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WO1993000427A2 - Facteurs regulateurs de la croissance des neurites - Google Patents

Facteurs regulateurs de la croissance des neurites Download PDF

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Publication number
WO1993000427A2
WO1993000427A2 PCT/EP1992/001384 EP9201384W WO9300427A2 WO 1993000427 A2 WO1993000427 A2 WO 1993000427A2 EP 9201384 W EP9201384 W EP 9201384W WO 9300427 A2 WO9300427 A2 WO 9300427A2
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WIPO (PCT)
Prior art keywords
cell
cells
factor
metalloprotease
myelin
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PCT/EP1992/001384
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English (en)
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WO1993000427A3 (fr
Inventor
Martin E. Schwab
Pierenrico W. Caroni
Paolo A. Paganetti
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Erziehungsdirektion Of The Canton Zurich
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Priority claimed from US07/719,692 external-priority patent/US5250414A/en
Application filed by Erziehungsdirektion Of The Canton Zurich filed Critical Erziehungsdirektion Of The Canton Zurich
Publication of WO1993000427A2 publication Critical patent/WO1993000427A2/fr
Publication of WO1993000427A3 publication Critical patent/WO1993000427A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • 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

Definitions

  • antibody to neurite growth inhibitor may be used to promote the regeneration of neurons over long distances following spinal cord damage.
  • the metalloproteases of the invention allow invasive growth of glioblastomas and allow neurite outgrowth in central nervous system tissue. They may have important uses in the treatment of central nervous system damage and degenerative nerve diseases. Inhibition of the metalloprotease can be therapeutic ⁇ ally useful in the treatment of malignant tumors. 2. BACKGROUND OF THE INVENTION
  • NGF nerve growth factor
  • the second form is stable at neutral pH and contains three different polypeptide chains, , ⁇ and (molecular weight -140,000).
  • the ⁇ chain is the biologically active chain and is identical to the first form of NGF.
  • the third form which is isolated primarily from mouse L cells, (see U.S. Patent No. 4,230,691, by Young, issued October 28, 1980, and references therein) has a molecular weight of about 160,000 but is unstable at neutral pH. NGF has thus far been isolated from the submandibullar glands of mice, mouse L cells, and the prostate gland of the guinea pig and bull (reviewed in Thoenen et al., 1982, supra) . No differences between the biological action of mouse, guinea pig and bull NGF have been detected. In addition, NGF isolated from mice have been found to bind to the human NGF receptor (Johnson et al., 1986, Cell 47:545-554) .
  • the differentiated central nervous system (CNS) of higher vertebrates is capable of only very limited regenerative neurite growth after lesions. Limited regeneration after lesion has been seen in the retina (McConnell and Berry, 1982, Brain Res. 241:362-365) and in aminergic unmyelinated fiber tracts after chemical (Bjorklund and Stenevi, 1979, Physiol. Rev. 59:62-95) but not mechanical lesions (Bregman, 1987, Dev. Brain Res. 34:265-279). Davis et al. (1987, Science 236:1101-1109) and Gage et al. (1988, Exp. Brain Res. 72:371-380) observed a limited ingrowth of fibers into the hippocampus at 8-12 weeks.
  • the differentiated CNS may lack cellular or substrate constituents that are conducive for neurite growth during development (Liesi, 1985, EMBO J. 4:2505-2511; and Carbonetto et al, 1987, J. Neurosci. 7:610-620), or it may contain components which are nonpermissive or inhibitory for nerve fiber regeneration (Schwab and Thoenen, 1985, J. Neurosci. 5:2415-2423) .
  • a growth (cell proliferation) inhibitory factor for mouse neuroblastoma cells was partially purified and characterized from the culture medium of fetal rat glioblasts as well as from C6 rat glio a cells (Sakazaki et al., 1983, Brain Res.
  • Rous sarcoma virus transformed chick embryo fibroblasts which degrades fibronectin and which was localized at adhesion sites and on "invadopodia" was described by Chen and Chen (1987, Cell 48:193-203).
  • Neurite outgrowth from normal mouse sensory ganglia can be enhanced by the addition of serine protease inhibitors, ovomucoid trypsin inhibitor, leupeptin, soybean trypsin inhibitor, or thrombin (Hawkins and Seeds, 1986, supra) .
