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WO1996011259A1 - ACTIVINE 1 ET ACTIVINE 3, DE LA FAMILLE DES RECEPTEURS DU FACTEUR TRANSFORMANT DE CROISSANCE TGF-β1 - Google Patents

ACTIVINE 1 ET ACTIVINE 3, DE LA FAMILLE DES RECEPTEURS DU FACTEUR TRANSFORMANT DE CROISSANCE TGF-β1 Download PDF

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Publication number
WO1996011259A1
WO1996011259A1 PCT/US1994/011328 US9411328W WO9611259A1 WO 1996011259 A1 WO1996011259 A1 WO 1996011259A1 US 9411328 W US9411328 W US 9411328W WO 9611259 A1 WO9611259 A1 WO 9611259A1
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Prior art keywords
tar
polypeptide
polynucleotide
receptor
leu
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PCT/US1994/011328
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English (en)
Inventor
Wei Wu He
Peter L. Hudson
Mark D. Adams
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Human Genome Sciences, Inc.
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Priority to PCT/US1994/011328 priority Critical patent/WO1996011259A1/fr
Priority to AU10390/95A priority patent/AU1039095A/en
Publication of WO1996011259A1 publication Critical patent/WO1996011259A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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 newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is TGF-01, Activin Receptors 1 and 3, sometimes hereinafter referred to as "TAR- 1 and TAR-3". The invention also relates to inhibiting the action of such polypeptides.
  • TGF-31 The cloning of TGF-31 and the resultant elucidation of its precursor structure (Derynck, et al., Nature. 316:701-705 (1985)) have led to the identification of at least four other forms of TGF-S and the definition of a larger gene family comprising many other structurally related, but functionally distinct, regulatory proteins.
  • polypeptides that belong to the TGF-3 supergene family by virtue of amino acid homologies, particularly with respect to the conservation of seven of the nine cysteine residues of TGF-S among all known family members.
  • these include the mammalian inhibins (Mason, et al., Nature. 318:659-663 (1985)) and activins fLing, et al., Nature. 779-782 (1986)), and ullerian inhibitory substance (Cate, et al., Cell. 45:685-698 (1986)), as well as the predicted products of both a pattern gene in Drosophila (Padgett, et al., Nature.
  • BMP ⁇ bone morphogenetic proteins
  • TGF-3 itself (Kimelman and Kirschner, Cell. 51:869-877 (1987)) has been shown to augment the ability of fibroblast growth factor to induce esoderm and plays a pivotal role in morphogenesis and organogenesi ⁇ in mammalian embryos.
  • FSH follicle-stimulating-hormone
  • TGF- / 3 has been shown to inhibit the growth of several human cancer cell lines (Roberts et al., PNAS, 82:119-123 (1985)). TGF-/3 has also been described as being able to inhibit production of the HIV virus and decrease syncytia formation, U.S. Pat. No. 5,236,905.
  • Activins have an extensive anatomical distribution and are implicated in the regulation of many biological processes, including the proliferation of many cell lines (Gonzalez-Manchon, C. and Vale, W., Endocrinology. 125:1666- 1672 (1989)), control of the secretion and expression of the anterior pituitary hormones FSH, GH, and ACTH (Vale, w. , et al., Nature. 321:776-779 (1986)), neuronal survival (Hashimoto, M., et al., Biochem. Biophvs. Res. Commun..
  • Activin has also been shown to suppress androgen production (Hsueh et al., PNAS, 84:5082-5086 (1987)), therefore activin decreases follicular size and may lead to female infertility (Woodruff et al., Endocrinol., 127:3196-3205 (1990)).
  • activins There are three activins (A, B, and AB), comprising different combinations of two closely-related beta sub-units, (Mason, A.J., et al., Nature. 318:659-663 (1985)). Activins impinge on a much broader spectrum of cells than inhibins, however, in those systems in which both proteins are functional, they have opposing biological effects. The mechanistic basis for this antagonism is unknown.
  • Type I and II TGF-S receptor components like most growth factor receptors, are glycoproteins.
  • the type III protein is a proteoglycan consisting predominantly of heparin sulfate glycosaminoglycan chains with a smaller amount of chondroitin or der atan sulfate attached to a core protein of about 100-140 Kd.
  • the binding site for TGF-S resides in this core protein (Cheifetz, et al., J. Biol. Chem.. 263:16904- 16991 (1988)).
