggin2 to inhibit the BMP signaling cascade was assessed by the ability of nuggin2 to suppress the expression of the main genetic target of this cascade - the Vent2 homeobox gene - in explants of embryonic embryonic ectoderm nuclei (AC explants) microinjected with a mixture of noggin2 mRNA and BMP7 BMP2, mRNA, mRNA.
  • nuggin mRNA was mixed for microinjections with mRNA of all of the above BMPs simultaneously. As a control, similar experiments were carried out with noggin mRNA.
  • Vent2 gene expression was analyzed by RT-PCR based on total RNA from AC explants as described in Example 2.
  • the following Vent2-specific primers were used: 5'-ASTGAACASAAGGASTAATASA and 5'-AGAGGCCAGAGACTGCCCAA.
  • nuggin2 is able to inhibit the activation of Vent2 expression induced by any of these BMPs, as well as their mixture.
  • nuggin2 protein To directly investigate the ability of the nuggin2 protein to bind BMP molecules in X.lavis embryos, the active forms of nuggin2 and BMP4 were expressed in the expression vectors pCS2- ⁇ Myc-noggin2 and pCS2- ⁇ Flag-BMP4, respectively.
  • the nucleotide sequence encoding the three amino acid sequences of the Myc protein epitope was inserted by PCR into the ngingin2 cDNA directly at the 3 'end of the sequence encoding the signal peptide n2.
  • 5 'and 3' respectively, overlapping parts of nuggin cDNA were obtained in PCR with 5'- primers
  • the nucleotide sequence encoding the three Flag amino acid sequences was inserted by PCR into BMP4 cDNA directly to the 3 'end of the sequence encoding the Heporis BMP4 protein region.
  • 5 'and 3' overlapping portions of BMP4 cDNA were obtained in PCR with primers 5'-AATTGGATCCGCCACCATGATTCCTGGTAACCGAATG-3 'and 5'- TTTGTCATCATCGTCTTTGTAGTCCTTATCGTCGTCATCCTTGTAATCCTGTTT TGGASTTSTTTTTGAS-W 1 5 1 -
  • the cDNA fragments were purified from not including primers, mixed together, denatured by heating, annealed and subjected to a second round of PCR with the primers 5'-end AATTGGATSSGSSASSATGATTSSTGGTAASSGAATG-W 1 and 5 1 - AATSTSGAGTSAASGGSASSSASASSSTTSSA-W 1.
  • the resulting full-length BMP4 cDNA containing the sequence encoding the three amino acid sequences of the Flag epitope was cloned into the pCS2 vector at the restriction sites Ncol (type) / Agel (type) and Xhol / Xhol.
  • 600 pg of synthetic 3Myc-noggin2 mRNA and 600 pg of synthetic 3Flag-BMP4 mRNA were microinjected into developing X. lavis nuclei at the stage of 2 blastomeres. Subsequently, these embryos were incubated in a solution of 0.1 MMR to the stage of early gastrula.
  • nuggin2 The ability of nuggin2 to inhibit the activin / delayed signaling cascade were evaluated by the ability of nuggin2 to suppress the expression of the main genetic target of this cascade, the broshuur gene, in explants of embryonic embryonic ectoderm of the embryos (AC explants) microinjected with a mixture of nuggin mRNA and mRNA Xrl, Xnr2, or Xnr4 (homologs of gene d). As a control, similar experiments were performed with mRNA and noggin.
  • the expression of the baskhuur gene was analyzed by RT-PCR for total RNA from AC explants as described in Example 2.
  • nuggin2 is able to suppress activation of expression of braskhuur induced by any of these Khpr.
  • nuggin2 protein In order to study the ability of the nuggin2 protein to bind the molecules of the antivip and Xpr proteins in X. lavis embryos, the active forms of nuggin2, activip and Xpr were expressed in the expression vectors pCS2- ⁇ Myc-noggin2, pCS2- ⁇ Flag-activin and pCS2- ⁇ Flag Xn-n.
  • the nucleotide sequence encoding the three Flag amino acid sequences (DYKDDDDKDYKDDDKKDYKDDDK) was inserted by PCR into the Antivip-B cDNA immediately behind the sequence encoding the protein region of activis-Xvisvis protein.
  • the nucleotide sequence encoding the three Flag amino acid sequences was inserted by PCR into the Xnr2 cDNA directly at the 3 'end of the sequence encoding the Xsritzpory protein region.
  • 5 'and 3' overlapping parts of BMP4 cDNA were obtained in PCR with primers 5'-AATTGGATSSGSSASSATGGCA ⁇ G ⁇ - ⁇ 1 and 5'-
  • AATSTSGAGTSTSGTTASATSSASASTSATSSATSA-Z 1, respectively.
  • these cDNA fragments were purified from unincorporated primers, mixed with each other, denatured by heating, annealed, and subjected to a second round of PCR with 5'- end primers
  • AATSTSGAGTSAASGGASSASSASASSTSTSSA-Z 1 The resulting full-length Xnr2 cDNA containing a sequence encoding the three amino acid sequences of the Flag epitope was cloned into the pCS2 vector at the restriction sites Ncol (type) / Agel (type) and Xhol / Xhol.
  • nuggin2 protein molecules to bind the protein molecules of the crypto cofactor from the epidermal family was investigated growth factors EGF-CFC.
  • EGF-CFC growth factors
  • the active forms of noggin2 and ⁇ rorto were expressed in the expression vectors pCS2- ⁇ Myc-noggin2, pCS2- ⁇ Flag-Cripto, respectively.
  • the mechanism of inhibition of the activin / delivered cascade by the factor of nuggin2 is different from that for the BMP cascade and is not carried out by direct binding of the activin molecules or by the binding of the nuggin2 molecules, but due to the ability of the nuggin2 to bind the crypto cofactor molecules, the presence of which is necessary for the normal functioning of this cascade (Fig. four).
  • the revealed ability of nuggins2 to bind the ⁇ r ⁇ 1958to factor can be of great biomedical significance.
  • Example 6 Simultaneous inhibition of BMP and activin / half signaling cascades and induction of head structures using nuggins2
  • BMP and activin / the differentiation of the structures of the main axis of the vertebral body is inhibited on the ventral side of the embryo as a result of the permanent activation of two main TGF- ⁇ -cascades: BMP and activin / by