  • proteases were found to inhibit such neurite outgrowth. Results from preliminary studies indicate that such proteases possess a thrombin or trypsin like activity (Hawkins and Seeds, 1986, supra) .
  • proteases have also been characterized though their functional role in neurite outgrowth is as yet unknown. These include a urokinase-like plasminogen activator and a calcium dependent metalloprotease released by sympathetic and sensory rat neurons (Pitt an, 1985, Dev. Biol. 110:911-101).
  • the metalloprotease was found to have a molecular weight of 62 kD, to require l mM Ca 2+ for calcium activity, and to degrade native and denatured collagen more readily than casein, albumin, or fibronectin.
  • the plasminogen activator was found to have a molecular weight of 51 kD, and was precipitated by a rabbit antiseru produced against human urokinase. It may be converted to its active form of 32 kD.
  • NEUROBLASTOMA Neuroblastoma arises from neuroectoderm and contains anaplastic sympathetic ganglion cells (reviewed in Pinkel and Howarth, 1985, In: Medical Oncology, Calabrese, P., Rosenberg, S.A., and Schein, P.S., eds., MacMillan, NY, pp. 1226-1257).
  • One interesting aspect of neuroblastoma is that it has one of the highest rates of spontaneous regression among human tumors (Everson, 1964, Ann. NY Acad. Sci.
  • Neuroblastoma cells have been found to retain the capacity for morphological maturation in culture.
  • the tumors may occur anywhere along the sympathetic chain, with 50% of such tumors originating in the adrenal medulla.
  • NGF neuroblastoma
  • GLIOBLASTOMA Glioblastoma is a highly malignant astrocytic tumor usually located in the cerebral hemisphere.
  • Astrocytes appear to be a supporting tissue for neurons and comprise the vast majority of the intraparenchymal cells of the brain (reviewed in Cutler, 1987, In: Scientific American Medicine V. 2, Rubenstein and Federman, eds.. Scientific American, Inc. , NY, pp. 1-7) .
  • the tumors may also involve multiple lobes and may rupture into the ventricular system or extend across the corpus collosu to the opposite hemisphere. Due to the resulting increase in intracranial pressure, symptoms of tumor growth include headache, nausea and vomiting, mental status changes, and disturbances of consciousness. Due to their highly invasive properties, glioblastomas are associated with a poor prognosis. Chemotherapeutic agents or radiotherapies may be used. However, patients generally do not survive longer than two years even with these therapies.
  • the present invention relates to genes and their encoded proteins which regulate neurite growth and the diagnostic and therapeutic uses of such proteins. Such proteins are termed herein neurite growth regulatory factors.
  • the neurite growth regulatory factors of the present invention include, in one embodiment, central nervous system myelin associated proteins which inhibit neurite outgrowth, and are termed herein neurite growth inhibitory factors.
  • Another embodiment of the invention is directed to neurite growth regulatory factors which are metalloproteases associated with malignant tumors, in particular, those tumors metastatic to the brain. Such metalloproteases enable the malignant cells to overcome the inhibitory CNS environment and invade large areas of brain and spinal cord.
  • the CNS myelin associated proteins inhibit neurite outgrowth in nerve cells and neuroblastoma cells and also inhibit the spreading of fibroblasts and melanoma cells.
  • Such inhibitory proteins include but are not limited to 35,000 dalton and a 250,000 dalton molecular weight proteins and analogs, derivatives, and fragments thereof.
  • the CNS myelin associated inhibitory proteins may be used in the treatment of patients with malignant tumors which include but are not limited to melanoma and nerve tissue tumors (e.g., neuroblastoma).
  • the absence of the myelin associated inhibitory proteins can be diagnostic for the presence of a malignant tumor such as those metastatic to the brain (e.g., glioblastoma).
  • the present invention also relates to antagonists of the CNS myelin associated inhibitory proteins, including, but not limited to, antibodies, i.e. antibodies IN-1 or IN-2.
  • antibodies i.e. antibodies IN-1 or IN-2.