  • TGF-S supergene family receptor polypeptide has been disclosed in U.S. Patent No. 5,216,126. However, this receptor polypeptide has approximately 75% sequence identity with the mature human inhibin/activin receptor sequence.
  • a protein designated as an "activin receptor” has been expression-cloned (Mathews and Vale, Cell. 65:1-20 (1991)), however, it does not appear to be a mammalian receptor in that it is a serine kinase.
  • novel polypeptides which have been identified as TAR-1 and TAR-3 receptors, as well as fragments, analogs and derivatives thereof.
  • the receptor polypeptides of the present invention are of human origin.
  • polynucleotides which encode such receptor polypeptides.
  • Figure 1 is a cDNA sequence and corresponding deduced amino acid sequence of the TAR-1 receptor polypeptide.
  • the signal sequence of the receptor polypeptide is the first 15 amino acids indicated and the transmembrane portion is underlined.
  • Figure 2 is the cDNA sequence and corresponding deduced amino acid sequence of the TAR-3 receptor polypeptide wherein the first 21 amino acids represent the signal sequence and the transmembrane portion is underlined.
  • the standard one- letter abbreviation for amino acids is used throughout the figures.
  • Figure 3 illustrates the amino acid sequence homology between TAR-1 and TAR-3 and rat type I TGF-3 receptor. Boxed areas indicate identical amino acids between the different sequences.
  • Sequencing inaccuracies are a common problem when attempting to determine polynucleotide sequences. Accordingly, the sequences of Figure 1 and 2 are based on several sequencing runs and the sequencing accuracy is considered to be at least 97%.
  • the receptors of the present invention have been putatively identified as a TAR-1 and TAR-3 receptor. This identification has been made as a result of amino acid sequence homology.
  • nucleic acid which encode for the mature receptor polypeptides having the deduced amino acid sequence of Figure 1 and Figure 2 or for the mature receptor polypeptides encoded by the cDNA of the clone deposited as ATCC Deposit No. 75843 and ATCC Deposit No. 75842 for TAR-1 and TAR-3, respectively, which were deposited on July 27, 1994.
  • the polynucleotide encoding for TAR-1 was discovered in a cDNA library derived from a human adult spleen. It is structurally related to the TGF-S receptor family.
  • TAR-1 exhibits the highest degree of homology to type I TGF-0 receptor with 49% identity and 67% similarity over a 503 amino acid stretch.
  • the polynucleotide encoding for TAR-3 was discovered in a cDNA library derived from a human placenta. It is also structurally related to the TGF-S receptor family. It contains an open reading frame encoding a protein of about 503 amino acid residues of which approximately the first 21 amino acids residues are the putative signal sequence such that the mature protein comprises 482 amino acids. TAR-3 also exhibits the highest degree of homology to type I TGF-/S receptor with 47% identity and 64% similarity over a 500 amino acid stretch.
  • the polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature receptor polypeptides may be identical to the coding sequence shown in Figure 1 and Figure 2 or that of the deposited clone( ⁇ ) or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature receptor polypeptides as the DNA of Figure 1 and Figure 2 or the deposited cDNA.
  • the polynucleotides which encode for the mature receptor polypeptides of Figure 1 and Figure 2 or for the mature receptor polypeptides encoded by the deposited cDNA( ⁇ ) may include: only the coding sequence for the mature receptor polypeptides; the coding sequence for the mature receptor polypeptides and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature receptor polypeptides (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5 ' and/or 3' of the coding sequence for the mature receptor polypeptides.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequence of Figure 1 and Figure 2 or the polypeptide encoded by the cDNA( ⁇ ) of the deposited clone(s).
  • the variant of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides.
  • the present invention includes polynucleotides encoding the same mature receptor polypeptides as shown in Figure 1 and Figure 2 or the same mature receptor polypeptides encoded by the cDNA(s) of the deposited clone( ⁇ ) a ⁇ well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the receptor polypeptides of Figure 1 and Figure 2 or the receptor polypeptides encoded by the cDNA(s) of the deposited clone( ⁇ ).
  • nucleotide variant ⁇ include deletion variant ⁇ , substitution variants and addition or insertion variants.
  • the polynucleotide ⁇ may have a coding sequence which is a naturally occurring allelic variant of the coding sequences shown in Figure 1 and Figure 2 or of the coding sequences of the deposited' " clone(s) .
