WO2000077195A1 - Acide nucleique codant pour de nouveaux facteurs de croissance du type egf - Google Patents

Acide nucleique codant pour de nouveaux facteurs de croissance du type egf Download PDF

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WO2000077195A1
WO2000077195A1 PCT/EP2000/005363 EP0005363W WO0077195A1 WO 2000077195 A1 WO2000077195 A1 WO 2000077195A1 EP 0005363 W EP0005363 W EP 0005363W WO 0077195 A1 WO0077195 A1 WO 0077195A1
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protein
nucleic acid
egf
anyone
seq
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PCT/EP2000/005363
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English (en)
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Michael Hanke
Jens Pohl
Rainer Ries
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Biopharm Gesellschaft Zur Biotechnologischen Entw Icklung Und Zum Vertrieb Von Pharmaka Mbh
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Priority to AU62627/00A priority Critical patent/AU6262700A/en
Publication of WO2000077195A1 publication Critical patent/WO2000077195A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to nucleic acids encoding a protein, which is an epidermal growth factor receptor (EGFR)-ligand having e.g. no heparin-binding site.
  • EGFR epidermal growth factor receptor
  • the protein is capable of stimulating astroglial cell maturation and/or has a selective survival promoting activity on dopaminergic (DAergic) and/or peripheral neurons and/or has a regenerative effect on peripheral and axonal neurons.
  • DAergic dopaminergic
  • the present invention further relates to antisense nucleic acids, ribozymes and antibodies directed to the nucleic acid or the protein, to methods of their production, to antagonists directed to the protein, to agonists which substitute the functional activity of the protein and to pharmaceutical compositions for the treatment as well as to diagnostic kits for the detection of disorders such as neurodegenerative diseases, cancer and AIDS.
  • EGF epidermal growth factor
  • porcine HB-EGF mRNA derived from 22 different tissues was investigated (Vaughan et al., Biochem. J. (1992) 287 681-684).
  • detection of HB-EGF via RT-PCR techniques shows expression in skin, midbrain, cerebellum, hypothalamus, cerebral cortex, bulbourethral gland, lung, heart (ventricel), kidney, prostate, seminal vesicle and testis.
  • Weak expression was found in lymph node, thymus and spleen, however no HB-EGF expression was found in pituitary, olfactory bulb, thyroid, duodenum, pancreas, liver or subma- xillary gland. Therefore, there is a great demand for novel EGF-like factors that relate to any diseases which can be influenced by binding of said EGF-like factors to the corresponding receptors.
  • the technical problem underlying the present invention ist to provide novel EGF-like factors and nucleic acids coding for such factors.
  • the present invention relates to a nucleic acid containing a nucleotide sequence encoding the primary amino acid sequence of a protein, e.g. derived from chromaffin granules, or a functionally active fragment or derivative or mutant or variant thereof, wherein the protein is an EGFR-ligand, preferably being capable of stimulating astroglial cell maturation and/or having a selective survival promoting activity on DAergic neurons and/or peripheral neurons.
  • the protein of the present invention has a regenerative effect on peripheral and axonal neurons.
  • the protein of the present invention may have a heparin-binding site or no heparin-binding site.
  • nucleic acid and nucleotide sequence refer to endogeneously expressed, semi-synthetic, synthetic or chemically modified nucleic acid molecules, preferably consisting substantially of deoxyribonucleotides and/or ribonucleotides and/or modified nucleotides. Further, the term “nucleotide sequence” may comprise exons, wherein the nucleotide sequence encodes the primary amino acid sequence of the protein and may be degenerated based on the genetic code.
  • primary amino acid sequence refers to the sequence of amino acids irrespective of tertiary and quaternary protein structure.
  • protein derived from chromaffin granules refers to single, defined proteins or functionally active fragments or derivatives or parts or mutants thereof that, when applied singly or in combinations, exert trophic, survival and differentiation promoting effects on DAergic and peripheral neurons.
  • selective survival promoting acitivity on DAergic and peripheral neurons refers to a proteinaceous activity that may confer, by itself or in combination with other factors present in chromaffin granules, survival and differentiation upon DAergic and peripheral neurons within the nanomolar range or below.
  • the protein encoded by the nucleotide sequence of the nucleic acid according to the present invention is an epidermal growth factor receptor (EGFR)-ligand.
  • EGF-receptors are e.g. HER, HER2, HER3 and HER4.
  • heparin-binding site refers to a three dimensional arrangement of atoms which is capable of interacting with heparin through any physical and/or chemical interaction. Such interactions comprise covalent binding, electrostatic interactions, hydrogen bonding, Van-der-Waals interactions and hydrophobic interactions.
  • An example of a heparin-binding site is a stretch of eleven amino acids, having a substantial basic character due to seven Lys and/or Arg residues, which is found in HB-EGF molecules.
  • a specific heparin-binding site comprises e.g. amino acids 91 to 101 of human HB-EGF (HUM-HB) as shown in Fig. 3.
  • the chromaffin granule-derived protein of the present invention is capable of promoting astroglial cell maturation.