  • Such antibodies can be used to neutralize the neurite growth inhibitory factors for regenerative repair after trauma, degeneration, or inflammation.
  • monoclonal antibody IN-1 may be used to promote regeneration of nerve fibers over long distances following spinal cord damage.
  • the present invention further relates to neurite growth regulatory factor receptors and fragments thereof as well as the nucleic acid sequences coding for such neurite growth regulatory factor receptors and fragments, and their therapeutic and diagnostic uses. Substances which function as either agonists or antagonists to neurite growth regulatory factor receptors are also envisioned and within the scope of the present invention.
  • the metalloproteases of the present invention can be found associated with malignant tumors, in particular, those capable of metastasizing to the brain.
  • the metalloprotease is associated with membranes of glioblastoma cells.
  • the metalloproteases, and analogs, derivatives, and fragments thereof can have value in the treatment of nerve damage resulting from trauma, stroke, degenera- tive disorders of the central nervous system, etc.
  • the metallo ⁇ protease may be used in combination with antibodies to the neurite growth inhibitory factors to treat nerve damage.
  • the present invention is also directed to inhibitors of and/or antibodies to the metallo ⁇ proteases of the invention.
  • Such inhibitors and/or antibodies can be used in the diagnosis and/or treatment of malignant tumors such as those which can metastasize to the brain, including but not limited to glioblastomas.
  • the metalloprotease inhibitors in combination with CNS myelin associated inhibitory protein or analogs, derivatives, or fragments thereof, may be used in the treatment and/or diagnosis of malignant tumors including but not limited to glioblastoma, neuroblastoma, and melanoma.
  • BSA bovine serum albumin
  • cbz-ala-phe-NH 2 carbobenzoxy-alanine- phenylalanine-amide
  • cbz-phe-phe-NH 2 carbobenzoxy-phenylalanine- phenylalanine-amide
  • cbz-gly-phe-phe-NH 2 carbobenzoxy-glycine-phenylalanine
  • EDTA ethylenediamine tetracetate
  • EGTA ethylene glycol-bis-(b-aminoethyl ether) - N,N,N'-N'-tetracetate
  • FCS fetal calf serum
  • GdNPF glial-derived neurite promoting factor
  • GFAP glial fibrillary acid protein
  • HBO highly branched oligodendrocyte
  • Hepes N-2-hydroxyethylpiperazine-N•-2- ethanesulfonic acid
  • IN-1 a monoclonal antibody against gel- purified 250 kD
  • IN-2 a monclonal antibody against gel- purified 35 kD CNS myelin associated inhibitory protein
  • Jl a cell adhesion molecule of molecular weight 160-180 kD
  • NGF nerve growth factor neurite growth CNS myelin associated 35 kD and regulatory factors: 250 kD inhibitory proteins, and a glioblastoma cell membrane associated metalloprotease
  • PNS peripheral nervous system
  • Tris Tris (hydroxymethyl) aminomethane
  • C6 but not 3T3 or B16 cells attach and spread on CNS white matter of rat cerebellar frozen sections.
  • C6 cells overcome the inhibitory substrate property of CNS myelin.
  • FIG. 6 Degradation of CNS inhibitory substrate by C6 plasma membranes is 1,10-phenan ⁇ throline sensitive. Spreading of 3T3 cells on CNS myelin was induced by pretreatment of myelin with C6 plasma membranes. 1,10-phenanthroline abolished this effect. Shown are phase contrast micrographs of 3T3 cells on polylysine (PLYS) (a) , on CNS myelin (b) , on CNS myelin pretreated with C6 plasma membranes, and on CNS myelin pretreated with C6 plasma membranes (C6-PM) in the presence of 1,10-phenanthroline.
  • PLYS polylysine
  • C6 cell attachment and spreading on CNS white matter of rat cerebellar frozen section is impaired by metalloprotease blockers.
  • Inhibition of attachment and spreading is particularly evident in the center of the white matter (asterisks) , but is also visible in the main white matter branches (arrows) .
  • FIG. 8 C6 cell infiltration into CNS explants is impaired by cbz-tyr-tyr.