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • the present invention also includes polynucleotides, wherein the coding sequence for the mature receptor polypeptides may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
  • the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotides may also encode for a proprotein which is the mature protein plu ⁇ additional 5' amino acid residues.
  • a mature protein having a prosequence is a proprotein and i ⁇ an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
  • the polynucleotide ⁇ of the present invention may encode or mature receptor proteins, or for a receptor protein having a prosequence or for a receptor protein having both a prosequence and a presequence (leader sequence) .
  • the polynucleotide ⁇ of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the receptor polypeptides of the present invention.
  • the marker sequence may be a hexa- hi ⁇ tidine tag ⁇ upplied by a pQE-9 vector to provide for purification of the mature receptor polypeptide ⁇ fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al.. Cell, 37:767 (1984)).
  • the present invention further relate ⁇ to polynucleotide ⁇ which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides .
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequence ⁇ .
  • polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode receptor polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA( ⁇ ) of Figure 1 and Figure 2 or the deposited cDNA(s).
  • the present invention further relates to TAR-1 and TAR-3 receptor polypeptides which have the deduced amino acid sequence of Figure 1 and Figure 2 or which have the amino acid sequences encoded by the deposited cDNA(s), as well as fragments, analogs and derivatives of such polypeptide.
  • fragment when referring to the receptor polypeptides of Figure 1 and Figure 2 or that encoded by the deposited cDNA(s), mean ⁇ receptor polypeptides which retain essentially the same biological function or activity a ⁇ such polypeptides.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce active mature receptor polypeptides.
  • polypeptides of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the receptor polypeptides of Figure 1 and Figure 2 or that encoded by the deposited cDNA( ⁇ ) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a ⁇ ub ⁇ tituent group, or (iii) one in which the mature polypeptide is fused with another compound, such a ⁇ a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vector ⁇ of the invention and the production of polypeptide ⁇ of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form cf a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the TAR-1 and TAR-3 genes.
  • the culture conditions such as temperature, pH and the like, are those previously u ⁇ ed with the ho ⁇ t cell ⁇ elected for expression, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing receptor polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expression vectors for expres ⁇ ing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox viru ⁇ , and pseudorabies.
  • any other vector may be used as long as it i ⁇ replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence i ⁇ inserted into an appropriate restriction endonuclease site(s) by procedure ⁇ known in the art.
  • procedure ⁇ known in the art.
  • Such procedure ⁇ and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoter A ⁇ repre ⁇ entative example ⁇ of ⁇ uch promoter ⁇ , there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P L promoter and other promoter ⁇ known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expres ⁇ ion vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells ⁇ uch a ⁇ dihydrofolate reductase or neomycin resi ⁇ tance for eukaryotic cell culture, or ⁇ uch a ⁇ tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA ⁇ equence a ⁇ hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the proteins.
  • bacterial cells such as E. coli. Streptom ⁇ ces, Salmonella typhimurium: fungal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cell ⁇ such as CHO, COS or Bowes melanoma; plant cells, etc.
  • fungal cells such as yeast
  • insect cells such as Drosophila and Sf9
  • animal cell ⁇ such as CHO, COS or Bowes melanoma
  • plant cells etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention also includes recombinant constructs comprising one or more of the sequences a ⁇ broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • suitable vectors and promoter ⁇ are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example.
  • Bacterial pQE70, pQE60, pOE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNHl ⁇ A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • any other plasmid or vector may be used a ⁇ long a ⁇ they are replicable and viable in the ho ⁇ t.
  • Promoter region ⁇ can be ⁇ elected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable marker ⁇ .
  • Two appropriate vector ⁇ are PKK232-8 and PCM7.
  • Particular named bacterial promoter ⁇ include lad, lacZ, T3, T7, gpt, lambda P justify, P L and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTR ⁇ from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described constructs.
  • the ho ⁇ t cell can be a higher eukaryotic celT, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the ho ⁇ t cell can be a prokaryotic cell, ⁇ uch as a bacterial cell.
  • Introduction of the construct into the ho ⁇ t cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L. , Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)).
  • constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthe ⁇ izers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic ho ⁇ t ⁇ are de ⁇ cribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the di ⁇ clo ⁇ ure of which i ⁇ hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription.
  • Example ⁇ including the SV40 enhancer on the late ⁇ ide of the replication origin bp 100 to 270, a cytomegaloviru ⁇ early promoter ennancer, the polyoma enhancer on the late ⁇ ide of the replication origin, and adenoviru ⁇ enhancers.
