WO2004035627A1 - Modulation de parcours de signalisation de type beta tgf - Google Patents

Modulation de parcours de signalisation de type beta tgf Download PDF

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WO2004035627A1
WO2004035627A1 PCT/GB2003/004535 GB0304535W WO2004035627A1 WO 2004035627 A1 WO2004035627 A1 WO 2004035627A1 GB 0304535 W GB0304535 W GB 0304535W WO 2004035627 A1 WO2004035627 A1 WO 2004035627A1
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polypeptide
tsk
nucleic acid
cancer
tgfβ superfamily
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PCT/GB2003/004535
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English (en)
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Shin-Ichi Ohnuma
Giuseppe Lupo
William Harris
Kunimasa Ohta
Sei Kuriyama
Hideaki Tanaka
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Cambridge University Technical Services Limited
Cancer Research Technology Limited
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Priority to AU2003271988A priority Critical patent/AU2003271988A1/en
Publication of WO2004035627A1 publication Critical patent/WO2004035627A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the modulation of Transforming Growth Factor (TGF ⁇ ) -like molecule signalling pathways, in particular, the modulation of the Bone Morphogenetic Protein (BMP) and TGF ⁇ signalling pathways and agents and methods for performing such modulation for a range of therapeutic purposes.
  • TGF ⁇ Transforming Growth Factor
  • BMP Bone Morphogenetic Protein
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • the superfamily can roughly be grouped into four families: the TGF- ⁇ s, the Activins, the Nodal-related factors, and the Bone Morphogenetic Proteins (BMP)s (Piek, E. et al (1999). Faseb J 13, 2105-24).
  • BMP Bone Morphogenetic Proteins
  • BMP2 subfamily BMP2, BMP4
  • BMP5 subfamily BMP5, BMP6, BMP7 , BMP8
  • GDF5 subfamily GDF5, GDF6, GDF7
  • Vgl subfamily Vgl/GDFl, GDF3
  • BMP3 subfamily BMP3, GDF10
  • Nodal subfamily Nodal, Dorsalin, GDF8, GDF9
  • Activin subfamily Activin- ⁇ A, Activin- ⁇ B, Activin- ⁇ C, Activin- ⁇ E
  • TGF- ⁇ subfamily TGF-fll, TGF- ⁇ 2, TGF- ⁇ 3
  • BMPs form the largest group within the TGF- ⁇ family and are broadly conserved across the animal kingdom, including vertebrates, arthropods and nematodes (von Bubnoff, h . and Cho, K. W. (2001) Dev Biol 239, 1-14) . BMPs act as instructive signals during embryogenesis, and are involved in the maintenance and repair of bone and other tissues in the adult (Hoffmann, A. and Gross, G. (2001) Cri t Rev Eukaryot Gene Expr 11, 23-45) . In particular, during vertebrate embryonic development, BMPs control the dorso- ventral patterning of the mesoderm and specification of epidermis (Altmann, C. R. and Brivanlou, A.
  • TGF- ⁇ superfamily signal transduction pathway involves two distinct kinds of transmembrane serine/threonine kinase receptors (type I and type II) and a family of receptor substrates (the Smad proteins) that move into the nucleus.
  • TGF- ⁇ ligands bind to a type II receptor, which then recruits a type I receptor.
  • the type II receptor phosphorylates serine and threonine residues within the intracellular domain of the type I receptor.
  • the activated type I receptor kinase phosphorylates particular members of the Smad family, called receptor-regulated Smads (R-Smads) , which are released from the receptor upon phosphorylation, and interact with Smad .
  • Phosphorylation also results in the nuclear accumulation of these otherwise cytoplasmic localized factors to permit the assembly of Smad/transcription factor complexes on the promoter of target genes (Hill, C. S. (2001) Curr Opin Genet Dev 11, 533-40; Kloos, D. U. et al (2002) Trends Genet 18, 96-103; Moustakas, A. et al (2001) J Cell Sci 114, 4359-69; von Bubnoff and Cho, 2001 supra; Whitman, M. (2001) . Dev Cell 1, 605-17) .
  • TGF ⁇ family signalling is regulated by multiple mechanisms at extracellular, cytoplasmic and nuclear levels to control access of TGF ⁇ family members to their receptors, the activity of their receptors and receptor substrates, and the nuclear function of the transcriptional complexes generated by this pathway.
  • Extra-cellular modulators have been a particular focus of investigation, since a major means of signal regulation for TGF ⁇ signalling is the modulation of ligand availability at the extra-cellular level.
  • One of the best studied paradigms for this kind of regulation is provided by the evolutionarily conserved mechanism of modulation of ligand activity for the BMP family of TGF ⁇ molecules.
  • BMP4 In amphibians and zebrafish, opposing activities of BMP4 and its antagonist Chordin have been implicated in patterning the embryonic dorso-ventral axis (Kishimoto, Y. et al (1997) Developmen t 124, 4457-66; Sasai, Y. et al (1995) Na ture 376, 333-6; Schmidt, J. et al (1995) Development 121, 4319-28).
  • BMP activity specifies ventral fates in the early embryo, and is antagonized by the secreted protein Chordin, which is expressed dorsally.
  • dpp BMP homologue decapentaplegic
  • salig Chordin homologue Short gastrulation
  • dpp specifies dorsal fates in the embryo and is antagonized by Sog, which is expressed ventrally (Holley, S. A. et al (1995) Na t ure 376, 249-53).
  • Noggin Another secreted protein, Noggin, is also expressed in the dorsal side of the amphibian embryo and is involved in specification of dorsal fates (Re ' em-Kalma, Y. et al (1995) Proc Na tl Acad Sci U S A 92, 12141-5; Zimmerman, L. B. et al (1996) Cell 86, 599-606) .
  • Chordin and Noggin are able to promote formation of dorsal tissues (such as notochord and muscle) within the mesoderm, and neural tissue within the ectoderm, at the same time inhibiting formation of ventral mesoderm and epidermis.
  • Chordin and Noggin bind specifically to BMPs, but not to Activin or TGF- ⁇ , and antagonize BMP signalling by blocking BMP interaction with their cognate receptors.
  • Antagonism of BMP activity by Noggin is also critical for proper skeletal development, since Noggin-null mice have excess cartilage and failed to initiate joint formation (Brunet, L. J. et al (1998) Science 280, 1455-7) .
  • TGF ⁇ -family signalling underlies many clinical disorders, including various forms of cancer, such as colorectal, gastric, ovarian, breast, pancreatic and biliary cancers, bone diseases, such as proximal symphalangism, multiple synostoses, chondrodysplasias and osteoporosis, vascular diseases such as hypertension and various heritable developmental disorders such as hereditary hemorrhagic telangiectasia, holoprosencephaly, MUllerian duct syndrome and familial primary pulmonary hypertension (Massague, J et al (2000) Cell 103, 295-309) .
  • cancer such as colorectal, gastric, ovarian, breast, pancreatic and biliary cancers
  • bone diseases such as proximal symphalangism, multiple synostoses, chondrodysplasias and osteoporosis
  • vascular diseases such as hypertension and various heritable developmental disorders such
  • TGF ⁇ s and BMPs extra-cellular modulators of members of the TGF ⁇ superfamily, including TGF ⁇ s and BMPs, and are involved in embryogenesis and the pathogenesis of human disorders resulting from abnormal TGF ⁇ signalling. These modulators are termed Tsukushi (TSK) polypeptides.
  • a first aspect of the invention provides an isolated nucleic acid encoding a TSK polypeptide which modulates TGF ⁇ superfamily signalling pathways and which has at least 45% sequence identity with the sequence shown in SEQ ID NO: 2.
  • Modulates of TGF ⁇ superfamily signalling pathways may include modulation of one or more activities of a member of the TGF ⁇ superfamily, for example a TGF ⁇ , such as TGF ⁇ l, TGF ⁇ 2 or TGF ⁇ 3, or a BMP, such as BMP1, BMP2, BMP3 or BMP4.
  • a TGF ⁇ such as TGF ⁇ l, TGF ⁇ 2 or TGF ⁇ 3, or a BMP, such as BMP1, BMP2, BMP3 or BMP4.
  • an isolated nucleic acid may comprise or consist of the nucleic acid sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 11, as shown in Figures 1, 3, 5, 7, 9 or 11 respectively, or may be an allele or variant thereof.
  • SEQ ID NOS: 1, 3, 5, 7, 9 and 11 are the Human TSK (h-TSK) , Xenopus TSK (x-TSK) , chick TSK-A (C-TSK-A) , chick TSK-B (C- TSK-B) , mouse TSK (m-TSK) , and zebrafish TSK (z-TSK) nucleic acid sequences respectively.
  • nucleic acid consisting of SEQ ID NO: 1 and/or the sequence of Genbank Ace No: BF717749 may be excluded.
  • An isolated nucleic acid may comprise or consist of an open reading frame encoding a TSK polypeptide.
  • an isolated nucleic acid may consist of the nucleic acid sequence of beginning at position 14 and position 1,072 of the nucleotide sequence of SEQ ID NO: 1 or may be an allele or variant thereof.
  • An isolated nucleic acid may comprise or consist of the polynucleotide beginning at position 1 and ending at position 1,062 of the nucleotide sequence of SEQ ID NO: 9, the polynucleotide beginning at position 142 and ending at position 1,248 of SEQ ID NO:5, the polynucleotide beginning at position 1 and ending at position 1,056 of SEQ ID NO:7, the polynucleotide beginning at position 220 and ending at position 1,272 of SEQ ID NO: 3 or the polynucleotide beginning at position 1 and ending at position 1,041 of SEQ ID NO: 11 or may be an allele or variant of any of these.