  • Cells were added to one tip of optic nerve explants (chamber cultures) in the presence of the metalloprotease inhibitor cbz-tyr-tyr, or of the control peptide cbz-ala-phe. 14 day old cultures were quantified. Infiltrated cells were counted in the first 1.3 mm of the explants. Each column represents the number of infiltrated cells per ° «1 TM& « Only the most central part of the explants was considered (0.25 mm). Values represent means ⁇ SEM of two sets of experiments for a total of 8 explants.
  • FIG. 10 Cell fractionation experiments localize a cbz-FAF[ 125 I]Y-NH 2 (SEQ ID NO:2) degradative activity to plasma membranes. Autoradiogram of thin layer chromatography of substrate incubated with plasma membranes (I) . For quantification the incubation medium was extracted with chloroform. The organic phase extracted completely unreacted substrate (II) , whereas degradation products were retained in the aqueous phase (III) . 0.8 mM phenanthroline completely blocked the degradation (IV-VI) .
  • FIG. 11 The substrate cbz-FAF[ 125 I]Y-NH 2 (SEQ ID NO:2) is degraded at one single site by a metallo- endoprotease. Autoradiogram of thin layer chromatography showing bestatin effect on Y-NH 2 formation, c. , plasma membranes; best, bestatin; phen, o-phenanthroline; o, undegraded substrate. Plasma membrane activity was measured as described for Figure 10.
  • SUBSTITUTE SHEET ISA EP 100 mM TRIS/HC1 (diamonds) , and contained 100 mM NaCl and 10 ⁇ M pepstatin.
  • FIG. 14 Schematic diagram of the hippocampus, showing the caudal (C) or lateral (L) directions of distances measured.
  • B extracellular matrix bridge;
  • E entry point of regenerating fibers into hippocampus;
  • R rostral hippocampus;
  • CdH caudal hippocampus.
  • Figure 15 Amino acid composition of the HPLC peak II derived from rat and bovine 35 kD neurite inhibitory factor (NI-35) . Values represent the ratio of each amino acid to the amount of aspartic acid.
  • N-terminal amino acid sequence of rat NI-35 derived of HPLC peak II The N-terminal amino acid sequence (SEQ ID NO:4) of NI-35 is shown, as well as a predicted mRNA sequence (SEQ ID NO:5) encoding the protein sequence, and the predicted sequence of a DNA (SEQ ID NO:6) complementary to the RNA which can be used as a hybridization probe for the cloning of NI-35.
  • Figure 17 Internal amino acid sequence of rat NI-35 derived of HPLC peak II. The amino acid sequence (SEQ ID NO:7) of an internal peptide of NI-35 derived from Endoproteinase-Lys C cleavage is shown.
  • RNA sequence (SEQ ID NO:8) encoding the protein sequence, and the predicted sequence of a DNA (SEQ ID NO:9) complementary to the RNA which can be used as a hybridization probe for the cloning of NI-35.
  • the present invention is directed to genes and their encoded proteins which regulate neurite growth and the diagnostic and therapeutic uses of such proteins.
  • the proteins of the present invention include proteins associated with central nervous system myelin with highly nonpermissive substrate properties, termed herein neurite growth inhibitory factors.
  • the neurite growth regulatory factors also include metalloproteases which can be found associated with malignant tumors, in particular, those tumors metastatic to the brain.
  • the CNS myelin associated proteins of the invention inhibit neurite outgrowth in nerve cells or neuroblastoma cells.
  • the protein can also inhibit fibroblast spreading and migration.
  • These inhibitory proteins are active cross-species and may be used in the treatment of patients with malignant tumors including but not limited to melanoma and tumors of nerve tissue (e.g. neuroblastoma) .
  • melanoma e.g. melanoma
  • tumors of nerve tissue e.g. neuroblastoma
  • a 35 kilodalton and a 250 kilodalton CNS myelin associated protein are described.
  • Section 8 infra f the 250 kD protein is a complex containing the 35 kD protein.
  • the present invention is also directed to antibodies to and peptide fragments and derivatives of the neurite growth inhibitory proteins and their therapeutic and diagnostic uses. These antibodies or peptides can be used in the treatment of nerve damage resulting from, e.g., trauma (e.g., spinal cord injuries) , stroke, degenerative disorders of the central nervous system, etc.
