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WO2001098345A1 - Facteurs de croissance, acide nucleique les codant et leurs methodes d'identification - Google Patents

Facteurs de croissance, acide nucleique les codant et leurs methodes d'identification Download PDF

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Publication number
WO2001098345A1
WO2001098345A1 PCT/SE2001/001362 SE0101362W WO0198345A1 WO 2001098345 A1 WO2001098345 A1 WO 2001098345A1 SE 0101362 W SE0101362 W SE 0101362W WO 0198345 A1 WO0198345 A1 WO 0198345A1
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Prior art keywords
nucleic acid
polypeptide
mdf451
mdf628
effects
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PCT/SE2001/001362
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English (en)
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Carlos IBÁÑEZ
Henrik JÖRNVALL
Peter LÖNNERBERG
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Karolinska Innovations Ab
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Priority to AU2001274753A priority Critical patent/AU2001274753A1/en
Publication of WO2001098345A1 publication Critical patent/WO2001098345A1/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]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to new polypeptide growth factors of the transforming growth factor- ⁇ (TGF- ⁇ ) superfamily.
  • the invention also relates to nucleic acid molecules encoding the said polypeptide growth factors, as well as to methods for identification of agents mimicking or modulating the effects of growth factors of the TGF- ⁇ superfamily.
  • TGF- ⁇ The transforming growth factor ⁇ (TGF- ⁇ ) superfamily constitutes the largest group of polypeptide growth factors known, including the TGF- ⁇ s, activins, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), and neurotrophic factors of the GDNF ligand subfamily.
  • BMPs bone morphogenetic proteins
  • GDFs growth and differentiation factors
  • TGF- ⁇ superfamily members are characterized by a common three-dimensional (3D) fold containing a cysteine-knot (McDonald and Hendrickson, 1993).
  • the pattern of cysteine residues is highly conserved in the primary sequence of different TGF- ⁇ superfamily members. So powerful is the structural constrain imposed by the conserved spacing of cysteine residues that even very distant members of the superfamily, such as TGF- ⁇ 2 and GDNF, which are completely divergent in the sequence segments in between the cysteines (Lin et al., 1993), have an almost indistinguishable 3D structure (Eigenbrot and Gerber, 1997; Schlunegger and Gr ⁇ ter, 1992). approximately 55 kDa, and the type II receptors of approximately 70 kDa.
  • Both receptors cooperate to ligand binding, type II receptors phosphorylate type I receptors, and the latter activate of the Smad family of signal transducers, which then translocate to the nucleus where they take part in a number of DNA binding complexes (for recent reviews, see e.g. Attisano and Wrana, 2000; Massague and Chen, 2000; ten Dijke et al., 2000; Wrana, 2000).
  • GDNF glial cell line- derived neurotrophic factor
  • Fig. 1 Alignment of the amino acid sequences for MDF451 and MDF628 with the corresponding region of mouse GDNF and TGF- ⁇ 2. Stars (*) indicate positions for conserved cysteine residues.
  • Fig. 2 Phylogenetic tree based on sequences of representative members of different TGF- ⁇ subfamilies, including MDF451 and MDF628.
  • MDF451 and MDF628 Two novel genes, designated MDF451 and MDF628 respectively, have been identified.
  • the new genes display sequence similarities to members of the TGF- ⁇ superfamily, primarily in the pattern of six conserved cysteine residues.
  • MDF451 and MDF628 have no match in EST and GenBank databases, indicating that these genes may be expressed at low levels or restricted to a specific developmental stage or tissue.
  • Phylogenetic tree analyses of these sequences together with representative members of different subfamilies of TGF- ⁇ molecules revealed that MDF451 and MDF628 constitute a novel and distinct subgroup within the TGF- ⁇ superfamily that appear to be more closely related to the GDNF subfamily of ligands.
  • the two new members of the TGF- ⁇ superfamily are likely to represent new signaling molecules important for the control of cell survival, differentiation, proliferation or fate, in particular in the central nervous system.