  • allelic or variant sequence may differ from that of SEQ ID NOS: 1, 3, 5, 7, 9 or 11 by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to a nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code.
  • nucleic acid may include a sequence different from the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 yet encode a polypeptide with the same amino acid sequence.
  • An isolated nucleic acid may share greater than about 55% sequence identity with a nucleic acid sequence as shown in SEQ ID NO: 3, 5, 7, 9 or 11, greater than 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%.
  • a nucleic acid may share greater than about 65% similarity, greater than about 70% similarity, greater than about 80% similarity, greater than about 90% similarity or greater than about 95% similarity.
  • the present invention also extends to nucleic acid that hybridizes with the sequence shown in SEQ ID NO: 1,3, 5, 7, 9 or 11 under stringent conditions.
  • nucleic acid may encode a TSK polypeptide as described herein or a fragment thereof.
  • suitable conditions include, e.g. for detection of sequences that are about 80-90% identical suitable conditions include hybridisation overnight at 42°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0.1 X SSC, 0.1% SDS.
  • suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1% SDS.
  • the nucleic acid consisting of SEQ ID NO: 1 may be excluded.
  • isolated requires that the material be removed from its original environment (e.g. the natural environment if it is naturally occurring) .
  • a naturally occurring polynucleotide or a peptide present in a living animal is not isolated, but the same polynucleotide or peptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • oligonucleotide "nucleic acid” and
  • polynucleotide include DNA, RNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.
  • a nucleotide may be a naturally occurring nucleotide or it may encompass modified nucleotides which comprise at least one of the following modifications: (a) an alternative linking group, (b) a purine analogue (c) a pyrimidine analogue, or (d) a sugar analogue.
  • modifications include (a) an alternative linking group, (b) a purine analogue (c) a pyrimidine analogue, or (d) a sugar analogue.
  • Examples of linking group, purine, pyrimidine, and sugar analogues are well known in the art (for example, WO95/04064).
  • the present invention also encompasses fragments of the sequences described above, for example a fragment of the nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 11.
  • Suitable fragments may consist of less than the full length TSK nucleotide sequence as described above, for example less than 2622 nucleotides or less than 1053 (x-TSK) , 1107 (c-TSK-A), 1126 (c-TSK-B), 1065 (m-TSK) , 1059 (h-TSK) or 1041 (z-TSK) nucleotides.
  • Such a fragment may, for example, consist of at least 10, 20, 30, 40 or 50 nucleotides, and for example less than 1000, 900, 800 or 700 nucleotides.
  • Such a fragment may encode a TSK polypeptide as described herein and may retain the TGF ⁇ superfamily modulation activity of the full-length protein (for example, BMP or TGF ⁇ modulatory activity) or it may be useful as an oligonucleotide probe or primer.
  • a fragment may exclude the nucleic acid consisting of the sequence of Genbank Accession No: BF717749.
  • a nucleotide sequence encoding a TSK polypeptide may be manipulated to express a mature TSK protein by deleting TSK propeptide sequences and replacing them with sequences encoding an amino acid sequence of a different non-TSK polypeptide (i.e. a heterogeneous sequence), for example a BMP protein or another member of the TGF ⁇ superfamily.
  • a different non-TSK polypeptide i.e. a heterogeneous sequence
  • Another aspect of the present invention provides a chimeric nucleic acid construct comprising a nucleotide sequence encoding an amino acid sequence from a polypeptide other than a TSK polypeptide (i.e. a non- ⁇ TSK' sequence) linked, preferably in the correct reading frame, to a nucleotide sequence encoding a TSK polypeptide .
  • a chimeric nucleic acid construct comprising a nucleotide sequence encoding an amino acid sequence from a polypeptide other than a TSK polypeptide (i.e. a non- ⁇ TSK' sequence) linked, preferably in the correct reading frame, to a nucleotide sequence encoding a TSK polypeptide .
  • TSK polypeptide with TGF ⁇ superfamily modulation activity which has at least 45% sequence identity with the sequence of SEQ ID NO: 2.
  • an isolated TSK polypeptide may comprise or consist of the amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 10 or 12 or may be an allele or variant thereof.
  • h-TSK Human TSK
  • x-TSK Xenopus TSK
  • c-TSK-A chick TSK-A
  • TSK (z-TSK) have the amino acid sequences specified in SEQ ID NO: 1
  • sequence of SEQ ID NOS: 2, 4, 6, 8, 10 and 12 respectively.
  • sequence of SEQ ID NO: 2 and/or the sequence of Genbank Ace. NO: CAC39777.1 may be excluded.
  • a polypeptide of the invention may be encoded by -a nucleic acid as described above.
  • a TSK polypeptide may comprise an amino acid sequence which shares greater than about 40% sequence identity with the sequence of SEQ ID NO: 2, greater than 45%, greater than about 50%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%.
  • the sequence may share greater than about 30% similarity with the sequence of SEQ ID NO: 2, greater than about 40% similarity, greater than about 50% similarity, greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity.
  • an amino acid sequence variant or allele of retains TSK activity i.e. it modulates TGF- ⁇ super family signalling.
  • TSK activity i.e. it modulates TGF- ⁇ super family signalling.
  • BMP or TGF ⁇ signalling may be modulated.
  • BMP signalling may be inhibited and/or TGF ⁇ signalling enhanced or increased.
  • TSK polypeptides may be readily identified in public domain databases, for example by Blast searching using these sequences.
  • GAP GAP
  • other algorithms e.g. BLAST (which uses the method of Altschul et al . (1990) J. Mol . Biol . 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444- 2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol . 147: 195-197), or the TBLASTN program, of Altschul et al . (1990) supra, generally employing default parameters.
  • the psi-Blast algorithm Nucl. Acids Res. (1997) 25 3389-3402) may be used.
  • conservative amino acid substitutions may be made among amino acids with basic side chains, such as lysine (Lys or K) , arginine (Arg or R) and histidine (His or H) ; amino acids with acidic side chains, such as aspartic acid (Asp or D) ) and glutamic acid (Glu or E) ; amino acids with uncharged polar side chains, such as asparagine (Asn or N) , glutamine (Gin or Q) , serine (Ser or S) , threonine (Thr or T) , and tyrosine (Tyr or Y) ; and amino acids with nonpolar side chains, such as alanine (Ala or A) , glycine (Gly or G) , valine (Val or V), leucine (Leu or L) ,
  • a residue in the L-form may be replaced by a residue in the D-form or a glutamic acid residue may be replaced by a pyro-glutamic acid compound.
  • the synthesis of peptides containing at least one residue in the D-form is, for example, described by Koch, Y. (1977) Biochem Biophys Res Commun 74, 488-491.
  • the activity of an allele or variant of a TSK protein can be readily determined, for example, by subjecting the variant to an assay as described below.
  • Particular amino acid sequence variants may differ from a known polypeptide sequence as described herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30, 30-50, or more than 50 amino acids .
  • a "allele” or “variant” of a polypeptide or nucleic acid may include a natural sequence variation which is found in one or more individual within a population. Alleles or variants of an amino acid sequence may involve one or more of insertion, addition, deletion or substitution of one or more amino acids, without fundamentally altering the qualitative nature of the activity of the wild-type TSK polypeptide, indeed the existence of a naturally occurring allelic variant presupposes that activity is retained.
  • a variant polypeptide also includes species homologues i.e. a TSK polypeptide as described above which is from a species other than mouse, Xenopus, chick, zebrafish, or human. Such variants may comprise one or more, for example 10, 20, 30 or more or all the conserved residues highlighted in figure 14.
  • a variant polypeptide may have TSK activity as described herein.
  • a TSK polypeptide preferably comprises one or more leucine rich repeats and a cysteine rich domain as shown in Figure 14. The presence of these conserved domains may be used to identify TSK polypeptides.
  • a TSK polypeptide may comprise one or both of the following motifs
  • Nucleic acid encoding a TSK polypeptide as described above may be provided as part of a replicable vector, particularly any expression vector from which the encoded polypeptide can be expressed under appropriate conditions, and a host cell containing any such vector or nucleic acid.
  • An expression vector in this context is a nucleic acid molecule including nucleic acid encoding a polypeptide of interest and appropriate regulatory sequences for expression of the polypeptide, in an in vi tro expression system, e.g. reticulocyte lysate, or in vivo, e.g. in eukaryotic cells such as COS or CHO cells or in prokaryotic cells such as E . coli . This is discussed further below.
  • Another aspect of the invention provides a method of producing a TSK polypeptide comprising: (a) causing expression from nucleic acid which encodes a TSK polypeptide in a suitable expression system to produce the polypeptide recombinantly;
  • a polypeptide may be tested for TSK activity by determining one or more of the following:
  • TGF ⁇ -like polypeptide 1) binding to a TGF ⁇ -like polypeptide, by immunoprecipitation of TSK proteins and other TGF- ⁇ like polypeptides with purified proteins, cell, embryo or adult tissue extracts;
  • TGF ⁇ -like polypeptide 2) determining antagonism or synergism of a TGF ⁇ -like polypeptide, by co-overexpression or double knock-out of TSK genes and other TGF- ⁇ s in cell lines or embryos;
  • Smads modulated by TSK signalling may include Smadl, 2, 3, 4, 5 and 8.