  • the expression "capable of astroglial cell maturation” means that within a culture system of embryonic mesencephalic cells or in embryonic and adult mesencephalon, the protein increases the number of astroglial cells visualized by expression of proteins that are specific for this cell type.
  • the chromaffin granule-derived protein which is encoded by the nucleotide sequence of the above-defined nucleic acid, is capable of modulating the activity and/or proliferation of non-DAergic cells which include e.g. neuronal cells such as glial progenitor cells as well as non-neuronal cells such as cells derived from adrenal gland, pancreas and other tissues.
  • non-DAergic cells include e.g. neuronal cells such as glial progenitor cells as well as non-neuronal cells such as cells derived from adrenal gland, pancreas and other tissues.
  • the expression “capable of modulating the activity and/or proliferation of non-DAergic cells” means that an inhibition or stimulation and/or an increase or decrease in the number of the affected cells is the effect initially caused by the "chromaffin granule-derived protein".
  • non-DAergic cells such as astroglial cells
  • this initial effect is supposed to be the prerequisite for the promotion of survival by a factor secrete
  • the nucleic acid according to the present invention is derived from a vertebrate.
  • Preferred vertebrates are mammals such as pigs, cattle, rodents, e.g. mice, rats, rabbits, and primates, e.g. humans.
  • the expression "derived from a vertebrate” means that the gene coding for the protein is transcribed and/or translated in cells of the vertebrate, e.g. the mammal, such that the mRNA and/or the protein is detectable by methods known in the art such as in situ hybridization, RT-PCR, Northern or Western blotting.
  • the functionally acitve form of the above-defined protein or fragment or derivative or part thereof may be a monomeric, dimeric or oligomeric form, or a heteromeric form.
  • a preferred nucleic acid according to the present invention contains at least the nucleotide sequence shown in Fig. 1A, 2A, 8A or 10A (SEQ ID NO 1 , SEQ ID NO 4, SEQ ID NO 6 or SEQ ID NO 8) or a fragment or mutant thereof.
  • sequences include allelic derivatives of the nucletide sequence shown in Fig. 1A, 2A, 8A or 10A and nucleotide sequences degenerated as a result of the genetic code for said sequences. They also include nucleotide sequences hybridizing with the nucleotide sequence as defined above. Furthermore, mutant sequences of the above nucleotide sequence may result from the insertion, deletion and/or substitution of one or more nucleotides.
  • mutant nucleotide sequence of the above-defined nucleic acid is a substitution of the thymidine nucleotide (T) at postion 152 of the nucleotide sequence shown in Fig. 1A (SEQ ID NO 1 ) by a cytosine nucleotide (C).
  • a further example of a mutant sequence results from a deletion of the guanine nucleotide (G) at position 270 of the nucleotide sequence shown in Fig. 1A (SEQ ID NO 1 ). The resulting amino acid sequence is shown in Fig. 1 C (SEQ ID NO 3).
  • Variants of the above-defined nucleic acid may result from alternative splicing of the primary transcript of the gene coding for the nucleotide sequence of the nucleic acid of the present invention.
  • An alternative splicing may result in an insertion, deletion and/or substitution of one or more nucleotides in the nucleotide sequence of the above- defined nucleic acid.
  • Other variants of the nucleic acid according to the present invention encode proteins which are derived from the EGFR-ligand by, e.g. post- translational, processing and/or modification.
  • allelic, degenerate and hybridizing sequences may have structural divergences due to naturally occurring mutations, such as small deletions or substitutions, they will usually still exhibit essentially the same useful properties, allowing their use in basically the same medical or diagnostic applications.
  • hybridization means conventional hybridization conditions, preferably conditions with a salt concentration of 6 x SSC at 62 °C to 66 °C followed by a one-hour wash with 0.6 x SSC, 0.1 % SDS at 62 °C to 66 °C.
  • Fig. 1A cDNA of AG-EGF, SEQ ID NO 1 ; deduced amino acid sequence see Fig. 1B,
  • Fig. 2A cDNA of the mature form of HB-EGF, SEQ ID NO 4; deduced amino acid sequence see Fig. 2B, SEQ ID NO 5 were obtained from bovine adrenal gland.
  • the nucleotide sequence shown in Fig. 8A full-length cDNA of (premature) HB-EGF, SEQ ID NO 6, deduced amino acid sequence see Fig. 8B, SEQ ID NO 7) was cloned from a bovine brain cDNA library whereas the nucleotide sequence shown in Fig. 10A (cDNA of PA-EGF, SEQ ID NO 8, deduced amino acid sequence see Fig. 10B, SEQ ID NO 7) was obtained from bovine pancreas. All proteins, bovine adrenal gland (AG)-EGF, bovine pancreas
  • PA-EGF as well as premature and mature bovine HB-EGF, encoded by the cloned nucleotide sequences show a surprisingly strong neurotrophic effect.
  • cloning was carried out according to the method described below.