  • this invention relates to isolated nucleic acid molecules selected from:
  • nucleic acid molecules comprising the nucleotide sequences set forth in SEQ ID NO: 1 and 3, respectively;
  • nucleic acid molecules comprising a nucleotide sequence capable of hybridizing, under stringent hybridization conditions, to nucleotide sequences complementary the polypeptide coding region of a nucleic acid molecule as defined in (a) and which codes for MDF451 or MDFR628 polypeptide, or modified forms thereof; and
  • nucleic acid molecules comprising a nucleic acid sequence which is degenerate as a result of the genetic code to a nucleotide sequence as defined in (a) or (b) and which codes for an MDF451 or MDF628 polypeptide, or modified forms thereof.
  • modified forms is intended to encompass polypeptides having substantially the same structural features and/or biological activities as the MDF451 or MDF628 polypeptide.
  • structural features is in particular referring to the pattern of six conserved cysteine residues illustrated in Fig. 1 and by the formula -Cys-(26)-Cys-(3)-Cys-(28)-Cys-Cys-(28)-Cys-(l)-Cys- wherein the figures within brackets represent the approximate number of amino acid residues between the conserved cysteine residues.
  • biological activities is intended to mean the ability to mediate the biological function characteristic for known members of the TGF- ⁇ superfamily (Massague & Chen, 2000; ten Dijke et al, 2000; Wrana, 2000; Attisano & Wrana, 2000).
  • stringent hybridization conditions is known in the art from standard protocols (e.g. Ausubel et al., supra) and could be understood as e.g. hybridization to filter-bound DNA in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
  • the invention encompasses DNA molecules carrying modifications like substitutions, small deletions, insertions or inversions, which nevertheless encode polypeptides having substantially the structural features and/or the biological activity of the MDF451or MDF628 polypeptides. Included in the invention are consequently DNA molecules, the nucleotide sequences of which are at least 90% homologous, preferably at least 95% homologous, with the nucleotide sequence set forth as SEQ ID NO: 1 or 3 in the Sequence Listing.
  • nucleotide sequences are degenerate, because of the genetic code, to the nucleotide sequence shown as SEQ ID NO: 1 or 3.
  • a sequential grouping of three nucleotides, a "codon”, codes for one amino acid. Since there are 64 possible codons, but only 20 natural amino acids, most amino acids are coded for by more than one codon. This natural "degeneracy", or
  • the invention provides isolated polypeptides encoded by the nucleic acid molecules defined above.
  • the said polypeptides have amino acid sequences set forth as SEQ ID NO: 2 and 4, respectively, of the Sequence Listing.
  • the polypeptides according to the invention are not to be limited strictly to polypeptides having amino acid sequences identical with SEQ ID NO: 2 or 4. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the structural features and/or biological activity of the MDF451 or MDF628 polypeptides.
  • the invention also embraces polypeptides that have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 10% identity and/or homology to the polypeptides having amino acid sequences set forth as SEQ ID NO: 2 and 4, respectively.
  • the invention provides a vector harboring a nucleic acid molecule according to the invention.
  • the said vector can e.g. be a replicable expression vector, which carries and is capable of mediating the expression of a nucleic acid molecule according to the invention.
  • replicable means that the vector is able to replicate in a given type of host cell into which is has been introduced.
  • vectors are viruses such as bacteriophages, cosmids, plasmids and other recombination vectors.
  • Nucleic acid molecules are inserted into vector genomes by methods well known in the art.
  • a cultured host cell harboring a vector according to the invention.
  • a host cell can be a prokaryotic cell, a unicellular eukaryotic cell or a cell derived from a multicellular organism.
  • the host cell can thus e.g. be a bacterial cell such as an E. coli cell, a yeast cell, or a mammalian cell.
  • the methods employed to effect introduction of the vector into the host cell are standard methods well known to a person familiar with recombinant DNA methods.
  • a further aspect of the invention is a process for production of a polypeptide, specifically an MDF451 or MDF628 polypeptide, which process comprises culturing a host cell as defined above under conditions whereby said polypeptide is produced, and recovering said polypeptide.
  • Expression of MDF451 and MDF 628 in the brain provides an indication that MDF polypeptides, as well as agents mimicking or modulating MDF activity, have utility for treating neurological disorders, such as schizophrenia, affective disorders, ADHD/ADD (i.e. Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.