  • TGF ⁇ -like molecules may include Vgl/Gdfl (Ace No: P27539) Activins (Ace No: CAA40805, CAA57890, NP113667), Nodal (Ace No: BAB62524)and TGF ⁇ s (Ace No: NP000651, AAA50404, P16047) and BMP polypeptides such as BMP1 (Ace No: P13497), BMP2 (P12643), BMP3 (NP058801), BMP4 (NP570912), BMP5 (NP066551), BMP6 (NP001709) and BMP7 (NP001710), (US5,108, 922; US5,013,649; US5, 116, 738 ; US5,106,748; US5,187,076; and US5,141,905) BMP8 (P34820) (WO91/18098 ) , BMP9 (NP057288) (WO93/00432) , BMP10 (NP055297)
  • the TGF ⁇ -like polypeptide is a BMP polypeptide, for example a BMP4 polypeptide and the BMP4 antagonistic activity is determined.
  • the TGF ⁇ -like polypeptide is a TGF ⁇ polypeptide, and the TGF ⁇ agonist activity is determined.
  • a TSK polypeptide or a fragment thereof as described above may be generated by chemical synthesis or by recombinant expression from encoding nucleic acid.
  • Peptide fragments may be generated wholly or partly by chemical synthesis.
  • the compounds of the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide
  • Alanine scans are commonly used to find and refine peptide motifs within polypeptides. This involves the systematic replacement of each residue in turn with the amino acid alanine, followed by an assessment of biological activity. This enables the residues responsible for the activity to be determined.
  • Nucleic acid sequences encoding a TSK polypeptide may be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Russell and Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press NY, (2001), and Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, 1992), given the nucleic acid sequence and clones available. These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g.
  • PCR polymerase chain reaction
  • DNA encoding TSK polypeptides may be generated and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers.
  • the sequences can be incorporated in a vector having one or more control sequences operably linked to the nucleic acid to control its expression.
  • the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide or peptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.
  • plasmids viral e.g. 'phage, or phagemid, as appropriate.
  • Polypeptide may be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the polypeptide is produced and recovering the polypeptide from the host cells or the surrounding medium.
  • Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others.
  • a common, preferred bacterial host is E. col i .
  • Bacterial cells may also be suitable hosts.
  • various strains of E . col i e.g., HB101, MC1061 are well- known as host cells.
  • Various strains of B . subtil is , Pseudomonas, and other bacill i may also be employed.
  • Yeast cells may also be available as host cells for expression of the polypeptides. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein.
  • Fusion proteins may be generated that incorporate six histidine residues at either the N-terminus or C-terminus of the recombinant protein.
  • a histidine tag may be used for purification of the protein by using commercially available columns which contain a metal ion, either nickel or cobalt (Clontech, Palo Alto, CA, USA) . These tags also serve for detecting the protein using commercially available monoclonal antibodies directed against the six histidine residues (Clontech, Palo Alto, CA, USA) .
  • TSK polypeptide or polypeptide fragment a nucleic acid encoding such a polypeptide or fragment, or a modulator as described above may be used in formulation of a composition, which may include at least one additional component, for example a pharmaceutical composition including a pharmaceutically acceptable excipient, vehicle or carrier.
  • a pharmaceutical composition including a pharmaceutically acceptable excipient, vehicle or carrier.
  • Such compositions may be used in prophylactic and/or therapeutic treatment as discussed below.
  • a method of producing a pharmaceutical composition may comprise :
  • the recombinantly produced polypeptide may be tested for modulation of one or more TGF ⁇ superfamily signalling pathways, for example, the polypeptide may be tested for BMP polypeptide antagonist or TGF ⁇ agonist activity.
  • a method may include the step of modifying a TSK polypeptide to optimise its pharmaceutical properties.
  • a method of producing a pharmaceutical may comprise; providing a TSK polypeptide as described above, modifying the polypeptide to optimise its pharmaceutical properties, determining the modulation of TGF ⁇ superfamily signalling pathways by said modified polypeptide, synthesising and/or manufacturing said modified polypeptide, and; formulating said modified polypeptide with a pharmaceutically acceptable excipient.
  • Another aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide or nucleic acid as described above and a pharmaceutically acceptable excipient .
  • a pharmaceutical composition may further comprise one or more other therapeutically useful agents including a growth factor such as epidermal growth factor (EGF) , fibroblast growth factor (FGF), transforming growth factor (TGF and TGF ⁇ ) , activin, Nodal, Vgl, inhibin, insulin-like growth factor (IGF) , a BMP polypeptide, for example any one of BMP1-16, GDFs (see e.g. W094/15965), BIP (WO94/01557 ) , HP00269 (JP7-250688) ; and MP52, (WO93/16099) .
  • a growth factor such as epidermal growth factor (EGF) , fibroblast growth factor (FGF), transforming growth factor (TGF and TGF ⁇ )
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • TGF and TGF ⁇ transforming growth factor
  • activin Nodal
  • Vgl transforming growth factor
  • IGF insulin-like growth factor
  • composition may also * include soluble antagonists of growth factors, such as Chordin, (US5, 986, 056) ; Noggin (US5,843,775) ; Follistatin (US5, 041, 538 ) ; Cerberus and Frzb-1 (US6, 133,232) .
  • Chordin (US5, 986, 056)
  • Noggin (US5,843,775)
  • Follistatin (US5, 041, 538 )
  • Cerberus and Frzb-1 US6, 133,232) .
  • a composition may comprise a matrix capable of delivering TSK-related or other BMP proteins to the site of bone and/or cartilage and/or other connective tissue damage, providing a structure for the developing bone and cartilage and other connective tissue and, preferably, capable of being resorbed into the body.
  • the matrix may provide slow release of TSK protein and/or other bone inductive factors, as well as an appropriate environment for cellular infiltration.
  • Such matrices may be formed of materials presently in use for other implanted medical applications (Kirker-Head, CA. (2000) Advanced Drug Delivery Reviews 43, 65-92) .
  • Matrices for the compositions may be biodegradable and may include calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyanhydrides and biological matrices such as bone, dermal collagen or pure proteins or extracellular matrix components.
  • Non-biodegradable matrices may include sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability .
  • compositions are described in more detail below. Methods and uses of TSK polypeptides and nucleic acids in therapy are also described below.
  • TSK polypeptide as described herein may also be used in the design of mimetics which retain TSK polypeptide activity.
  • a method of designing a mimetic of a TSK polypeptide wherein said mimetic modulates TGF ⁇ superfamily signalling pathways and/or antagonises BMP activity may comprise (i) analysing a TSK polypeptide as described above to determine the amino acid residues essential and important for the biological activity (i.e. the modulation of TGF ⁇ superfamily signalling) to define a pharmacophore; and, (ii) modelling the pharmacophore to design and/or screen candidate mimetics having the biological activity.
  • Another aspect of the present invention provides a method of identifying a variant or homologue of a TSK nucleic acid comprising; providing a cDNA or genomic library, screening said library with a TSK nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOS 1, 3, 5, 7, 9 or 11 or a fragment thereof; and, identifying a sequence in said library which hybridises to said nucleic acid molecule.
  • a cDNA or genomic library may be provided from cells of any suitable source, including human or other mammalian cells, or cells from another species.
  • Screening may be carried out by hybridisation with the nucleic acid molecule under appropriate hybridisation conditions, as described above and in the examples below.
  • a sequence which is a member of the library which is found to hybridise to the TSK nucleic acid molecule may be subjected to further analysis, for example it may be cloned and/or sequenced.
  • a method of identifying a variant or homologue of a TSK nucleic acid may comprise; providing a cDNA or genomic sequence database, screening said database with a TSK sequence selected from the group consisting of SEQ ID NOS 1, 3, 5, 7, 9 or 11 or a fragment thereof; and, identifying a sequence in said database which has greater then 45% identity to said TSK sequence.
  • TSK polypeptides are useful in methods of identifying and/or obtaining agents and compounds which modulate TGF ⁇ superfamily signalling, such as TGF ⁇ and/or BMP signalling, for example by modulating the activity of a TSK polypeptide or its binding to a member of the TGF ⁇ superfamily.
  • agents and compounds may be useful in a range of therapeutic applications.
  • An aspect of the invention provides the use of a TSK polypeptide as described herein, or a nucleic acid molecule encoding such a TSK polypeptide, for screening for or obtaining test compounds which (a) share a TSK polypeptide biological activity or (b) bind to the TSK polypeptide, or (c) inhibit a biological activity of a TSK polypeptide polypeptide .
  • Another aspect of the invention provides a method for identifying and/or obtaining a modulator of TSK, which method comprises:
  • the TSK polypeptide and the test compound may be contacted 10 in the presence of a TGF ⁇ -like polypeptide (i.e. a member of the TGF ⁇ superfamily) , and; the activity of said TGF ⁇ -like polypeptide may be determined.
  • a TGF ⁇ -like polypeptide i.e. a member of the TGF ⁇ superfamily
  • Activity may be determined in the presence and absence of the test compound. An increase or decrease in the activity of the TGF ⁇ -like polypeptide in the presence relative to the absence of test compound is indicative that the compound is a modulator of TSK activity, for example, an 0 enhancer (i.e. an agonist) or inhibitor (i.e. an antagonist) of TSK activity.
  • an 0 enhancer i.e. an agonist
  • inhibitor i.e. an antagonist
  • a TGF ⁇ -like polypeptide may include a member of the TGF ⁇ superfamily, such as BMP polypeptide, 5 for example a polypeptide comprising the sequence of any one of BMP 1 to 16, or Actinin, Vgl, Nodal, or TGF ⁇ or an allele or variant of any of these.
  • ⁇ the TGF ⁇ -like polypeptide is a BMP 4 polypeptide or allele or variant thereof.
  • the TGF ⁇ -like polypeptide is a TGF ⁇ polypeptide or allele or variant thereof.