  • the preparation of host cells capable of producing e.g. the AG-EGF protein, which has no heparin- binding site, and the production of said protein can be easily accomplished using known recombinant DNA techniques comprising constructing the expression plasmids encoding said protein and transforming a host cell with said expression plasmid, cultivating the transformant in a suitable culture medium, and recovering the product having AG-EGF activity.
  • diseases which are associated with the expression of the nucleotide sequence of the above-defined nucleic acid containing e.g.
  • nucleotide sequence encoding AG-EGF can be treated either by increasing the amount or activity of AG-EGF or by suppressing the amount or activity of AG-EGF.
  • further embodiments of the present invention relate to an antisense nucleic acid directed to the above-defined nucleic acid and to a ribozyme which is capable of cleaving the above-defined nucleic acid. The inhibition may therefore achieved by masking the mRNA with the antisense nucleic acid or by cleaving the mRNA with the ribozyme.
  • antisense nucleic acids The production of antisense nucleic acids is well known (see e.g. Weintraub, H. M. 1990, Scientific American 262: 40).
  • the antisense nucleic acids hybridize with the respective mRNA and form a double-stranded molecule which can then no longer be translated.
  • the use of antisense nucleic acids is, for example known from Marcus-Sekura, C. J. 1988 (Anal. Biochem. 172: 289-295).
  • Ribozymes are RNA molecules which are able to specifically cleave other single-stranded RNA molecules. The production of ribozymes is described for example in Czech, J. 1988, Amer. Med. Assn. 260: 3030.
  • a further embodiment of the present invention relates to a vector containing at least the nucleic acid or the antisense nucleic acid or the ribozyme as defined above.
  • the term "vector” refers to a DNA and/or RNA replicon that can be used for the amplification and/or expression of the nucleotide sequence of the nucleic acid or the antisense nucleic acid or the ribozyme as defined above.
  • the vector may contain any useful control units such as promoters, enhancers, or other stretches of sequence within the 5' and/or 3' regions of the nucleotide sequence serving for the control of its expression.
  • the vector may additionally contain sequences within the 5' and/or 3' region of the nucleotide sequence, that encode amino acid sequences which are useful for the detection and/or isolation of the protein which may be encoded by the nucleotide sequence.
  • the vector contains further elements that enable the stable integration of the above-defined nucleic acids into the genome of a host organism and/or the transient expression of the nucleotide sequence of the above-defined nucleic acids. It is also prefered i to use vectors containing selectable marker genes which can be easily selected for transformed cells. The necessary operations are well known to the person skilled in the art.
  • a further embodiment of the present invention relates to a host organism containing at least the nucleic acid or the antisense nucleic acid or the ribozyme or the vector as defined above.
  • suitable host organisms include various eucaryotic and procaryotic cells, such as Bacillus spec, or E. coli, insect cells, plant cells, such as tobacco, potato, or Arabidopsis, animal cells such as verte- brate cell lines, e.g. mammalian cell lines such as the Mo, COS or CHO cell line, and fungi such as yeast.
  • a further embodiment of the present invention relates to the protein itself, which is encoded by the nucleic acid as defined above.
  • Preferred examples of the primary amino acid sequence of the protein according to the present invention include the amino acid sequence shown in Fig. 1 B, 1 C, 2B, 8B or 10B (SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7 or SEQ ID NO 9) as well as functionally active fragments or derivatives or mutants or variants thereof.
  • a mutation leading to a functionally active mutant of the protein according to the present invention may comprise an insertion, deletion or substitution of one or more amino acids.
  • An example of such a mutant amino acid sequence comprises a substitution of the leucine residue (L) at position 51 of the amino acid sequence shown in Fig.
  • a mutant amino acid sequence is derived from a deletion of nucleotide 270 in the cDNA encoding AG- EGF (cf. Fig. 1A) and results in the amino acid sequence shown in Fig. 1C (SEQ ID NO 3).
  • Functionally acitve variants of the protein according to the present invention include e.g. splice variants such as splice variants of the mature form of the above-defined proteins.
  • Variants of the protein according to the present invention may include an insertion, deletion or substitution of one or more amino acids.
  • Other functionally active variants of the protein according to the present invention include proteins which are derived by, e.g. post-translational, processing and/or modification of the above-defined protein.
  • a further embodiment of the present invention relates to an antagonist which is directed to the above-defined protein.
  • Another embodiment of the present invention relates to an agonist as a substitute for the functional activity of the above-defined protein.
  • an antibody which may be monoclonal or polyclonal, or a functional fragment thereof directed against the protein or a functional derivative or part thereof as defined above.
  • a further embodiment of the present invention relates to a method for the production of the nucleic acid, the antisense nucleic acid, the ribozyme, the vector, or the protein as defined above, comprising the steps of:
  • a further embodiment of the present invention relates to a pharmaceutical composition containing the nucleic acids and/or the antisense nucleic acid and/or the ribozyme and/or the vector and/or the protein and/or the antagonist and/or the agonist and/or the antibody as defined above, in a pharmaceutically effective amount, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.
  • the pharmaceutical composition may be used for the prevention and/or treatment of diseases influenced by initiation of protein biosynthesis, especially diseases which interfere with the adrenal gland, kidney (i.e. renal failure), and diseases influenced by changes of cell proliferation and/or differentiation, e.g.