  • MDF polypeptides or agents mimicking or modulating MDF activity, may have utility
  • diseases for which MDF polypeptides, or agents mimicking or modulating MDF activity, may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, metabolic diseases like diabetes type 2, obesity, neuroendocrine disorders such as growth hormone deficiency, inflammatory disorders, cancers, and the like.
  • MDF polypeptides is intended to include MDF451 and MDF628, as well as modified forms thereof having essentially similar biological activities.
  • MDF activity is intended to mean the biological activities of MDF polypeptides, such as the biological activities characteristic for known members of the TGF- ⁇ superfamily, which activities can mediate effects comprising neurotrophic, cell proliferation, tissue repair, wound healing, trauma treatment, cartilage inducing, bone inducing, connective tissue deposition, anti-inflammatory, lymphoid cell proliferation inhibition, hematopoietic, lymphopoietic, immunosuppressive, immunoregulatory, or epidermal cell proliferation inhibition effects.
  • the invention includes a pharmaceutical composition comprising an MDF polypeptide and a pharmacologically acceptable carrier.
  • the invention also includes a method for the treatment of disorders such as e.g. neurological disorders, comprising administering to a patient in need thereof an effective amount of an MDF polypeptide.
  • the term "treatment" is intended to include prophylaxis or attenuation of an existing condition.
  • the invention further comprises a method of mimicking the effects, such as e.g. neurological effects, in particular effects relating to neuron survival and neurite outgrowth,, of TGF- ⁇ superfamily ligands in a mammalian subject, comprising administering to said subject an effective amount of the polypeptide according to any one of claims 3 to 6.
  • a method of mimicking the effects such as e.g. neurological effects, in particular effects relating to neuron survival and neurite outgrowth, of TGF- ⁇ superfamily ligands in a mammalian subject, comprising administering to said subject an effective amount of the polypeptide according to any one of claims 3 to 6.
  • the invention provides a diagnostic method comprising determining the amounts of an MDF polypeptide, or alternatively, a nucleic acid molecule encoding an MDF polypeptide, in a tissue or fluid sample from a human patient.
  • the said diagnostic method is useful for diagnosis of medical conditions relating to effects characteristic for known members of the TGF- ⁇ superfamily.
  • the MDF polypeptides of the present invention may also be used to raise polyclonal or monoclonal antibodies.
  • Such antibodies may be prepared by conventional techniques; see e.g. Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, (1988); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980).
  • Antibodies of the invention are useful for e.g. therapeutic purposes (by modulating activity of MDF451 or MDF628), diagnostic purposes to detect or quantitate MDF451 or MDF628, and purification of MDF451 or MDF628.
  • Kits comprising an antibody of the invention for any of the purposes described herein are also included in the invention.
  • a kit of the invention also includes a control antigen for which the antibody is immunospecific.
  • the MDF polypeptides according to the invention can be utilized in methods for identification of agents mimicking or modulating the effects, such as neurological effects, of TGF- ⁇ superfamily ligands.
  • agent means a biological or chemical compound such as a simple or complex organic molecule, a peptide, a protein or an oligonucleotide.
  • the invention provides a method for the identification of an agent mimicking the effects of TGF- ⁇ superfamily ligands, comprising (i) contacting a test agent with a mammalian cell; (ii) comparing the effect of the said test agent on the said mammalian cell with the effect of an MDF polypeptide according to the invention, whereby a similar activity indicates that said test agent is mimicking the effects of TGF- ⁇ superfamily ligands.
  • Also included in the invention is a method for identifying an agent useful for decreasing or inhibiting the biological activities of an MDF polypeptide, or decreasing or inhibiting the expression of a nucleic acid molecule encoding an MDF polypeptide, said method comprising the steps (i) contacting a test agent with an MDF polypeptide or with a nucleic acid molecule encoding an MDF polypeptide; and
  • test agent decreases or inhibits the biological activities of the said MDF polypeptide, or decreases or inhibits the expression of the said nucleic acid molecule.
  • Motifer J ⁇ rnvall, 1999 was used. Motifer is a software tool able to find directly in nucleotide databases very distant homologues to an amino acid query sequence.