  • an activity of a TGF ⁇ polypeptide may include TGF ⁇ mediated migration, apoptosis or cell cycle arrest. Methods described herein may further comprise the step of determining the ability of said test compound to modulate, for example inhibit TGF ⁇ superfamily signalling pathways.
  • Polypeptide fragments as described herein which retain all or part of the activity of the full-length protein may be generated and used in any suitable way known to those of skill in the art. Suitable ways of generating fragments include, but are not limited to, recombinant expression of a fragment from encoding DNA. Such fragments may be generated by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Small fragments (e.g. up to about 20 or 30 amino acids) may also be generated using peptide synthesis methods, which are well known in the art.
  • Suitable detectable labels include 35 S-methionine, which may be incorporated into recombinantly produced peptides and polypeptides. Recombinantly produced peptides and polypeptides may also be expressed as a fusion protein containing an epitope, which can be labelled with an antibody.
  • the polypeptide which is immobilized on a solid support may be immobilized using an antibody against that polypeptide bound to a solid support or via other technologies which are known per se .
  • a preferred in vi tro interaction may utilise a fusion protein including glutathione-S- transferase (GST) . This may be immobilized on glutathione agarose beads.
  • GST glutathione-S- transferase
  • a test compound can be assayed by determining its ability to diminish the amount of labelled peptide or polypeptide which binds to the immobilized GST- fusion polypeptide. This may be determined by fractionating the glutathione-agarose beads by SDS-polyacrylamide gel electrophoresis .
  • the beads may be rinsed to remove unbound protein and the amount of protein which has bound can be determined by counting the amount of label present in, for example, a suitable scintillation counter.
  • assays according to the present invention take the form of in vivo assays.
  • In vivo assays may be performed in a cell line such as a yeast strain, insect or mammalian cell line in which the relevant polypeptides or peptides are expressed from one or more vectors introduced into the cell.
  • TSK polypeptide and a TGF ⁇ -like polypeptide i.e. a member of the TGF ⁇ super family such as Actinin, Nodal, Vgl, TGF ⁇ or a BMP polypeptide such as BMP4 polypeptide
  • a polypeptide or peptide containing a fragment of a TSK polypeptide or a TGF ⁇ -like polypeptide as the case may be, or a peptidyl analogue or variant thereof as disclosed may be fused to a DNA binding domain such as that of the yeast transcription factor GAL 4.
  • the GAL 4 transcription factor includes two functional domains.
  • GAL4DBD DNA binding domain
  • GAL4TAD GAL4 transcriptional activation domain
  • LexA DNA binding domain and the VP16 transcriptional activation domain LexA DNA binding domain and the VP16 transcriptional activation domain.
  • the TSK or TGF ⁇ -like polypeptide or peptide When looking for peptides or other substances which interfere with interaction between a TSK polypeptide or peptide and a TGF ⁇ -like polypeptide or peptide, the TSK or
  • TGF ⁇ -like polypeptide or peptide may be employed as a fusion with (e.g.) the LexA DNA binding domain, and the counterpart (e.g.) BMP or TSK polypeptide or peptide as a fusion with (e.g.) VP16, and involves a third expression cassette, which may be on a separate expression vector, from which a peptide or a library of peptides of diverse and/or random sequence may be expressed.
  • a reduction in reporter gene expression e.g.
  • test substance is not peptidyl and may not be expressed from encoding nucleic acid within a said third expression cassette
  • a similar system may be employed with the test substance supplied exogenously.
  • Combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide.
  • test substances Prior to or as well as being screened for modulation of activity, test substances may be screened for ability to interact with the polypeptide, e.g. in a yeast two-hybrid system (which requires that both the polypeptide and the test substance can be expressed in yeast from encoding nucleic acid) . This may be used as a coarse screen prior to testing a substance for actual ability to modulate activity of the polypeptide.
  • test substance or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.01 to 100 nM concentrations of putative inhibitor compound may be used, for example from 0.1 to 10 nM.
  • Test compounds may be natural or synthetic chemical compounds used in drug screening programmes . Extracts of plants which contain several characterised or uncharacterised components may also be used.
  • One class of putative inhibitor compounds can be derived from the TSK polypeptide and/or a ligand which binds to a TSK polypeptide such as a TGF ⁇ -like polypeptide, for example a BMP or TGF ⁇ polypeptide.
  • the inhibitory properties of a peptide fragment as described above may be increased by the addition of one of the following groups to the C terminal: chloromethyl ketone, aldehyde and boronic acid. These groups are transition state analogues for serine, cysteine and threonine proteases .
  • the N terminus of a peptide fragment may be blocked with carbobenzyl to inhibit aminopeptidases and improve stability (Proteolytic Enzymes 2nd Ed, Edited by R. Beynon and J. Bond, Oxford University Press 2001) .
  • Antibodies directed to the site of interaction in either the TSK polypeptide or TGF ⁇ -like polypeptide form a further class of putative inhibitor compounds.
  • Candidate inhibitor antibodies may be characterised and their binding regions determined to provide single chain antibodies and fragments thereof which are responsible for disrupting the interaction.
  • Another aspect of the invention provides an antibody molecule, which binds specifically to a TSK polypeptide as described.
  • Such an antibody may be a candidate inhibitor of TSK polypeptide activity as described herein.
  • an antibody which specifically binds to an antigen may not show any significant binding to molecules other than the antigen.
  • an antibody may specifically bind to a particular epitope which is carried by a number of antigens, in which case the antibody will be able to bind to the various antigens carrying the epitope
  • Antibodies may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al . , 1992, Nature 357: 80-82).
  • Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal .
  • an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • the library may be naive, that is constructed from sequences obtained from an organism which has not been - immunised with any of the proteins (or fragments), or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.
  • Antibodies according to the present invention may be modified in a number of ways. Indeed, the term “antibody” should be construed as covering any binding substance having a binding domain with the required specificity. Thus the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimicks that of an antibody enabling it to bind an antigen or epitope.
  • Example antibody fragments which are capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region.
  • Single chain Fv fragments are also included.
  • Antibodies may also be used in purifying and/or isolating a polypeptide or peptide for use in the present methods, for instance following production of the polypeptide or peptide by expression from encoding nucleic acid therefor.
  • Antibodies may be useful in a therapeutic context (which may include prophylaxis) to disrupt TSK interactions with TGF ⁇ -like polypeptides with a view to inhibiting or reducing TSK activity.
  • Antibodies can for instance be micro-injected into tissues, e.g. at a tumour site, subject to radio- and/or chemo-therapy (as discussed already above) .
  • Antibodies may be employed in accordance with the present invention for other therapeutic and non-therapeutic purposes.
  • a method for detecting the presence of a TSK protein in a sample may comprise:
  • candidate modulator compounds may be based on modelling the 3-dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential modulator (for example, inhibitor) compounds with particular molecular shape, size and charge characteristics .
  • a potential modulator compound may be a "functional analogue" of a peptide or other compound which modulates TSK polypeptide/TGF ⁇ -like polypeptide binding in a method of the invention.
  • a functional analogue has the same functional activity as the peptide or other compound in question, i.e. it may interfere with the binding between a TSK polypeptide and a TGF ⁇ -like polypeptide, such as a BMP polypeptide.
  • Examples of such analogues include chemical compounds which are modelled to resemble the three dimensional structure of the TSK domain in the contact area, and in particular the arrangement of the key amino acid residues as they appear in TSK.
  • TSK activity or function of TSK may be inhibited, as noted, by means of a substance that interferes in some way with the interaction of TSK with other factors described herein.
  • An alternative approach to inhibition employs regulation at the nucleic acid level to inhibit activity or function by down-regulating production of the TSK polypeptide.
  • expression of a gene may be inhibited using anti-sense or RNAi technology.
  • anti-sense or RNAi technology The use of these approaches to down-regulate gene expression is now well-established in the art.
  • Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of TSK polypeptide so that its expression is reduced or completely or substantially completely prevented.
  • antisense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the antisense oligonucleotides can interfere with expression control sequences.
  • the construction of antisense sequences and their use is described for example in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990) and Crooke, Ann. Rev. Pharmacol. Toxicol . , 32:329-376, (1992).
  • Oligonucleotides may be generated in vi tro or ex vivo for administration or anti-sense RNA may be generated in vivo within cells in which down-regulation is desired.
  • double-stranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene.
  • the complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works.
  • the complete sequence corresponding to the coding sequence in reverse orientation may be used.
  • a fragment of this sequence may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon.
  • the antisense nucleic acids are chosen among the polynucleotides of 15-200bp long that are complementary to the 5' end of a nucleic acid encoding a TSK protein.
  • a suitable fragment may for example have about 14-23 nucleotides, e.g. about 15, 16 or 17.
  • Preferred antisense nucleic acids are complementary to a sequence of a TSK mRNA that contains the translational initiation codon ATG.
  • the antisense nucleic acid may also be complementary to a sequence in the 3' or 5' untranslated regions or to sequences in the splice sites of the pre-mRNA precursor.
  • Anti-sense oligonucleotides can be made from deoxyribonucleotides or ribonucleotides and can also contain chemical modifications which prevent degradation by endogenous nucleases such as phosphorothioate oligonucleotides or morpholino oligonucleotides (Heasman et al (2000), Developmen tal Biology, 222, 124-134).
  • RNA interference RNA interference
  • RNA interference is a two-step process.
  • dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs ( ⁇ 2nt) .
  • siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore P.D. Na ture Structural Biology, 8, 9, 746-750, (2001) )
  • RNAi may be also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3'- overhang ends (Zamore PD et al. Cell 101 25-33, (2000)). Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologous genes in a wide range of mammalian cell lines (Elbashir SM. et al . Na ture, 411, 494-498, (2001)).
  • nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific site - thus also useful in influencing gene expression.
  • Background references for ribozymes include Kashani-Sabet and Scanlon, 1995, Cancer Gene Therapy, 2(3): 213-223, and Mercola and Cohen, 1995, Cancer Gene Therapy, 2(1), 47-59.
  • a modulator of TSK activity and thus a modulator of TGF ⁇ superfamily signalling pathways may comprise a nucleic acid molecule comprising all or part of the TSK coding sequence and/or the complement thereof
  • Such a molecule may suppress the expression of TSK polypeptide and may comprise a sense or anti-sense TSK coding sequence or may be a TSK specific ribozyme, according to the type of suppression to be employed.
  • the type of suppression will also determine whether the molecule is double or single stranded and whether it is RNA or DNA. Examples of the use of siRNA to reduce or abolish TSK polypeptide expression are provided below.
  • Methods of the invention may comprise the step of identifying a test compound, for example a candidate modulator as described above, as a modulator of binding between the TSK polypeptide and a TGF ⁇ -like polypeptide as discussed above.
  • the test compound may be further identified as a modulator of TSK activity and thus a modulator of the activity of TGF ⁇ superfamily signalling pathways.
  • a modulator may include an inhibitor (i.e. an antagonist) of TSK activity or an enhancer (i.e. an agonist) of TSK activity.
  • a method may comprise, for example, isolating and/or purifying said test compound.
  • a method may further comprise synthesising and/or manufacturing the compound.
  • a method may further comprise modifying the compound identified as described above to optimise the pharmaceutical properties thereof.
  • a ⁇ lead' compound identified as biologically active is a known approach to the development of pharmaceuticals and may be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Modification of a known active compound may be used to avoid randomly screening large number of molecules for a target property.
  • Modification of a ⁇ lead' compound to optimise its pharmaceutical properties commonly comprises several steps. Firstly, the particular parts of the compound that are critical and/or important in determining the target property may be determined. In the case of a peptide, this ' can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore".
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X- ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.
  • a range of sources e.g. spectroscopic techniques, X- ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms
  • other techniques can be used in this modelling process.
  • a method of modifying a compound found to inhibit the interaction between TSK polypeptide and a TGF ⁇ - like polypeptide, modulate TSK polypeptide activity and/or modulate the activity of TGF ⁇ superfamily signalling pathways such as the BMP signalling pathway may comprise: (i) analysing a compound identified as having the biological activity to determine the amino acid residues essential and important for this activity to define a pharmacophore; and, (ii) modelling the pharmacophore to design and/or screen candidate molecules having the biological activity.
  • Suitable modelling techniques are known in the art. This includes the design of so-called “mimetics” which involves the study of the functional interactions of the molecules and the design of compounds which contain functional groups arranged in such a manner that they could reproduced those interactions .
  • the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the optimisation of the lead compound.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the modified compound is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the modified compounds found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Modified compounds include mimetics of the lead compound.
  • test compound may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, e.g. for any of the purposes discussed elsewhere herein.
  • a method of the invention may comprise formulating said test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier as discussed further below.
  • Another aspect of the invention provides method of producing a pharmaceutical comprising;
  • test compound (d) synthesising and/or manufacturing said test compound, and; (e) formulating said test compound with a pharmaceutically acceptable excipient.
  • a method may comprise the step of modifying the test compound to optimise its pharmaceutical properties.
  • Another aspect of the present invention provides a method of producing a pharmaceutical composition
  • a method of producing a pharmaceutical composition comprising; identifying a compound which modulates the activity of a TSK polypeptide using a method described herein; and, admixing the compound identified thereby with a pharmaceutically acceptable carrier.
  • compositions with pharmaceutically acceptable carriers is described further below.
  • Another aspect of the invention provides a method for preparing a pharmaceutical composition, for example, for the treatment of a condition which is ameliorated by the modulation (i.e. inhibition or enhancement) of TGF ⁇ superfamily signalling pathways, comprising; i) identifying a compound which is an agonist/antagonist of a TSK polypeptide ii) synthesising the identified compound, and; iii) incorporating the compound into a pharmaceutical composition .
  • the identified compound may be synthesised using conventional chemical synthesis methodologies. Methods for the development and optimisation of synthetic routes are well known to a skilled person.
  • the compound may be modified and/or optimised as described above .
  • Incorporating the compound into a pharmaceutical composition may include admixing the synthesised compound with a pharmaceutically acceptable carrier or excipient.
  • compositions for example, pharmaceutically acceptable excipients, carriers, buffers and stabilisers are well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required .
  • Another aspect of the invention provides a modulator, for example an inhibitor, of TSK activity, or composition comprising such a modulator, which is isolated and/or obtained by a method described herein.
  • Modulators may include small chemical entities, polypeptides, peptide fragments, nucleic acids or antibodies, as described above.
  • TSK polypeptides, nucleic acids and modulators as described above may be useful in treating disease conditions associated with aberrant TGF ⁇ superfamily signalling or which may be ameliorated by an increase or decrease in TSK polypeptide activity.
  • the invention extends in various aspects not only to a TSK polypeptide or nucleic acid or a compound identified as a modulator of TSK activity (e.g. an anti-TSK antibody), in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a TSK polypeptide or nucleic acid or modulator, a method comprising administration of such a composition to a patient, e.g. for treatment (which may include preventative treatment) of a condition associated with TGF ⁇ superfamily signalling, for example aberrant TGF ⁇ superfamily signalling, use of such a compound in manufacture of a composition for administration, e.g.
  • a modulator of TSK activity e.g. an anti-TSK antibody
  • compositions for treatment of a condition associated with TGF ⁇ superfamily signalling and a method of making a pharmaceutical composition comprising admixing such a compound with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients .
  • Pharmaceutical compositions are discussed in more detail above .
  • an active polypeptide, nucleic acid or compound may be used in combination with any other active substance, e.g. for anti-tumour therapy, another anti-tumour compound or therapy, such as radiotherapy or chemotherapy.
  • a method when conducted in vivo, may comprise determining the effect of the polypeptide, nucleic acid or modulator of TSK activity on TGF ⁇ superfamily signalling and/or its cellular effects.
  • TSK polypeptides nucleic acids as described above may be useful in treating disease conditions associated or mediated by TGF ⁇ superfamily signalling, in particular aberrant TGF ⁇ superfamily signalling or which may be ameliorated by an increase or decrease in TGF ⁇ superfamily signalling or TSK polypeptide activity.
  • TGF ⁇ superfamily signalling may include BMP or TGF ⁇ signalling .
  • An isolated polypeptide with TGF ⁇ superfamily regulatory activity which has at least 45% sequence identity with the sequence of SEQ ID NO: 2 as described above or an isolated nucleic acid encoding such a polypeptide may be useful in a method of treatment of the human or animal body.
  • a polypeptide with TGF ⁇ superfamily regulatory activity which has at least 45% sequence identity with the sequence of SEQ ID NO: 2 or a nucleic acid encoding such a polypeptide in the manufacture of a medicament for use in a method selected from the group consisting of wound healing or tissue repair, bone and/or cartilage tissue formation or the treatment of fibrosis, cancer and other disorders associated with or mediated by TGF ⁇ superfamily signalling.
  • Also provided is a method of treatment comprising administering a TSK polypeptide which has at least 45% sequence identity with the sequence of SEQ ID NO: 2 or a nucleic acid encoding said polypeptide to an individual in need thereof.
  • An individual may be in need of wound healing or tissue repair, bone and/or cartilage tissue formation, and/or be suffering from a disorder associated with or mediated by TGF ⁇ superfamily signalling, such as fibrosis or cancer.
  • a nucleic acid may have a sequence selected from the group consisting of SEQ ID NOS 1, 3, 5, 7, 9 and 11.
  • a polypeptide may have a sequence selected from the group consisting of SEQ ID NOS 2, 4, 6, 8, 10 and 12.
  • a TSK polypeptide may induce or influence cartilage and/or bone and/or other connective tissue formation and may be used in the healing of bone fractures and cartilage or other connective tissue defects.
  • a TSK protein may, for example, be used prophylactically in both closed and open fracture reduction and in the improved fixation of artificial joints.
  • a TSK polypeptide may also be used in the treatment of periodontal disease, and in other tooth repair processes.
  • TSK polypeptides may also be used for wound healing, reduction of fibrosis and reduction of scar tissue formation, and also in the prevention or therapy of cancer.
  • Nucleic acid encoding a TSK polypeptide may be used in methods of gene therapy, for example by insertion of the fully functional gene by a vector delivery system that would result in the repair of a damaged area.
  • the nucleic acid In order to achieve expression of the encoding nucleic acid, the nucleic acid must be delivered into a cell. Delivery may be accomplished in vitro, using laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain disease states, as described herein.
  • Non-viral methods for the transfer of polynucleotides into cultured mammalian cells may also be used, including calcium phosphate precipitation (Chen, C. and Okayama, H. (1987). Mol Cell Biol 7, 2745-52.; Graham and van der Eb, (1973). Virology 52, 456-67); DEAE-dextran (Gopal, T. V. (1985). Mol Cell Biol 5, 1188-90); electroporation (Potter, H., Weir, L. and Leder, P.
  • the nucleic acid to be expressed may be stably integrated into the genome of the recipient cell. This integration may be in the right location and orientation via homologous recombination (gene replacement) or it may be in a random, non-specific location. Alternatively, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or episomes encode sequences sufficient to permit maintenance and replication independent of, or in synchronization with the host cell cycle .