  • cancers such as breast, colorectal, liver, kidney, prostate, ovarian, brain and pancreatic tumors
  • neurological diseases such as Parkinson ' s disease, Huntington ' s chorea, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer ' s disease and pathogenesis of traumatic, toxic, inflammatory and metabolic neuropathies, and it may be used in wound healing, preferably corneal wound healing.
  • the pharmaceutical composition according to the present invention can also be therapeutically applied for common diseases such as diabetes, ischemia, trauma, haemotopoietic diseases and rheumatoid arthitis.
  • the pharmaceutical composition according to the present invention may also be used for the treatment of microbial or viral infections such as AIDS.
  • a method of treating a patient in order to apply the above-defined pharmaceutical compostion may comprise the transfection of, e.g. the above-defined vector in vitro or in vivo into patient cells.
  • the method may comprise, as a first step, the transfection of the vector in vitro into cells and the subsequent implantation of said transfected cells in a patient.
  • the application of the pharmaceutical composition is not limited to humans but can include animals, in particular domestic animals, as well.
  • the pharmaceutical composition according to the present invention further contains one or more growth factors and/or cytokines such as other EGF-like factors, insuline-like growth factors (e.g. IGF-I, IGF-II), TGF- ⁇ -like factors (e.g. TGF, BMP, GDF) and neurofactors (e.g. FGF, NGF, BDNF, neurotrophins).
  • growth factors and/or cytokines such as other EGF-like factors, insuline-like growth factors (e.g. IGF-I, IGF-II), TGF- ⁇ -like factors (e.g. TGF, BMP, GDF) and neurofactors (e.g. FGF, NGF, BDNF, neurotrophins).
  • a further embodiment of the present invention relates to a diagnostic kit containing the nucleic acid and/or the antisense nucleic acid and/or the ribozyme and/or the vector and/or the protein and/or and/or the antagonist and/or the agonist and/or the antibody as defined above.
  • the diagnostic kit according to the present invention may preferably be used for the detection of the disorders as defined above.
  • a further embodiment of the present invention relates to a cell culture medium at least containing the above-defined protein and/or the above-defined agonist.
  • the medium according to the present invention may be used e.g. for the stimulation of cultured cells.
  • a further embodiment of the present invention relates to a process for the preparation of the above defined chromaffin granule-derived protein, comprising the steps of isolating chromaffin granules from chromaffin cells and extracting the aqueous-soluble protein content containing the chromaffin granule-derived protein, from the chromaffin granules in a buffer solution.
  • isolated chromaffin granules from chromaffin cells comprises subcellular fractionation of chromaffin cell organelles by density gradient centrifugation at e.g. 1.7M sucrose.
  • extracting the aqueous-soluble protein content containing the chromaffin granule-derived protein comprises lysis of the organelles obtained as a pellet of the examplified 1.7M sucrose centrifugation in e.g. a 10 mM phosphate buffer at pH 7.0 following twenty minutes freezing at - 80°C or below.
  • Further subject-matter of the present invention relates to the use of a protein or a functionally active fragment or derivative or mutant or variant thereof, wherein the protein is an EGFR-ligand, preferably being capable of stimulating astroglial cell maturation and/or preferably having a selective survival promoting activity on DAergic and/or peripheral neurons, for the preparation of a pharmceutical composition for the prevention and/or treatment of diseases selected from the group consisting of diseases influenced by initiation of protein biosynthesis, diseases influenced by changes of cell proliferation and/or differentiation and neurological diseases.
  • diseases in the use as defined above may be selected from the diseases as defined above for the pharmaceutical composition of the present invention.
  • Fig. 1 (A) shows the nucleotide sequence of the cDNA encoding AG-EGF
  • (SEQ ID NO 1 ) derived from bovine adrenal gland and (B) shows the amino acid sequence of AG-EGF (SEQ ID NO 2) as deduced from the nucleotide sequence shown in (A).
  • (C) shows the amino acid sequence of a mutant AG-EGF (SEQ ID NO 3) resulting from the deletion of nucleotide 270 of the nucleotide sequence shown in (A).
  • Fig. 2 (A) shows the nucleotide sequence of the cDNA encoding mature
  • ⁇ HB-EGF (SEQ ID NO 4) derived from bovine adrenal gland and (B) shows the amino acid sequence of HB-EGF (SEQ ID NO 4) as deduced from the nucleotide sequence shown in (A).
  • Fig. 3 shows the alignment of the amino acid sequence of AG-EGF with some of the related HB-EGF members. The mature regions are from positions 63 to positions 149. The dashed line from position 91 to 101 in the sequence denoted BOV-AG (SEQ ID NO 2) shows the region of the absent heparin binding-site.