  • the publicly available database of the Human Genome Project (available via http://www.ncbi.nlm.nih.gov) was searched using Motifer and the above query sequence. Sequences of known members of the TGF- ⁇ superfamily were included in the search. The first 300 positions of the obtained output file corresponded to previously described genes of the TGF- ⁇ superfamily. In addition, two novel sequences were identified displaying conservation of all but the first Cys of the query. The two sequences were provisionally named MDF (Motifer-Derived Factor) 451 (SEQ ID NOs: 1 and 2) and MDF 628 (SEQ ID NOs: 3 and 4), respectively.
  • MDF Motifer-Derived Factor
  • MDF451 nor MDF628 could be identified applying other algorithms, including BLAST, to the same database.
  • the genes contain a stop codon after 1 and 3 amino acid residues, respectively, downstream of the last cysteine, a feature that is highly conserved in all members of the TGF- ⁇ superfamily. No other cysteine in addition to those conforming to the query pattern could be found in either MDF451 or MDF628. This fact, together with the high conservation of the observed Cys pattern and the lack of internal stop codons, suggest that MDF451 and MDF628 are not pseudogenes.
  • Bootstrap numbers are indicated in the nodes of the tree, and give an estimate of the probability that the corresponding node represent to a true distinct branch in the tree. Nodes with a low bootstrap value are not well supported by the data and suggest that alternative relationships may also be possible.
  • the two MDFs appear in a distinct branch of the tree leading to the GDNF ligand subfamily. This branch has the relatively high bootstrap value of 993, indicating that it is well supported by the data and suggesting that MDF451 and MDF 628 may represent distant relatives of GDNF subfamily ligands.
  • EXAMPLE 3 Expression of MDF451 and MDF628 mRNA in human tissues
  • MDF451 and MDF628 mRNA were assayed by RNase protection assay according to standard procedures.
  • Human RNAs were purchased from Clontech.
  • RNase protection assays PCR fragments of MDF451 and MDF628 were subcloned into pBSKS+, linearized, and used as template for T7 RNA polymerase using a kit from Promega. 10 ⁇ g of total RNA was hybridized to [ ⁇ - 3 P]CTP-labeled cRNA probes using a kit from Ambion according to the manufacturer's instructions. Protected bands were visualized and quantified using a STORM840 phosphorimager and ImageQuant software (Molecular Dynamics).
  • MDF451 and MDF 628 appear to be predominantly expressed in nervous tissue, including fetal and adult brain and cerebellum (Fig. 3). Weak expression of MDF451 can also be seen in skeletal muscle and spinal cord and of MDF628 in testis. These data argues strongly that both MDFs are indeed expressed genes, and indicate that they may represent neuron survival or differentiation factors.
  • Full length clones for human MDF451 and MDF 628 are obtained by Rapid Amplification of cDNA Ends (RACE) using human cerebellum cDNA (Clontech) and specific upstream primers according to standard procedures and manufacturers' instructions.
  • RACE Rapid Amplification of cDNA Ends
  • the extended products obtained from RACE are verified by automatic DNA sequencing and ligated to the partial MDF fragments to obtain full-length clones.
  • MDF451 and MDF628 are subcloned in the mammalian expression vector pcDNA3 for transient expression in COS cells according to standard procedures.
  • An epitope tag is introduced at the time of cloning to monitor protein during purification stages.
  • MDF protein is harvested from COS cell supernatants and purified using conventional chromatography techniques, including ion exchange, size-exclusion and reverse phase chromatography (RPC). Purification is monitored by Western blotting using antibodies specific to the tagged MDF proteins. Final purity is assessed by silver staining of SDS/PAGE gels.
  • EXAMPLE 6 Cloning of rat and mouse homologues of human MDF451 and MDF628
  • phage libraries of rat and mouse brain are screened by low hybridization procedures according to standard procedures.
  • cDNAs corresponding to rodent MDFs are isolated, sequenced and extended by RACE to obtain ull-length clones as needed.
  • Standard techniques are employed to generate polyclonal or monoclonal antibodies to MDF451 and MDF628, and to generate useful antigen-binding fragments thereof or variants thereof, including "humanized” variants.