  • the selective inhibition of the BMP cascade leads by default to the development of structures characteristic of the trunk and posterior part of the body axis.
  • the simultaneous inhibition of both BMP and activin / dip cascades causes the induction of anterior-headed structures, including eye and forebrain.
  • nuggin2 The ability of nuggin2 to inhibit both the BMP cascade and the activin / short cascade simultaneously indicates the potential ability of this factor to induce forehead structures.
  • a similar effect could be masked by the strong neuralizing effect of nuggin2, caused by relatively high concentrations of microinjectable mRNA.
  • nuggin2 the ability of low concentrations of nuggin2 to induce the development of additional anterior-headed structures in whole embryos was studied.
  • synthetic nuggin mRNA obtained in the same manner as described in Example 2 was microinjected into one of the ventral vegetative blastomeres of Xeporus lavis embryos at the stage of 8-16 blastomeres in the amount of 2-5 pg.
  • the development of an additional anterior-head section was observed.
  • the hybridization method of ip siti with specific probes for mRNA of the forehead markers BF-I and Pax-6 such additional brain structures included the end brain and eyes (Fig. 3 B and B 1 ; D and D 1 ).
  • Example 7 Obtaining the recombinant protein nuggin2 To obtain the active homginimer nuggin2, expression of nuggin2 was carried out in a culture of green monkey monkey epithelial cells CV-I as part of the pcDNAZ-6His-noggin2 expression vector. To obtain the pcDNAZ-bHis-noggin2 vector, a nucleotide sequence encoding 6 amino acid residues of histidine was inserted by PCR into the ngingin2 cDNA immediately behind the sequence encoding the ngingin2 signal peptide. In the first cloning step, 5 'and 3' overlapping parts of nuggin cDNA were obtained in PCR with primers 5'-ATTASSGGTGGGAGAASSTTGTTSTTSATT- 1 and 5'-
  • the resulting full-length noggin2 cDNA containing a sequence encoding 6 histidines was cloned into the pcDNAZ vector at the restriction sites Ncol (dull) and Hol. Green monkey epithelial cells (CV-I) were cultured in
  • DMEM medium (Sigma) with 10% serum (Biolot), 100 U / ml penicillin and 100 mg / ml streptomycin at 37 0 C and 1 atm.
  • these cells were transfected with pcDNA3-6His-noggin2 vector.
  • Subsequent selection was carried out in a medium containing 1 ⁇ g / ml geneticin (Gibso BRL).
  • Secreted recombinant 6His-Noggin2 was obtained from the culture medium by affinity chromatography on Ni-NTA agarose (Qiagep). Protein yield was estimated as 50 ⁇ g / million cells per day. The activity of the protein was confirmed by its ability activate neurogenesis in explants of embryonic ectoderm.
  • Example 8 Obtaining polyclonal. antibodies against nuggin2 cDNA containing the complete coding region of nuggin2 was reamplified as described in Example 1, subcloned into the pQECO vector and used to transform Escherichia coli. A recombinant protein containing six histidine residues was purified using affinity chromatography on Talop Resip (Clop Lab Laboratories, Ips.) Under denaturing conditions and was used to immunize rabbits using standard technology.
  • Polyclonal serum was tested against recombinant protein using ELISA and Western blot methods.
  • nuggin2 proteins from gallis gallis having the amino acid sequences of SEQ ID NO: 6 and 8 encoded by nucleic acids having the sequences of SEQ ID NO: 5 and 7, respectively. It has been found that these nuggin2 proteins from Gallis gallis exhibit the same biological properties as nuggin2 X.lavis.
  • nuggin2 Since the genomic sequence of nuggin2 is devoid of introns, the full-length coding region of chicken nuggin2 cDNA (Gallis gallis) was obtained from the genomic DNA of chicken using PCR with primers: 5'-
  • nuggin2 cDNA fragment was cloned into the plasmid pSP64T (Protega) at the restriction sites HindSh (type) / Age (type) and Xhol / Sall.
  • nuggin2 The nucleotide sequence of nuggin2 was confirmed by sequencing. To obtain the nuggin2 protein, nuggin2 was expressed in the culture of the CV-I green monkey epithelial cells as part of the pcDNAZ-6His-noggin2 expression vector. To obtain the pcDNAZ-bHis-noggin2 vector, the nucleotide sequence encoding 6 histidine amino acid residues were inserted by PCR into the nuggin2 cDNA immediately behind the sequence encoding the ngingin2 signal peptide. In the first cloning step, 5 'and 3' overlapping parts of nuggin cDNA were obtained in PCR with 5'-ATTACCGGTTGGATGCGTCCAGGGCCGGGAAG-3 'and 5'- primers
  • these cDNA fragments were purified from unincorporated primers, mixed with each other, denatured by heating, annealed, and subjected to a second round of PCR with primers 5'-ATTASSGGTTGGATG ⁇ G ⁇ GGGCCCCGG ⁇ G- ⁇ 1 , and 5'-
  • the resulting full-length chicken nuggin2 cDNA containing a sequence encoding 6 histidines was cloned into the pcDNAZ vector at the restriction sites Ncol (dull) and Xhol.
  • Green monkey epithelial cells were cultured in DMEM medium (Sigma) with 10% serum (Biolot), 100 U / ml penicillin and 100 mg / ml streptomycin at 37 ° C and 1 atm. To obtain a constant cell line producing 6His-noggin2, these cells were transfected with pcDNA3-6His-noggin2 vector. Subsequent selection was carried out in a medium containing 1 ⁇ g / ml geneticin (Gibso BRL). Secreted recombinant 6His-Noggin2 was obtained from the culture medium by affinity chromatography on Ni-NTA agarose (Qiagep). Protein yield was estimated as 70 ⁇ g / million cells per day. Protein activity was confirmed by its ability to activate neurogenesis in explants of the embryonic ectoderm Heporus lavis.