  • BOV-AG amino acid sequence of AG-EGF (SEQ ID NO 2); HUM-HB: amino acid sequence of human HB-EGF (SEQ ID NO 10, Swiss Prot Accession no. Q99075); PIG-HB: amino acid sequence of porcine HB-EGF (SEQ ID NO 11 , Swiss Prot Accession No. Q01580); MUS-HB: amino acid sequence of mouse HB-EGF (SEQ ID NO 12, Swiss Prot Accession No. Q06186); RAT-HB: amino acid sequence of rat HB-
  • EGF (SEQ ID NO 13, Swiss Prot Accession No. Q06175).
  • Fig. 4 (A) shows an alignment of the amino acid sequences of the mature regions of the following EGF family proteins: BOV-MAT.AMI: amino acid sequence of AG-EGF (SEQ ID NO 2); BHB-MAT.AMI: amino acid sequence of the mature form of bovine HB-EGF (SEQ ID NO 5); HUM-MAT.AMI: amino acid sequence of the mature form of human HB-EGF (SEQ ID NO 10; Swiss Prot Accession No. Q99075); PIG-MAT.AMI: amino acid sequence of the mature form of porcine HB-EGF (SEQ ID NO 11 , Swiss Prot Accession No.
  • TGF-MAT.AMI mature form of human TGF-alpha (Swiss Prot Accession No. P01135); BET-MAT.AMI: mature form of human betacellulin (Swiss Prot Accession No. P35070); EGF-MAT.AMI: mature form of human EGF (Swiss Prot Accession No. P001133).
  • Fig. 5 shows a plot of an elution profile of an affinity-chromatography used for the purification of AG-EGF from chromaffin granules.
  • Left abscissa absorption at 280 nm (arbitrary units); right abscissa: concentration of buffer B (%); ordinate: time (min) and fraction numbers, respectively.
  • Fig. 6 shows the image of a Western blot analysis of fractions of the chromatographic run shown in Fig. 5 after SDS-gel electrophoresis (15%) under non-reducing conditions followed by immunostaining using an anti-HB-EGF antibody.
  • Fig. 7 (A) is a diagram demonstrating the survival promoting effect of AG-
  • EGF and HB-EGF from bovine chromaffin granules (VP 1 :20). Each bar represents the mean number of TH-positive cells counted in triplicate cultures +/- SEM from two experiments. It is also shown that this neurotrophic effect is inhibited using anti HB-EGF antibody (VP+a-HB-EGF).
  • C control.
  • the table in (B) shows results of appearance of GFAP positive astroglial cells induced by AG-EGF and HB-EGF (GFAP+ cells) and proliferation of non DAergic cells
  • Fig. 8 (A) shows the nucleotide sequence of the full-length cDNA encoding
  • HB-EGF (SEQ ID NO 6) derived from a bovine brain cDNA library.
  • the coding sequence starts at bp 71.
  • (B) shows the amino ac sequence of HB-EGF (SEQ ID NO 7) as deduced from the coding sequence (nucleotides 71 to 697) shown in (A).
  • Fig. 9 shows photographic images of agarose gels for the analysis of the distribution of AG/PA-EGF and HB-EGF mRNA in bovine tissues using AG-/HB-EGF specific primers (lanes a) and using ⁇ -actin specific primers as positive control (lanes b).
  • M 100 bp standard ladder; 1 : heart; 2: pancreas; 3: kidney; 4: liver; 5: brain; 6: testis; 7: adrenal gland; 8: blood.
  • Fig. 10 (A) shows the nucleotide sequence of the cDNA encoding PA-EGF
  • (SEQ ID NO 8) derived from bovine pancreas via RT-PCR.
  • (B) shows the amino acid sequence of PA-EGF (SEQ ID NO 9) as deduced from the nucleotide sequence shown in (A).
  • RNA polymerase chain reaction
  • STRATAGENE Polymerase chain reaction
  • the PCR reaction contained 1 ⁇ l (1 ⁇ g/ ⁇ l) poly A + RNA from bovine adrenal gland.
  • the reaction mixture was incubated for 120 s at 94°C and subjected to 10 cycles (50 s at 94°C, 50 s at 45°C, 50 s at 68°C), followed by 25 cycles (50 s at 94°C, 50 s at 50°C, 50 s at 68°C) with an additional extension for 120 s at 68°C in the thermocycler.
  • a 10 ⁇ l sample from the RT-PCR amplification was fractionated by electrophoresis using a 2 % agarose gel in TBE buffer.
  • Plasmid DNA from positive clones was isolated using the QIAwell 8 Plus Plasmid Kit (QIAGEN, Cat. no. 16142) and sequenced with an automatic DNA sequencer (ALFexpress, PHARMACIA). The resulting DNA sequence was analyzed by a homology search with the Blast program (Genetics Computer Group, Wisconsin Package 9.0, Madison, Wl, USA, 1997).
  • the amino acid sequence alignment of AG-EGF with sequences of different HB- EGF molecules (Figs. 3 and 4A) and the corresponding phylogenetic tree of AG- EGF and other members of the EGF family (Fig. 4B) demonstrate the homology of AG-EGF to the EGF family.
  • the homology of the derived AG-EGF mature amino acid sequence (amino acid 1 to amino acid 75) to the mature form of bovine HB- EGF (amino acid 1 to amino acid 86) displays a sequence homology of 83.7%.