  • a TSK polypeptide or nucleic acid may be used in conjunction with at least one BMP protein or growth factor including EGF, FGFs, TGF ⁇ , TGF ⁇ , activin, inhibin, nodal, vgl and IGF, or an antagonist of a growth factor including Chordin, Noggin, Cerberus or Frzb-1.
  • Disorders associated with TGF ⁇ superfamily signalling may include cancer, such as lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carcinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi ' s sarcoma, melanoma, lymphoma or leukaemia, bone diseases, such as proximal symphalangism, multiple synostoses, chondrodysplasias, osteoarthritis and osteoporosis, fibrotic conditions, vascular diseases such as hypertension and various heritable developmental disorders such as hereditary hemorrhagic telangiectasia, holoprosencephaly, M ⁇ llerian duct syndrome and familial primary pulmonary hypertension
  • cancer such as lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carcinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer,
  • a cancer condition may include an early stage or localised cancer, for example a stage 1 or stage 2 cancer, such as an early stage colorectal, gastric, prostate, lung, ovarian, breast, pancreatic or biliary cancer.
  • a stage 1 or stage 2 cancer such as an early stage colorectal, gastric, prostate, lung, ovarian, breast, pancreatic or biliary cancer.
  • administration of TSK polypeptide (or a mimetic or agonist of TSK) to a primary tumour site may enhance the cell-cycle arrest activity of TGF ⁇ and inhibit proliferation of tumour cells.
  • An aspect of the invention therefore encompasses a method of treating an early stage cancer condition in an individual comprising; administering a TSK polypeptide or nucleic acid or a mimetic or agonist thereof to the individual, and the use of a TSK polypeptide or nucleic acid or a mimetic or agonist thereof in the manufacture of a medicament for use in the treatment of an early stage cancer condition.
  • a cancer condition may include a late stage or metastatic cancer, for example a stage 3 or stage 4 cancer, such as a late stage colorectal, gastric, prostate, lung, ovarian, breast, pancreatic or biliary cancer.
  • Administration of a TSK antagonist such as an anti- TSK antibody may reduce the metastatic activity of TGF ⁇ and inhibit metastasis of tumour cells.
  • An aspect of the invention therefore encompasses a method of treating a late stage cancer condition in an individual comprising; administering a TSK antagonist, for example an anti-TSK antibody to the individual, and the use of such a TSK antagonist in the manufacture of a medicament for use in the treatment of a late stage or metastatic cancer condition.
  • compositions as described above may also be useful in regulating the formation of bone, cartilage, or other connective tissue, including tendon, ligament, meniscus and other connective tissue, as well as combinations of the above, including, for example, for regeneration of the tendon-to-bone attachment apparatus.
  • the compositions may be useful for the induction, growth, differentiation, maintenance and/or repair of tissues such as brain, liver, kidney, lung, heart, muscle, epidermis, pancreas, nerve, and other organs.
  • administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • a prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • the dosage regimen will be determined by the attending physician considering various factors which modify the action of TSK proteins, e.g. amount of bone or other tissue weight desired to be formed, the site of bone or tissue damage, the condition of the damaged bone tissue, the size of a wound, type of damaged tissue, the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and the types of TGF ⁇ -like proteins in the composition.
  • systemic or injectable administration will be initiated at a dose which is minimally effective, and the dose will be increased over a pre-selected time course until a positive effect is observed.
  • IGF I insulin like growth factor I
  • Progress can be monitored by periodic assessment of bone or tissue growth and/or repair.
  • the progress can be monitored, for example, x-rays, histomorphometric determinations and tetracycline labeling.
  • TSK The expression of TSK is shown herein to be related to disease conditions.
  • Various aspects of the invention relate to the diagnosis of disease conditions by measuring the expression of TSK polypeptides.
  • a method of assessing a cancer condition in an individual may comprise; determining the level or amount of TSK expression in a sample obtained from the individual.
  • a cancer condition is preferably a non-oestrogen dependent cancer condition i.e. a cancer which is not responsive to the presence or level of oestrogen, for example lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma, lymphoma or leukaemia.
  • a cancer condition may be non-hormone dependent (i.e. not responsive to the presence or level of hormones) .
  • the sample may be a tissue biopsy sample, for example from tissue suspected of malignancy, or may be a biological fluid sample, for example from blood, serum or plasma.
  • An increase in the level of amount of TSK expression in the sample relative to a control is indicative that the individual has a cancer condition.
  • the level or amount of TSK expression may be determining by determining the level of TSK mRNA in said sample, for example by RT-PCR or Northern Blotting, using conventional techniques .
  • the level or amount of TSK expression may be determining by determining the level of TSK polypeptide in said sample, for example using anti-TSK antibodies.
  • a method of assessing a cancer condition in an individual may comprise; contacting a sample obtained from the individual with an antibody molecule which specifically binds to TSK polypeptide, and; determining the binding of said antibody.
  • the level or amount of binding of said antibody to the sample is indicative of whether the individual has a cancer condition .
  • the binding of antibodies to a sample may be determined by any appropriate means. Tagging with individual reporter molecules is one possibility.
  • the reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals.
  • the linkage of reporter molecules may be direct or indirect, covalent, e.g. via a peptide bond, or non-covalent . Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion, encoding antibody and reporter molecule.
  • the antibody may be labelled with a fluorophore such as FITC or rhodamine, a radioisotope, or a non-isotopic-labelling reagent such as biotin or digoxigenin; antibodies • containing biotin may be detected using "detection reagents" such as avidin conjugated to any desirable label such as a fluorochrome .
  • a fluorophore such as FITC or rhodamine
  • a radioisotope such as a radioisotope
  • a non-isotopic-labelling reagent such as biotin or digoxigenin
  • biotin or digoxigenin antibodies • containing biotin may be detected using "detection reagents" such as avidin conjugated to any desirable label such as a fluorochrome .
  • Another possibility is detect the binding of antibodies to TSK using a second antibody, for example in an ELISA assay system.
  • the second antibody may
  • a labelled third antibody may be used to detect the binding of the second antibody.
  • the mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.
  • Suitable approaches include Western Blotting, immunofluorescence, enzyme linked immunosorbent assays (ELISA) , radioimmunoassays (RIA) , immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA) , including sandwich assays using monoclonal and/or polyclonal antibodies. All of these approaches are well known in the art.
  • An antibody for use in a method described herein may be immobilised or non-immobilised i.e. free in solution.
  • An antibody may be immobilised, for example, by attachment to a solid support for use in an immunoassay.
  • the support may be in particulate or solid form and may include a plate, a test tube, beads, a ball, a filter or a membrane.
  • a antibody may, for example, be fixed to an insoluble support that is suitable for use in affinity chromatography. Methods for fixing antibodies to insoluble supports are known to those skilled in the art.
  • An immobilised antibody may be preferred, for example, in assay formats such as ELISA.
  • Another aspect of the invention provides an immunoassay solid support comprising an antibody which specifically binds a TSK polypeptide as described herein.
  • Preferred immunoassay solid supports are described above and include microtiter plates.
  • An immunoassay solid support may be produced by a method comprising :
  • a method of assessing a cancer condition .in an individual may comprise: (a) providing an immunoassay solid support comprising a antibody which specifically binds a TSK polypeptide,
  • Complexes may be detected as described above by adding to the solid support from step (b) under complex forming conditions (i) a second detectably labeled antibody, wherein said second detectably labeled antibody binds to antibodies from said sample, for example an anti-IgG antibody.
  • methods of the invention may be performed in an ELISA format.
  • the wells of a microtiter plate may be coated with the peptide and a biological sample containing or suspected of containing antibody molecules is then added to the coated wells .
  • the plate (s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule (e.g. labeled anti-IgG antibody) added.
  • a detectably labeled secondary binding molecule e.g. labeled anti-IgG antibody
  • kits for use in assessing a cancer condition in an individual are provided.
  • kits may comprise an antibody which specifically binds to a TSK polypeptide.
  • the antibody may be immobilised on a solid support, for example, a microtitre plate.
  • a kit may include one or more other reagents required for the method, such as secondary antibodies, detection reagents, buffer solutions etc.
  • the secondary antibody may be labelled.
  • kits may comprise an oligonucleotide probe and/or primers which specifically hybridise to a TSK nucleic acid sequence. Suitable oligonucleotides may be complementary to the coding sequence of a TSK nucleic acid.
  • a kit may include one or more other reagents required for the method, which may for example be an RT-PCR or Northern blotting method. Suitable reagents include reverse transcriptase, thermostable polymerase, dNTPs, ATP, labels and detection reagents .
  • the kit may include instructions for use in a method of the invention.
  • a kit may include one or more other reagents required for the method, such as detection reagents, buffer solutions, wash solutions etc.
  • a kit may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself, e.g. a syringe for removing a sample and sample handling containers (such components generally being sterile) .
  • Figure 1 shows the nucleic acid sequence of human TSK (SEQ ID NO:l) .
  • Figure 2 shows the amino acid sequence of human TSK (SEQ ID NO: 2) .
  • Figure 3 shows the nucleic acid sequence of Xenopus TSK (SEQ ID NO:3) .
  • Figure 4 shows the amino acid sequence of Xenopus TSK (SEQ ID N0:4) .
  • Figure 5 shows the nucleic acid sequence of chick TSK-A (SEQ ID NO:5) .
  • Figure 6 shows the amino acid sequence of chick TSK-A (SEQ ID NO:6) .
  • Figure 7 shows the nucleic acid sequence of chick TSK-B (SEQ ID NO:7) .
  • Figure 8 shows the amino acid sequence of chick TSK-B (SEQ ID NO:8) .
  • Figure 9 shows the nucleic acid sequence of mouse TSK (SEQ ID NO:9) .