  • recombinant expre ⁇ sion vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such a ⁇ 3-phosphoglycerate kinase (PGK), o-factor, acid phosphatase, or heat shock proteins, among others.
  • the heterologou ⁇ structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus ⁇ ubtili ⁇ . Salmonella tvphimurium and variou ⁇ ⁇ pecie ⁇ within the genera P ⁇ eudomona ⁇ , Streptomyce ⁇ , and Staphylococcu ⁇ , although other ⁇ may al ⁇ o be employed a ⁇ a matter of choice.
  • useful expres ⁇ ion vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" section ⁇ are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expres ⁇ ion of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
  • mammalian cell culture sy ⁇ tem ⁇ can al ⁇ o be employed to express recombinant protein.
  • mammalian expression sy ⁇ tem ⁇ include the COS-7 lines of monkey kidney fibroblast ⁇ , described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation ⁇ ite, ⁇ plice donor and acceptor site ⁇ , transcriptional termination sequence ⁇ , and 5' flanking nontranscribed ⁇ equence ⁇ .
  • DNA ⁇ equence ⁇ derived from the SV40 splice, and polyadenylation ⁇ ite ⁇ may be u ⁇ ed to provide the required nontranscribed genetic elements.
  • the TAR-1 and TAR-3 receptor polypeptides can be recovered and purified from recombinant cell culture ⁇ by method ⁇ including ammonium ⁇ ulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • the receptor polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • the polypeptides may also include an initial methionine residue.
  • the present invention also relates to detecting altered levels of expression of mRNA encoding TAR-1 and TAR-3 receptors.
  • the overexpression of these genes or loss of expression is indicative of cancer or the risk of cancer.
  • Assays employed to detect the expression of these nucleic acid sequence ⁇ are well-known by those skilled in the art and include, for example, nucleic acid probe hybridization, PCR and Southern blot analysis. In the hybridization technique, a sample is derived from a host and analyzed to determine whether the sample contains altered levels of TAR receptor gene expression.
  • TAR-1 and TAR-3 receptor polypeptide ⁇ are useful in radio-receptor assays to measure all bindable (and active) forms of a TGF supergene family member such as activin.
  • Such an assay would be specific for activin and would be conducted using the naturally purified or recombinant TAR-1 and TAR-3 receptors a ⁇ the receptor element.
  • TAR-1 and TAR-3 receptor polypeptide ⁇ are useful for screening for compound ⁇ that bind to them and have TGF supergene family biological activity as defined above.
  • these compounds are small molecules ⁇ uch a ⁇ organic or peptide molecule ⁇ that exhibit one or more of the desired activities of a TGF supergene family member such a ⁇ activin. Screening assays of this kind are conventional in the art, and any such screening procedure may be employed, whereby the test sample i ⁇ contacted with the TAR-1 or TAR-3 receptor herein and the extent of binding and biological activity of the bound molecule ⁇ i ⁇ determined.
  • TAR-1 and TAR-3 receptor polypeptides are additionally useful in affinity purification of TGF supergene family member ⁇ that bind TAR-1 and TAR-3 receptor ⁇ and in purifying antibodie ⁇ thereto.
  • the receptor i ⁇ typically coupled to an immobilized resin such as Affi-Gel 10 (Bio- ad, Richmond, CA) or other such resins (support matrices) by means well-known in the art.
  • the resin is equilibrated in a buffer and the preparation to be purified is placed in contact with the resin, whereby the molecules are selectively adsorbed to the receptor on the resin.
  • the resin is then sequentially washed with suitable buffers to remove non-adsorbed material, including unwanted contaminants, from the mixture to be purified, u ⁇ ing, for an activin mixture, e.g., lOOmM glycine, pH 3, and 0.1% octyl 0-glucoside.
  • an activin mixture e.g., lOOmM glycine, pH 3, and 0.1% octyl 0-glucoside.
  • the resin is then treated so a ⁇ to elute the compound using a buffer that will break the bond between the compound and receptor.
  • the TAR-1 and TAR-3 receptor ⁇ of the present invention may also be employed in a proces ⁇ for ⁇ creening for antagoni ⁇ t ⁇ and/or agonists for the receptors.
  • mammalian cells or membrane preparations expressing either the TAR-1 or TAR-3 receptor would be incubated with labeled ligand in the presence of compound. The ability of the compound to enhance or block this interaction could then be measured.