  • the chromaffin granules In order to purify large dense core vesicles, the chromaffin granules, the resuspended pellet (0.3 M sucrose, 10 mM phosphate, pH 7.0) was loaded on a 1.7 M sucrose cushion. The chromaffin granules were obtained as a sediment after centrifugation at 100,000 g for 90 min. The sediment was resuspended in 10 mM phosphate pH 7.0, frozen in liquid nitrogen for twenty minutes and subsequently thawed, in order to lyze the vesicles and to extract the soluble protein content. Membrane fragments were collected by centrifugation at 100,000g for 30 min.
  • the supematent containing the soluble protein mixture from chromaffin granules was dialysed (cutoff 3,500 MW) over night against several batches of 100-fold excess of 10 mM phosphate buffer pH 7.0 to seperate catecholamines and other low molecular weight components from proteins.
  • To quantify protein concentrations the Bradford (Bio-Rad) protein assay was employed using bovine gamma globulin as a standard (Bio-Rad). The protein solution was diluted to give a final protein concentration of 20 mg/ml. This protein solution was then sterile filtered (0.22 ⁇ M) and stored in aliquots at -70°C.
  • Bovine AG-EGF and HB-EGF by affinity-chromatography using heparin-sepharose.
  • Bovine adrenal gland EGF-like activity (AG-EGF and HB- EGF) was further purified from bovine chromaffin granules using affinity- chromatography.
  • the chromatographic purification was realized using the AKTA- Explorer system (Pharmacia Biotech, Germany).
  • affinity-chromatography a 5 ml Heparin-Sepharose-cartridge (Econo-Pac, BioRad, Germany) was used.
  • the first step of the chromatographic procedure comprises a column-equilibration with three column volumes (CV) of loading buffer (10 mM TRIS, pH 7.3, 0.1 % CHAPS). Then the column was loaded with 125 ml bovine adrenal chromaffin granule pool, using the sample pump with a flow rate of 1.5 ml/min.
  • CV column volumes
  • FIG. 5 A typical profile of the affinity chromatography is shown in Fig. 5: AG-EGF shows an elution pattern using heparin-sepharose chormatography which is different from that of HB-EGF molecules.
  • HB-EGF binds strongly to heparin and is eluted at about 1 M NaCI (Raab, G. et al. (1997) Biochim. Biophys. Ada 1333: F179-F199).
  • main fractions containing AG-EGF purified from bovine chromaffin granules eluted between 300 and 600 mM NaCI.
  • AG-EGF and HB-EGF purified from bovine chromaffin granules exhibited a strong survival promoting effect on DAergic neurons (Fig. 7A). This neurotrophic effect is inhibited using anti HB-EGF antibody. Furthermore, AG-EGF and HB-EGF exhibited a strong stimulation of astroglial cell maturation as well as a stimulation of proliferation of non-DAergic cells as demonstrated by the appearance of GFAP positive astroglial cells (GFAP+ cells) and the proliferation of PCNA+ cells induced by AG-EGF and HB-EGF (Fig. 7B). The astroglial cell maturation effected by AGEGF and HB-EGF was inhibited using anti-HB-EGF.
  • Tissue culture Mesencephalic cell cultures were essentially established as described by Krieglstein and Unsicker (Neuroscience (1994) 63: 1189-1196). In brief, the ventral midbrain floor was dissected from embryonic day (E) 14 Wistar rat fetuses of two litters (20-25 embryos) and collected in CMF. Tissue pieces were enzymatically dissociated using 0.25% trypsin (BioWhittaker) in CMF for 15 min at 37°C. After addition of an equal volume of ice-cold horse serum and 1 mg DNAse, cells were triturated with fire-polished and siliconized pasteur pipettes and subsequently washed with DMEM/F12.
  • the single cell suspension (100 ⁇ l) was seeded on polyornithine (0.1 mg/ml in 15mM borate buffer, pH 8.4, Sigma)- laminin (5 ⁇ g/ml; Sigma) coated glass cover slips at a density of 200.000 cells/cm 2 .
  • Coverslips were incubated in a humified 5% C0 2 /95% air atmosphere to allow cells to attach. After two hours coverslips were transferred to 24-well plates (Falcon) containing 750 ⁇ l medium. On the following day, and subsequently every three days, 500 ⁇ l of the medium was replaced and neurotrophic factors were added at the same time at the given concentrations.
  • Bovine chromaffin cells were isolated by collagenase perfusion and digestion as previously described and enriched to >95% purity employing Percoll gradient centrifugation (Unsicker et al. (1980) Neuroscience 5: 1445-1460; Bieger et al. (1995) J. Neurochem. 64: 1521-1527). Chromaffin cells were seeded at 200,000 cells/cm 2 on plastic culture flasks (Falcon; 5x10 6 cells per 25cm 2 ) and maintained in 5 ml of DMEM with N1 supplements for 40h. After washing of cells with prewarmed medium cells were exposed to 2ml DMEM/N1 containing the cholinergic agonist carbachol (10 "5 M) for 15 min, while control cultures were
  • Conditioned medium from stimulated and unstimu- lated cells was stored in aliquots at -80°C to avoid repeated freezing and thawing and applied at 1 :4 dilution to cultures of mesencephalic DAergic neurons.