  • Figure 10 shows the amino acid sequence of mouse TSK (SEQ ID NO:10) .
  • Figure 11 shows the nucleic acid sequence of zebrafish TSK (SEQ ID NO:ll) .
  • Figure 12 shows the amino acid sequence of zebrafish TSK (SEQ ID NO:12) .
  • Figure 13 shows the sequence alignment of the x-TSK, c-TSK- A, c-TSK-B, m-TSK, h -TSK, and z-TSK coding sequences.
  • the overall cDNA sequence identity is 63.0% between Xenopus and chick, 56.4% between Xenopus and mouse, 56.7% between Xenopus and human, 55.9% between Xenopus and zebrafish, 59.4% between chick and mouse, 59.4% between chick and human, 58.4% between chick and zebrafish, 82.1% between mouse and human, 55.3% between mouse and zebrafish, 54.3% between human and zebrafish. Residues that are identical among all members are black shaded and residues that differ in only one are grey shaded.
  • Figure 14 shows the sequence alignment of x-TSK, c-TSK-A, c-TSK-B, m-TSK, h-TSK and z-TSK proteins.
  • the overall amino acid sequence identity is 58.7% between Xenopus and chick, 48.7% between Xenopus and mouse, 48.3% between Xenopus and human, 49.4% between Xenopus and zebrafish, 55.2% between chick and mouse, 56.1% between chick and human, 53.9% between chick and zebrafish, 85.4% between mouse and human, 44.5% between mouse and zebrafish, 46.3% between human and zebrafish.
  • Residues that are identical among all members are black shaded and residues that differ in only one are grey shaded.
  • FIG 15 shows the results of a luciferase assay showing activation of TGF- ⁇ signalling by TSK (TSK) .
  • Figure 16 shows the results of a BrdU incorporation assay which show enhancement of TGF- ⁇ mediated cell cycle arrest by TSK.
  • Figure 17 shows the results of Hoechst staining experiments showing enhancement of TGF- ⁇ mediated apoptosis by TSK.
  • Figure 18 shows that TSK treatment enhances MCF-7 cell migration .
  • FIG. 19 shows that treatment of MCF-7 cells with anti-TSK antibodies inhibits TGF ⁇ l-stimulated migration in vi tro
  • FIG. 20A shows TSK gene expression in breast tumour biopsies.
  • Figure 20B shows TSK gene expression in prostate tumour biopsies .
  • Figure 21A shows TSK gene expression in lung tumour biopsies .
  • Figure 21B shows TSK. gene expression in colon tumour biopsies .
  • the signal sequence trap (SST) method was performed as described by Klein et al (Klein, R. D. et al (1996) . Proc
  • the cDNAs were ligated to Notl/Xhol adaptors and size selected to isolate cDNAs between 0.5 and 1.2 kb by agarose gel electrophoresis.
  • the cDNAs were ligated into the pYEsuc2 vector, which contains an invertase gene lacking the signal peptide and initiator methionine.
  • the constructed cDNA library was transformed into yeast strain YT455, and plasmids were isolated from colonies that survived the invertase selection. Positive clones were sequenced randomly and analyzed for similarity to known genes.
  • C-TSK-A was isolated from E7 lens cDNA library using a cDNA fragment obtained by SST.
  • C-TSK-A full-length cDNA was used to screen a Xenopus embryo cDNA library (stage 13; No. 546, RZPD) by low-stringency hybridization according to the instruction manuals.
  • m-TSK was isolated from the screening of the mouse liver genomic library (CLONTECH) with a cDNA fragment obtained by degenerate primers DSFLXXVDC and PLRYLXLX.
  • a zebrafish EST cDNA fragment accession NO. BF717749 was used as a probe for screening the 1-month-old embryonic cDNA library (CLONTECH) .
  • Xenopus embryos and in si tu hybridiza tions Embryos were obtained as previously described by Newport, J. and Kirschner, M. (1982) Cell 30 675-86, and staged according to conventional techniques. Embryos and explants were fixed in MEMFA and processed for whole-mount in situ hybridization as previously described, except that proteinase K treatment was omitted for explants hybridization. Pigmented embryos and explants were bleached after the colour reaction as Mayor, R. et al (1995) Developmen t 121, 767-77.
  • RNAs were in vitro synthesized from linearized plasmid templates as described in Krieg, P. A. and Melton, D. A. (1984) Nucleic Acids Res 12, 7057-70. Embryos were injected at the 2- and 4-cell stage in O.lx MMR, 4% Ficoll, and then transferred after a few hours into O.lx MMR for subsequent culturing.
  • mice were injected in the animal pole of 2-cell stage embryos.
  • Animal caps were dissected out of stage 8-9 embryos (either uninjected or control) in IxMBS; after healing, caps were cultured in 0.5xMBS until early tailbud stage 21/22 alongside with sibling embryos.
  • VMZ/DMZ experiments embryos were marginally injected at the 4-cell stage in either the ventral or the dorsal blastomeres. VMZ or DMZ explants were dissected in IxMBS at stage 10-10.25 and cultured in 0.5xMBS until stage 24-27 alongside with sibling embryos.
  • the expression plasmid for TSK-Fc was constructed with the full-length of TSK and the Fc region of the human immunoglobulin gene in the pEF-BOS expression vector.
  • the TSK-Fc fusion protein was transiently produced in COS-7 cells and purified from culture supematants using a protein A-Sepharose column.
  • COS-7 cells were transfected at 90% confluence with Myc- tagged c-TSK-A or chick chordin in pEFl/Myc-HisA (Invitrogen) and Flag-tagged BMP2, BMP-4, or BMP-7 in pFLAG-CMV-5a (SIGMA) in DEME/Haml2 containing 10% fetal calf serum by LipofetAMINE 2000 (Invitrogen) . 16 hours after transfection, cells were washed twice with PBS and cultured in serum-free Opti-MEM (Invitrogen) for an additional 48 hours. The conditioned media were concentrated by Centriplus-10 (Amicon) , analyzed for protein levels by Western blotting and used for co- immunoprecipitation experiments.
  • SIGMA pFLAG-CMV-5a
  • c-TSK-A and BMP2, BMP4, or BMP7 proteins were incubated for 16 hours at 4°C in 1ml of saline buffer containing 150 mM NaCI, 20 mM Tris-HCl pH 7.5, 1.5 mM CaCl 2 , 1.5 mM MgCl 2 , 0.1% Triton X-100, 0.1% CHAPS, 5% glycerol and 0.1% BSA (IP buffer) .
  • IP buffer IP buffer
  • TSK cDNAs Over-expression experiments were performed by microinjection of in vitro transcribed clone TSK mRNAs in early Xenopus embryos. These experiments showed that TSK genes are endowed with a potent dorsalizing activity in the mesoderm, which transforms ventral mesoderm to dorsal fates, and with cement gland and neural inducing activity in the ectoderm. These activities are indicative of inhibition of the BMP signalling pathway. Confirmation that the TSK gene works as a BMP antagonist was obtained by co-injection of TSK and BMP RNAs in Xenopus embryos, and by immuno-precipitation experiments (see below example 4).
  • VMZ Ventral Marginal Zone
  • VMZ from uninjected embryos did not show convergent extension movements or expression of muscle actin, since they normally differentiate into ventral mesoderm.
  • VMZ explants from embryos injected with c-TSK-A or x-TSK mRNA showed extensive elongation, indicating the induction of dorsal mesoderm in the explants. This induction was confirmed by strong labelling of the explants after in si tu hybridization with a muscle actin probe.
  • mice ectodermal explants (animal caps) from late blastula embryos, either uninjected or injected at the 2 cell stage with either 800 pg of c- TSK-A mRNA or 1.5 ng of x-TSK mRNA per blastomere. Dissected caps were cultured until stage 21 ⁇ 22 and scored for the expression of ectodermal or mesodermal molecular markers .
  • c-TSK-A and x-TSK were able to trigger cement gland and neural differentiation, as indicated by the activation of XAG-1 , Xotx ⁇ b and Sox2.
  • the induced neural tissue showed expression of Xotx2, indicative of forebrain identity.
  • Cement gland and neural induction by c-TSK-A and x-TSK was direct and not due to the presence of dorsal mesoderm, as shown by the lack of muscle actin expression in the injected caps.
  • TSK Genes work as Extra-cellular BMP Antagonists
  • D-V Dorso-Ventral polarity of the vertebrate embryo
  • BMP molecules are expressed in ventral regions of the embryo and specify ventral developmental fates.
  • dorsal fates is achieved by antagonism of BMP signalling by a cocktail of secreted factors (e.g. chordin, noggin, follistatin) that bind to BMP in the extra-cellular space and prevent their binding to the cognate receptors (reviewed in De Robertis, E. M. et al (2000) . Nat Rev Genet 1, 171-81) .
  • a cocktail of secreted factors e.g. chordin, noggin, follistatin
  • x-TSK was able to counteract BMP4 ventralizing activity, since co-injection of 2 ng of x-TSK mRNA along with 1 ng of BMP4 mRNA rescued elongation and actin expression in the explants.
  • TSK genes antagonize BMP4 function in the extra-cellular space (possibly by direct inhibitory binding of TSK proteins to BMP4 or the BMP receptors) or intracellularly (through possible activation of a specific signal transduction pathway and regulation of the BMP4 transcriptional targets) .
  • a constitutively active form of the BMP4 type I receptor (CA-Alk3) was used, which is able to actively transduce signal independently from the binding with BMP4 (Onichtchouk, D. et al (1999) Nature 401, 480-5) .