  • the response of a known second messenger system following interaction of ligand and receptor would be measured compared in the presence or absence of the compound.
  • second me ⁇ enger systems include but are not limited to, cAMP guanylate cycla ⁇ e, ion channels or phosphoino ⁇ itide hydrolysis.
  • screening techniques include the use of cells which express the receptor ⁇ (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, a ⁇ described in Science, volume 246, pages 181-296 (October 1989)'.
  • potential agonists or antagonists may be contacted with a cell which expresses the receptors and a second messenger response, e.g. signal transduction or pH changes, may be measured to determine whether the potential agonist or antagoni ⁇ t is effective.
  • Another such screening technique involves introducing RNA encoding the receptors into xenopu ⁇ oocyte ⁇ to transiently express the receptor.
  • the receptor oocytes may then be contacted in the case of antagonist screening with the receptor ligand and a compound to be screened, followed by detection of inhibition of a calcium signal.
  • Another screening technique involves expres ⁇ ing the TAR- 1 or TAR-3 receptor in which the receptor i ⁇ linked to a pho ⁇ pholipase C or D.
  • a pho ⁇ pholipase C or D As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embryonic kidney cells, etc.
  • the screening for an antagoni ⁇ t or agonist may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.
  • a potential antagonist i ⁇ an antibody, or in some cases an oligonucleotide, which binds to the TAR-1 and TAR-3 receptors but does not elicit a second messenger response such that the activity of the receptors is prevented.
  • Potential antagonists also include proteins which are closely related to the ligand of the receptors, i.e. a fragment of the ligand, which have lost biological function and when binding to the TAR-1 and TAR-3 receptor ⁇ , elicit no response.
  • a potential antagonist also includes an antisense construct prepared through the use of antisense technology.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • the antisense RNA oligonucleotide hybridizes to the mRNA n vivo and blocks translation of the mRNA molecule into the receptor ⁇ (antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides a ⁇ Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • the oligonucleotide ⁇ de ⁇ cribed above can al ⁇ o be delivered to cell ⁇ such that the antisen ⁇ e RNA or DNA may be expre ⁇ ed in vivo to inhibit production of the receptor ⁇ .
  • Another potential antagoni ⁇ t i ⁇ a small molecule which binds to the TAR-1 and/or TAR-3 receptor ⁇ , making them inaccessible to ligands such that normal biological activity is prevented.
  • ⁇ mall molecule ⁇ include but are not limited to ⁇ mall peptide ⁇ or peptide-like molecule ⁇ .
  • Potential antagonists also include a soluble form of a TAR-1 and TAR-3 receptors, e.g. a fragment of the receptor, which binds to the ligand and prevents the ligand from interacting with membrane bound receptor ⁇ .
  • the ⁇ e ⁇ oluble fragments bind to TGF-/5" and/or activin and prevent binding to membrane-bound receptor ⁇ .
  • the antagonists may also be employed to treat female sterility, ⁇ ince activin decreases follicle size and may lead to this disorder.
  • the antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
  • Agonists to the TAR-1 and TAR-3 receptors may be employed to activate these receptors and upregulate the functional application of TGF-S. Since TGF- / S has been implicated in suppressing growth of cancerous cells, the activation of the TAR-1 and TAR-3 receptor may be employed to treat cancer. TGF-)8 also inhibits growth and proliferation of the HIV virus and accordingly stimulating the TAR-1 and TAR-3 receptors treats HIV infection.
  • Agonist ⁇ which stimulate these receptors also will upregulate activins and stimulate spermatogonial proliferation to treat male infertility.
  • compositions comprise a therapeutically effective amount of the agonist or antagonist and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredient ⁇ of the pharmaceutical compo ⁇ ition ⁇ of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredient ⁇ of the pharmaceutical compo ⁇ ition ⁇ of the invention.
  • Associated with such container( ⁇ ) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological product ⁇ , which notice reflect ⁇ approval by the agency of manufacture, use or sale for human administration.
  • the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.
  • the pharmaceutical composition ⁇ may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes.
  • the pharmaceutical composition ⁇ are administered in an amount which is effective for treating and/or prophylaxis of the specific indication.
  • the pharmaceutical compositions will be administered in an amount of at least about 10 ⁇ g/kg body weight and in most cases they will be administered in an amount not in excess of about 3 mg/Kg body weight per day.