  • Such protocols can be found, for example, in Sambrook et al. (1989) and Harlow et al. (Eds.), Antibodies, A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988).
  • Peptides obtained from hydrophilic regions of the mouse and rat MDF451 and MDF628 sequences can be synthesized, conjugated to Keyhole Limpet Hemocyanin (KLH) and used to immunize rabbits for antisera preparation. Titer of the subsequent boostings can be monitored by Western blotting using purified recombinant MDF protein.
  • KLH Keyhole Limpet Hemocyanin
  • MDFs riboprobes are labeled with 35 S-UTP or 33 P-UTP using linearized template DNA fragments and reagents for in vitro transcription from Promega.
  • 14 mm sections are thawed onto 3- ⁇ aminopropyl ethoxysilane coated slides for hybridization with radiolabeled probes as follows. Following fixation in 4% paraformaldehyde for 15 min, slides are rinsed once in PBS and twice in distilled water.
  • Tissue is de-proteinated in 0.2 M HC1 for 10 min, acetylated with 0.25% acetic anhydride in 0.1M ethanolamine for twenty minutes and dehydrated with increasing concentrations of ethanol.
  • Slides are incubated 16 h in a humidified chamber at +58°C with 8 xlO 5 cpm of probe in 300 ml of hybridization cocktail (50% formamide, 20 mM Tris-HCl (pH 7.6), 1 mM EDTA pH 8.0, 0.3 M NaCl, 0.1 M dithiothreitol, 0.5 mg/ml yeast tRNA, 0.1 mg/ml polyA-RNA, IX Denhardt's solution and 10% dextran sulphate).
  • hybridization cocktail 50% formamide, 20 mM Tris-HCl (pH 7.6), 1 mM EDTA pH 8.0, 0.3 M NaCl, 0.1 M dithiothreitol, 0.5 mg/ml yeast tRNA,
  • RNAse treatment (10 mg/ml) for 30 min at +37°C in 0.5 M NaCl, 20 mM Tris-HCl (pH 7.5), 2mM EDTA.
  • Tissue is washed twice with lxSSC at +65°C for 30 min before dehydration in ethanol and air drying.
  • GDNF a glial cell line-derived neurotrophic factor for midbrain dopaminergic neruons.

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Abstract

La présente invention concerne de nouveaux facteurs de croissance polypeptidiques, désignés MDF451 et MDF628, de la superfamille des facteurs de croissance transformants-β(TGF-β). L'invention concerne également des molécules d'acide nucléique codant lesdits polypeptides MDF451 et MDF628, ainsi que des méthodes d'identification d'agents mimétiques ou modulateurs des effets des facteurs de croissance de la superfamille des TGF-β.
PCT/SE2001/001362 2000-06-22 2001-06-15 Facteurs de croissance, acide nucleique les codant et leurs methodes d'identification WO2001098345A1 (fr)

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AU2001274753A AU2001274753A1 (en) 2000-06-22 2001-06-15 Growth factors, nucleic acid encoding them and methods for identification

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SE0002364A SE0002364D0 (sv) 2000-06-22 2000-06-22 Growth Factors

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997000958A1 (fr) * 1995-06-22 1997-01-09 St. Vincent's Hospital Sydney Limited NOUVELLE CYTOKINE DE TYPE FACTEUR DE CROISSANCE TRANSFORMANT-β (TGF-β)
WO2000002916A2 (fr) * 1998-07-10 2000-01-20 The Regents Of The University Of California COMPOSITIONS PEPTIDIQUES IMITANT L'ACTIVITE DE TGF-$g(b)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997000958A1 (fr) * 1995-06-22 1997-01-09 St. Vincent's Hospital Sydney Limited NOUVELLE CYTOKINE DE TYPE FACTEUR DE CROISSANCE TRANSFORMANT-β (TGF-β)
WO2000002916A2 (fr) * 1998-07-10 2000-01-20 The Regents Of The University Of California COMPOSITIONS PEPTIDIQUES IMITANT L'ACTIVITE DE TGF-$g(b)

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