  • c-TSK-A The ability of c-TSK-A to antagonize the action of CA-Alk3, was analysed by comparing the effects of the over- expression of c- TSK-A, c-TSK-A + CA-Alk3 or c-TSK-A + BMP4 on the expression of the cement gland marker XAG-1 in animal caps.
  • stage 21 ⁇ 22 animal caps from uninjected embryos did not show any XAG-1 induction, while caps from embryos injected with 1.6 ng of c-TSK-A mRNA (0.4 ng of c-TSK-A mRNA per blastomere in all 4 animal blastomeres at the 8 cell stage) were strongly induced.
  • XAG-1 activation was completely prevented by coinjection of c-TSK-A + CA-Alk3 in either 4:1 (3.2 ng of c-TSK-A + 0.8 ng of CA-Alk3 mRNA) or 6:1 (4.8 ng of c- TSK-A + 0.8 ng of CA-Alk3 mRNA) ratios.
  • XAG-1 expression was still significantly, though more weakly, induced by a 5:1 ratio of c-TSK-A + BMP4 (2.5 ng of c-TSK-A + 0.5 ng of BMP4 mRNA), when compared to c- TSfC-A-injected and control caps.
  • Whole embryos injected with 0.5 ng of BMP4 mRNA showed strong ventralization, indicating that this BMP4 dosage was sufficient to elicit a robust biological activity.
  • c-TSK-A is able to antagonize the ventralizing activity of BMP4 , but not of CA-Alk3, demonstrating that c-TSK-A works by antagonizing BMP signalling in the extra-cellular space.
  • TSK genes are expressed during embryonic development.
  • c-TSK-A expression is detectable in the Hensen's node and in the anterior region of the primitive streak at the early gastrula stages.
  • c-TSK-A expression becomes localized to the prospective pre-somitic mesoderm, and precedes segmentation of the paraxial mesoderm.
  • c-TSK-A expression correlates to the process of somitogenesis, and progressively moves posteriorly, so that c-TSK-A is down- regulated in the newly formed somites and turned on in the adjacent non-segmented paraxial mesoderm.
  • c-TSK-A expression is also detectable in the ectoderm flanking the anterior part of the neural tube.
  • c-TSK-A is also expressed in the olfactory placodes, branchial arches and in the limb buds .
  • x-TSK is expressed throughout the marginal zone and the animal pole of the embryo.
  • x-TSK expression is down-regulated on the dorsal side of the embryo during gastrulation (e.g. stage 10.5X11).
  • x-TSK is expressed in the ventro-lateral ectoderm, the prospective epidermis, but is excluded from the neural plate (stages 13 to 17).
  • x-TSK expression still overlaps with that of BMP4 in the tailbud region, in the branchial arches and in the dorsal part of the eye (st.22 ⁇ 23;).
  • x-TSK expression is dependent on functional BMP signalling.
  • Animal caps dissected from normal, un-injected, embryos give rise to epidermis and accordingly show expression of x-TSK, while injection of both 500pg of Chordin mRNA and Ing of Dominant-Negative BMP Receptor (tBR) mRNA strongly reduce x-TSK expression in animal caps.
  • tBR Dominant-Negative BMP Receptor
  • Both c-TSK-A and x-TSK are expressed during embryonic development in structures which are known to require modulation of BMP signalling, and x-TSK expression is regulated by BMP signalling.
  • c-TSK-A and x-TSK are expressed during embryonic development in structures which are known to require modulation of BMP signalling
  • x-TSK expression is regulated by BMP signalling.
  • the expression pattern of c-TSK-A and x-TSK, x-TSK regulation by BMP signalling and the observation that c-TSK-A and x-TSK over-expression can antagonize BMP signalling extra-cellularly by direct binding to BMPs, provide indication that c-TSK-A and x-TSK are extracellular modulators of BMP signalling during development.
  • TSK TGF- ⁇ signalling by TSK
  • TSK is an activator of TGF- ⁇ signalling.
  • TSK Enhancement of TGF- ⁇ mediated cell cycle arrest by TSK
  • TGF- ⁇ is known to down-regulate proliferation of non-transformed cells.
  • TSK effect of TSK on TGF- ⁇ mediated down-regulation of the cell cycle was investigated using the BrdU Incorporation assay.
  • NIH 3T3 cells were treated with 0.5 ng/ml TGF- ⁇ and TSK conditioned medium (CM) produced from TSK transfected COS-7 cells.
  • Control CM was produced from pCS2+ transfected COS-7 cells.
  • TGF- ⁇ 0.5 ng/ml TGF- ⁇ to NIH 3T3 cells results in a dramatic decrease of BrdU positive cells over 5-fold relative to control CM, indicative of cell cycle arrest.
  • BrdU positive cells decrease further to 45-fold less than the control.
  • Application of TSK CM alone to the cells did not result in a decrease of BrdU positive cells in comparison to control CM. See figure 16. This data is indicative of TSK enhancing TGF- ⁇ mediated cell cycle arrest.
  • TSK is expressed in a variety of cancer cell lines RT-PCR ( i O cycles of PCR) was performed to test for TSK mRNA expression in a variety of cancer cell lines. The PCR products were sequenced to demonstrate that the bands on the gel were TSK DNA.
  • TSK was found to be expressed in breast cancer cell lines (MCF-7, ZR-75-1), prostate cancer cell lines (LNCAP, PC3), cervical cancer cells (Hela) , ovarian cancer cells (OVCAR3) and lung cancer cells (A549) but was not expressed in glioblastoma cells (U373) .
  • TSK mRNA has been identified as. one of four mRNAs which is upregulated in MCF-7 cells in response oestrogen treatment (Charpentier et al. 2000 Cancer Research 60 5977-5983) .
  • TSK expression was observed in the present investigation to be constitutively expressed in OVCAR3 cells (ovarian cancer cell line) . TSK expression was not upregulated in these cells after 3hr oestrogen treatment.
  • TSK protein is secreted by the SEG-1 adenocarcinoma cell line
  • Opti-MEM (supernatant) was collected from the cells and concentrated using Vivaspin centrifugal concentrators. The resulting concentrated supernatant was subject to Western blotting, using an anti- TSK antibody.
  • the adenocarcinoma cell line, SEG-1 was found to endogenously secrete TSK protein into the culture medium, independent of oestrogen treatment.
  • MCF-7 cell migration in vi tro was measured using migration chambers (8 ⁇ m pore inserts) according to manufacturers instructions (Becton Dickinson) .
  • MCF-7 cells were seeded at 5 xlO 5 cells per upper chamber. MCF-7 cells were allowed to migrate to
  • Opti-MEM media containing 10% FBS in the lower chamber for 20 h were incubated in conditioned media from mock-transfected COS-7 cells; TSK treated cells were incubated in conditioned media from COS-7 cells transfected with a TSK construct (NB. presence of TSK in the media was confirmed by western blotting (data not shown)).
  • TSK treatment was observed to enhance MCF-7 cell migration (Figure 18; 1.4-fold). MCF-7 cell migration was further enhanced by treatment with a combination of TGF ⁇ l (lOng/mL) and TSK ( Figure 18; 1.6-fold).
  • Anti-TSK treatment of MCF-7 cells inhibits TGF ⁇ l- stimulated migration of MCF-7 cells in vi tro
  • the effect of anti-TSK on TGF ⁇ l-stimulated MCF-7 cell migration in vi tro was measured using migration chambers (8 ⁇ m pore inserts) according to manufacturers instructions (Becton Dickinson) .
  • MCF-7 cells were seeded at 5 xlO 5 cells per upper chamber in OptiMEM media.
  • TGF ⁇ l treatment of cells results in an increase in the migratory capacity of MCF-7 cells (figure 19; 1.25-fold). Furthermore, TGF ⁇ l-stimulated MCF-7 cell migration was inhibited by the presence of an anti-TSK polyclonal antibody (figure 19; migration was inhibited to control levels) . These data provide indication that TSK plays a role in TGF ⁇ l-stimulated MCF-7 cell migration.
  • RNA was DNase-1 treated and normalised such that 0.5ug was used per assay.
  • the ABI Prism 7900HT Sequence Detection System was used to quantify TSK RNA levels using forward and reverse primers and a probe optimally designed using ABI .software.
  • Each profiling assay for TSK was performed in parallel with an assay for endogenous 18S (ribosomal RNA) expression. All samples were assayed in triplicate.
  • Gene expression is represented graphically in Figures 20A, 20B, 21A and 21B, according to tissue type. The mean expression level of TSK ⁇ S.E.M. is shown.
  • TSK mRNA was present in uniformly low levels in normal tissue biopsies from breast (E12, F12, G12, H12), prostate (E9, F9, G9, H9) , lung (E6, F6, G6) and colon (E3, F3 , G3, H3) .
  • TSK expression levels were elevated above normal tissue levels in a substantial number of tumour samples from each tissue type (see Table 1).

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Abstract

L'invention concerne une nouvelle famille de polypeptides qui sont des modulateurs extra-cellulaires d'éléments de la superfamille du facteur de croissance transformant bêta (TGFβ), y compris des TGFβ et des BMP, et qui sont impliqués dans l'embryogenèse et la pathogenèse de troubles humains à médiation par signalisation de superfamille TGFβ. Ces modulateurs sont dénommés polypeptides Tsukushi (TSK). L'invention concerne en outre des agents et des procédés de modulation de parcours de signalisation de molécules du type (TGFβ) utilisant des polypeptides TSK.
PCT/GB2003/004535 2002-10-21 2003-10-21 Modulation de parcours de signalisation de type beta tgf WO2004035627A1 (fr)

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WO2023210410A1 (fr) * 2022-04-27 2023-11-02 国立大学法人 大分大学 Agent de cicatrisation de plaie

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