  • the do ⁇ age is from about 10 ⁇ g/kg to about 1 g/kg body weight daily, taking into account the routes of administration, symptom ⁇ , etc.
  • TAR-1 and TAR-3 receptor polypeptides and agoni ⁇ t ⁇ and antagoni ⁇ t ⁇ which are polypeptide ⁇ may al ⁇ o be employed in accordance with the invention by expre ⁇ sion of the polypeptides in vivo , which is often referred to as "gene therapy" .
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding the TAR-1 and TAR-3 polypeptides ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptides.
  • a polynucleotide DNA or RNA
  • cells may be engineered by procedure ⁇ known in the art by u ⁇ e of a retroviral particle containing RNA encoding a polypeptide of the pre ⁇ ent invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo .
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA( ⁇ ).
  • Computer analysis of the cDNA( ⁇ ) i ⁇ u ⁇ ed to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process.
  • These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosome ⁇ and pre ⁇ election by hybridization to construct chromosome specific-cDNA(s) libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA(s) clone ⁇ to a metapha ⁇ e chromosomal spread can be u ⁇ ed to provide a preci ⁇ e chromosomal location in one step.
  • Thi ⁇ technique can be used with cDNA(s) as short as 500 or 600 bases; however, clone ⁇ larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • FISH requirs ⁇ use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 i ⁇ better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time.
  • the physical po ⁇ ition of the ⁇ equence on the chromosome can be correlated with genetic map data.
  • genetic map data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkin ⁇ Univer ⁇ ity Welch Medical Library).
  • the relation ⁇ hip between gene ⁇ and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
  • a cDNA(s) precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
  • the polypeptides, their fragments or other derivative ⁇ , or analogs thereof, or cells expressing them can be used a ⁇ an immunogen to produce antibodie ⁇ thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well a ⁇ Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and ragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptide ⁇ to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide ⁇ itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodie ⁇ can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provide ⁇ antibodie ⁇ produced by continuou ⁇ cell line culture ⁇ can be used.
  • Examples include the hybrido a technique (Kohler and Mil ⁇ tein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybriBoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Lies, Inc., pp. 77-96).
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basi ⁇ . or can be constructed from available plasmids in accord with published procedures.
  • equivalent pla ⁇ mid ⁇ to tho ⁇ e de ⁇ cribed are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequence ⁇ in the DNA.
  • the variou ⁇ restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used a ⁇ would be known to the ordinarily skilled artisan.
  • For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • For the purpose of isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and ⁇ ubstrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 * C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
  • Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980).
  • Oligonucleotide ⁇ refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a pho ⁇ phate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers to the process of forming phosphodiester bonds between two double ⁇ tranded nucleic acid fragments (Maniati ⁇ , T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • plasmid, TAR-1 HA i ⁇ derived from a vector pcDNA( ⁇ )I/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire TAR-1 precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter.
  • the HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
  • HA tag to our target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the DNA sequence encoding TAR-1, ATCC # 75843, was constructed by PCR on the original EST cloned using two primers: the 5' primer 5' GCGCGAAGCTTTACAATGGTAGATGG AGTGAT 3' contains a Hind III site followed by 21 nucleotides of TAR-1 coding sequence including the initiation codon; the 3' sequence 5' GCGCGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGG GTAACAGTCAGTTTTCAATTTGT 3' contains complementary sequences to an Xho site, translation stop codon, HA tag and the last 20 nucleotides of the TAR-1 coding sequence (not including the stop codon).
  • the PCR product contains a Hind III site, TAR-1 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xho site.
  • the PCR amplified DNA fragment and the vector, pcDNA(s)I/Amp were dige ⁇ ted with Hind III and Xho I restriction enzyme and ligated.
  • the ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning System ⁇ , 11099 North Torrey Pine ⁇ Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and examined by restriction analysi ⁇ for the presence of the correct fragment.
  • COS cells were transfeeted with the expression vector by DEAE-DEXTRAN method.
  • the expression of the TAR-1 HA protein was detected by radiolabelling and immunoprecipitation method.
  • Cells were labelled for 8 hours with 3S S-cysteine two days post transfection.
  • plasmid, TAR-3 HA is derived from a vector pcDNA( ⁇ )I/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire TAR-3 precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter.
  • the HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previou ⁇ ly described (I. Wilson, H.