  • coverslips were stained with a monoclonal antibody to rat TH (1 :200; Boehringer Mannheim; diluted in 1 % horse serum) or with an antibody against serotonin (1 :50; DAKO) for 1 h at 37°C. Specific staining was anonymized using the anti-mouse Vectastain ABC kit in combination with DAB (Camon, Germany).
  • GFAP glial fibrillary acidic protein
  • BrdU was identified using anti-BrdU detection Kit I (Boehringer, Mannheim). BrdU/TH double detection was achieved by first applying the protocol for BrdU followed after another five washes with PBS and by the procedure for TH staining using TRITC anti-mouse-lgG as a secondary antibody. Nuclei were stained with propidium iodide (20 s, 0.1 ⁇ g/ml). Coverslips were mounted using Aquatex (Merck, Darmstadt).
  • the amplification was performed in 1 x PCR-buffer (Qiagen, Germany), 1 mM of dATP, 1 mM of dCTP, 1 mM of dGTP, 0.6 mM of dTTP (Pharmacia, Germany), 0.4 mM of digoxigenin-11-dUTP (Boehringer Mannheim, Germany), 100 pmol of each oligonucleotide bo-AG/HB- mat-N (5 ' -GACTTGGAAGAGGCAAACCTGG-3 ' ; SEQ ID NO 18) and bo-AG/HB- mat-R (5 ' -GAGGCTCAGCCCATGGCACC-3 ' ; SEQ ID NO 19) and 1 U of Taq DNA- polymerase (Qiagen, Germany).
  • the PCR mixture was overlaid with 40 ⁇ l of paraffin, incubated for 180 s at 94 °C and subjected to 25 cycles (50 s at 94°C, 50 s at 60°C, 50 s at 72°C) with an additional extension for 180 s at 72°C.
  • Prehybridization of plaque lift filters from cDNA library was performed at 60°C for 4 h in 0.25 M Na 2 HP0 4 , 7 % SDS, 1 % BSA, 1 mM EDTA, pH 7.2.
  • Hybridization was carried out with 50 ng of labelled HB-EGF PCR probe for 15 h under the same buffer conditions as described above for prehybridization. Filters were washed 3 times (5 min, 10 min and 30 min) with 30 mM Na 2 HP0 4l 0.1 % SDS at 60°C. Detection of signals was performed using the DIG Luminescent detection kit from Boehringer Mannheim, Germany (Cat. no. 1363514). According to positive signals, 7 clones were isolated and sequenced.
  • Fig. 8A SEQ ID NO 5
  • Fig. 8B SEQ ID NO 6
  • RNA isolation was performed using the Rneasy Kit (Qiagen) following the instructions by the manufacturer.
  • RT-PCRs were performed with 1 ⁇ g of total RNA using the Ready-To-Go RT-PCR Beads (Pharmacia).
  • HB-EGF bovine HB-EGF mRNA was found in all tissues which were investigated (Fig. 9). Main expression of PA/AG-EGF was detected in pancreas, liver, brain and adrenal gland. Low expression of PA/AG-EGF was detected in heart, testis and kidney. No expression of PA/AG-EGF was found in the blood sample, but this sample showed an additional amplification product which is larger than the HB-EGF PCR product.
  • the mature forms (Sphl/Pstl fragments) of bovine AG-EGF, bovine HB-EGF and human HB-EGF were each subcloned into the corresponding sites of the expression plasmid pQE-31 (Qiagen, Germany). This cloning strategy resulted in a tag of 6 additional histidine residues at the N-terminus of the mature AG-/HB- EGF molecules. Resulting plasmids were transformed into E.coli strain SG13009rep4 from Qiagen (Germany).
  • LB amp, kan
  • 500 ml of LB amp, kan were inoculated with a 5 ml overnight culture, and grown for 180 min at 37°C and 120 rpm until the OD 6 oo reached between 0.7 and 0.8. After induction with IPTG (1 mM final concentration), the cultures were further incubated for 4 h under the same growth conditions.
  • the cells were harvested by centrifugation at 6000 rpm for 20 min and the pellets were frozen at -20 °C until further purification.
  • Recombinantly expressed His-tagged AG-/HB-EGF proteins were purified using metalchelate chromatography (Ni-NTA).
  • Ni-NTA metalchelate chromatography
  • Frozen E. coli material which contained the recombinant protein was resuspended in 20 ml 8 M urea, 0.1 M NaH 2 P0 , 0.01 M Tris/HCI, pH 8.0 and agitated on a horizontal shaker for 1 h at room temperature (RT). The suspension was subjected to centrifugation at 10000 x g for 20 min and the supernatant incubated with 2 ml of Ni-NTA-agarose (Qiagen) for 1 h with slow shaking. The whole material was transferred into a suitable column and flowthroughs were collected for further analysis.