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Abstract

La présente invention concerne des protéines et notamment la protéine noggin2 régulant la différentiation cellulaire par l'inhibition des protéines de la superfamille TGF-β et des procédés de fabrication correspondants.
PCT/RU2006/000382 2005-07-21 2006-07-18 Procede de blocage d'activite des cibles specifiques a noggin2 WO2007011265A1 (fr)

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RU2391352C1 (ru) * 2008-10-24 2010-06-10 Учреждение Российской академии наук Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова РАН СПОСОБ БЛОКИРОВАНИЯ СИГНАЛЬНОГО ПУТИ, АКТИВИРУЕМОГО TGF-beta ФАКТОРОМ VgI В КЛЕТКАХ ЖИВОТНЫХ
RU2407799C1 (ru) * 2009-06-18 2010-12-27 Учреждение Российской академии наук Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова РАН СПОСОБ БЛОКИРОВАНИЯ СИГНАЛЬНОГО ПУТИ, АКТИВИРУЕМОГО TGF-beta ФАКТОРОМ DERRIERE В КЛЕТКАХ ЖИВОТНЫХ

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DATABASE MEDLINE [online] FLETCHER R.B. ET AL.: "Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod", XP003007557, Database accession no. (NLM15567718) *
DATABASE MEDLINE [online] FURTHAUER M. ET AL.: "Three different noggin genes antagonize the activity of bone morphogenetic proteins in the zebrafish embryo", XP003007556, Database accession no. (NLM10491267) *
DEV. BIOL., vol. 214, no. 1, 1 October 1999 (1999-10-01), pages 181 - 196 *
GENE EXPR. PATTERNS, vol. 5, no. 2, 2004, pages 225 - 230 *
LANGENFELD E.M. ET AL.: "The mature bone morphogenetic protein-2 is aberrantly expressed in non-small cell lung carcinomas and stimulates tumor growth of A549 cells", CARCINOGENESIS, vol. 24, no. 9, 2003, pages 1445 - 1454, XP003007558 *

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