  • HA tag to our target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the plasmid construction strategy is described as follows: The DNA ⁇ equence encoding TAR-3, ATCC No. 75842, wa ⁇ constructed by PCR on the Full-length TAR-3 clone using two primers: the 5' primer is 5' GCGCGAAGCTTACCATGACC TTGGGCTCCCCCA 3' contains a Hind III site followed by 22 nucleotide ⁇ of TAR-3 coding ⁇ equence starting from the initiation codon; the 3' sequence 5' GCGCGTCTAGATCAAGCGTAG TCTGGGACGTCGTATGGGTAGTGAATCACTTTGGGCTTCTC 3' contains complementary sequences to an Xba I site, translation stop codon, HA tag and the last 21 nucleotides of the TAR-3 coding sequence (not including the stop codon).
  • the PCR product contains a Hind III site, TAR-3 coding sequence followed by HA tag fused in frame, a translation termination ⁇ top codon next to the HA tag, and an Xba I site.
  • the PCR amplified DNA fragment and the vector, pcDNA(s)I/Amp were digested with Hind III and Xba I restriction enzyme and ligated.
  • the ligation mixture wa ⁇ transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and re ⁇ i ⁇ tant colonies were selected.
  • COS cell ⁇ were tran ⁇ fected with the expre ⁇ sion vector by DEAE-DEXTRAN method.
  • the expression of the TAR-3 HA protein was detected by radiolabelling and immunoprecipitation method.
  • Cells were labelled for 8 hours with 3S S-cysteine two days post tran ⁇ fection.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,

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Abstract

L'invention concerne des polynucléotides codant les récepteurs polypeptidiques TAR-1 et TAR-3, ainsi que les polypeptides, et des anticorps et antagonistes dirigés contre ces polypeptides. On décrit également un procédé pour produire de tels polypeptides par des techniques de recombinaison, ainsi que l'utilisation d'agonistes et d'antagonistes à des fins thérapeutiques, par exemple pour inhiber la croissance de cellules et pour traiter les fibroses hépatiques et pulmonaires.
PCT/US1994/011328 1994-10-05 1994-10-05 ACTIVINE 1 ET ACTIVINE 3, DE LA FAMILLE DES RECEPTEURS DU FACTEUR TRANSFORMANT DE CROISSANCE TGF-β1 WO1996011259A1 (fr)

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AU10390/95A AU1039095A (en) 1994-10-05 1994-10-05 Tgf-beta 1, activin receptors 1 and 3

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Cited By (2)

* Cited by examiner, † Cited by third party
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WO2003006057A1 (fr) * 2001-07-13 2003-01-23 Bioa Pty Limited Composition et procede pour le traitement d'une maladie
US8920806B2 (en) 2003-10-06 2014-12-30 Paranta Biosciences Limited Method of modulating inflammatory response by downregulation of activin

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CELL, Vol. 68, issued 21 February 1992, H. LIN et al., "Expression Cloning of the TGF-beta Type II Receptor, a Functional Transmembrane Serine/Threonine Kinase", pages 775-785. *
CELL, Vol. 75, issued 19 November 1993, L. ATTISANO et al., "Identification of Human Activin and Tgfbeta Type I Receptors That form Heteromeric Kinase Complexes with Type II Receptors", pages 671-680. *
DEVELOPMENTAL DYNAMICS, Vol. 196, issued 1993, W. HE et al., "Developmental Expression of Four Novel Serine. Threonine Kinase Receptors Homologous to the Activin/Transforming Growth Factor-beta type II Receptor Family", pages 133-142. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 268, No. 17, issued 15 June 1993, K. MATSUZAKI et al., "A Widely Expressed Transmembrane Serine/Threonine Kinase That Does Not Bind Activin, Inhibin, Transforming Growth Factor beta, or Bone Morphogenic Factor", pages 12719-12723. *
ONCOGENE, Vol. 8, Number 10, issued 1993, P. TEN DIJKE et al., "Activin Receptor-Like Kinases: A Novel Subclass of Cell-Surface Receptors with Predicted Serine/Threonine Kinase Activity", pages 2879-2887. *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006057A1 (fr) * 2001-07-13 2003-01-23 Bioa Pty Limited Composition et procede pour le traitement d'une maladie
US8920806B2 (en) 2003-10-06 2014-12-30 Paranta Biosciences Limited Method of modulating inflammatory response by downregulation of activin

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