  • Method I a single step method used to renature AG-/HB-EGF molecules, utilizes high concentrations of urea. This method shows significant refolding amounts after a short incubation time of 1 hour on ice using 5 mg protein in 8 M urea, pH 4.5 (total volume: 0.5 ml). Refolding efficiency was increased considerably by incubation at 4 °C for a longer time. For example, an incubation period of more than 4 months was examined showing no loss of biological activity. After refolding, protein was desalted using Microcon 10 columns (Amicon) and washed twice with PBS.
  • Method II a redox system, utilizes glutathione/glutathione disulfide (GSH/GSSG) in order to renature recombinant AG-/HB-EGF.
  • the refolding mixture is composed of 1 ng/ ⁇ l protein in 1.2 M urea, 120 mM NaCI, 20 mM Tris/HCI pH 7.5, 5 mM EDTA and glutathione, glutathione disulfide in concentrations of 6 mM and 1 mM, respectively.
  • the mixture was incubated at 4 °C for 48 hours.
  • Immunological detection of 6xHis-tagged AG-/HB-EGF molecules was performed by western blotting using a commercially available RGS-His antibody (Qiagen) against the His-tag in combination with Western Light chemoluminescent detection system using the goat anti-mouse-AP antibody (Tropix, U.S.A.).
  • EP170.7 cells undergo apoptosis in the absence of interleukin-3 (IL-3), but as they express the EGF-receptor, they can survive in the presence of EGF receptor ligand by using a different pathway.
  • IL-3 interleukin-3
  • Cells were grown in IL3-supplemented RPMI 1640 medium (Gibco) with 10% FCS at 37 °C and 5 % C0 2 to a density of 1.5 x 10 6 /ml and washed once in IL3-free medium.
  • 0.5 ml of cell suspension were added to 2.5 ml IL3-free medium in 3 cm dishes (6-well plate) and the proteins of interest were given directly to the wells.
  • results of the activity assay of bovine AG-EGF, bovine HB-EGF and human HB- EGF are shown in Table 1.
  • bovine HB-EGF and AG-EGF proteins which had been subjected to the above-described refolding methods I or II, respectively, were applied in the apoptosis assay.
  • Human HB-EGF was obtained from R&D systems (USA, Cat. No. 259-HE).
  • IL-3 was applied in the form of conditioned media produced by WEHI cells.
  • GDF-5 was manufactured by the applicant.

Abstract

La présente invention concerne des acides nucléiques codant pour une protéine qui constitue un ligand du récepteur du facteur de croissance de l'épiderme (EGFR) ne comportant p. ex. pas de site de liaison à l'héparine. De préférence, la protéine est capable de stimuler la maturation de cellules d'astroglie et/ou possède un effet activant la survie sélective de neurones dopaminergiques (DAergic) et/ou périphériques et/ou a un effet régénérateur sur des neurones périphériques et axonaux. La présente invention concerne en outre des acides nucléiques antisens, des ribozymes et des anticorps dirigés contre l'acide nucléique ou contre la protéine, des procédés de production de ceux-ci, des antagonistes dirigés contre la protéine, des agonistes remplaçant l'activité fonctionnelle de la protéine et des compositions pharmaceutiques pour traiter des affections telles que des maladies neutodégénératives, le cancer et le SIDA, ainsi que des trousses diagnostiques pour détecter lesdites affections.
PCT/EP2000/005363 1999-06-09 2000-06-09 Acide nucleique codant pour de nouveaux facteurs de croissance du type egf WO2000077195A1 (fr)

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WO2002063037A2 (fr) * 2001-02-02 2002-08-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede d'identification d'acides nucleiques fonctionnels
AU2008249216B2 (en) * 2002-03-08 2012-01-19 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Use of EGFR transactivation inhibitors in human cancer

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WO1992006705A1 (fr) * 1990-10-16 1992-04-30 The Children's Medical Center Corporation Mitogene de liaison d'heparine presentant une homologie avec le facteur de croissance epidermique (egf)
WO1998004688A1 (fr) * 1996-07-29 1998-02-05 Biopharm Gmbh Facteur semblable a egf derive de granules de chromaffine et facteur neutrotrophique derive de cellules glia presentant une activite favorisant la survie de neurones daergiques

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WO1992006705A1 (fr) * 1990-10-16 1992-04-30 The Children's Medical Center Corporation Mitogene de liaison d'heparine presentant une homologie avec le facteur de croissance epidermique (egf)
WO1998004688A1 (fr) * 1996-07-29 1998-02-05 Biopharm Gmbh Facteur semblable a egf derive de granules de chromaffine et facteur neutrotrophique derive de cellules glia presentant une activite favorisant la survie de neurones daergiques

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002063037A2 (fr) * 2001-02-02 2002-08-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede d'identification d'acides nucleiques fonctionnels
WO2002063037A3 (fr) * 2001-02-02 2003-10-02 Max Planck Gesellschaft Procede d'identification d'acides nucleiques fonctionnels
AU2008249216B2 (en) * 2002-03-08 2012-01-19 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Use of EGFR transactivation inhibitors in human cancer

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