  • antibodies to CNS myelin associated proteins may be used to promote regeneration of nerve fibers.
  • monoclonal antibody IN-1 may be used to promote the regeneration of nerve fibers over long distances following spinal cord damage.
  • the present invention further relates to neurite growth regulatory factor receptors and peptide fragments thereof as well as the nucleic acid sequences coding for neurite growth regulatory factor receptors and fragments, and their therapeutic and diagnostic uses.
  • Antibodies to neurite growth regulatory factor receptors are also envisioned and within the scope of the present invention.
  • the present invention is also directed to metalloproteases associated with malignant tumors, in particular, those metastatic to the brain.
  • the metalloprotease is associated with glioblastoma cells.
  • the metalloproteases of the invention are associated with the CNS infiltration activity of malignant cells, and can neutralize the inhibitory substrate properties of the CNS myelin- associated proteins.
  • the metalloproteases can have therapeutic value in the treatment of nerve damage such as that resulting from traumatic injury (e.g. spinal cord injuries) , stroke, degenerative disorders of the central nervous system, etc.
  • the metalloprotease may be used in combination with antibodies directed against myelin associated inhibitory proteins to treat nerve damage.
  • the present invention is also directed to inhibitors of the metalloproteases.
  • Such inhibitors can impair malignant cell spreading and infiltration. and can be used in the treatment of malignant tumors (e.g. glioblastoma) .
  • the metalloprotease inhibitors in combination with CNS myelin associated inhibitory proteins such as the 35,000 dalton and/or the 250,000 dalton molecular weight proteins, may be used in the diagnosis and/or treatment of malignant tumors which include but are not limited to glioblastomas, neuroblastomas, and melanomas.
  • the method of the invention can be divided into the following stages, solely for the purpose of description: (1) isolation and purification of neurite growth regulatory factors; (2) characteriza- tion of neurite growth regulatory factors; (3) molecular cloning of genes or gene fragment encoding neurite growth regulatory factors; (4) production of antibodies against neurite growth regulatory factors; and (5) generation of neurite growth regulatory factor related derivatives, analogs, and peptides.
  • the method further encompasses the diagnostic and therapeutic uses of neurite growth regulatory factors and their antibodies.
  • the present invention relates to CNS myelin associated inhibitory proteins of neurite growth, receptors of CNS myelin associated inhibitory proteins of neurite growth, and to metalloproteases such as that associated with membranes of glioblastoma cells.
  • the CNS myelin associated inhibitory proteins of the invention may be isolated by first isolating myelin and subsequent purification therefrom.
  • the metalloprotease may be obtained by isolation from mammalian glioblastoma cells. Isolation procedures which may be employed are described more fully in the sections which follow.
  • the CNS myelin associated inhibitory proteins or the metalloprotease may be obtained from a recombinant expression system (see Section 5.3., infra) . Procedures for the isolation and purification of receptors for the CNS myelin associated inhibitory proteins are described in Section 5.1.2., infra.
  • CNS myelin associated inhibitory proteins can be isolated from the CNS myelin of higher vertebrates including, but not limited to, birds or mammals (both human and nonhuman such as bovine, rat, porcine, chick, etc.) (Caroni and Schwab, 1988, J. Cell Biol. 106:1281-1288).
  • Myelin can be obtained from the optic nerve or from central nervous system tissue that includes but is not limited to spinal cords or brain stems. The tissue may be homogenized using procedures described in the art (Col an et al., 1982, J. Cell Biol. 95:598-608) .
  • the myelin fraction can be isolated subsequently also using procedures described (Colman et al. , 1982, supra) .
  • the solubilized proteins can be subjected to one dimensional electrophoresis, followed by isoelectric focussing and elution from the focussing gel.
  • Gel-eluted proteins can be acetone- precipitated, redissolved in 10% formic acid and chromatographed on a C 4 reverse phase HPLC column (see Section 8, infra) .
  • the CNS myelin associated inhibitory proteins may be isolated and purified using immunological procedures.
  • the proteins can first be solubilized using detergent (e.g., Nonidet P-40TM, sodium deoxycholate) .
  • the proteins may then be isolated by immunoprecipitation with antibodies to the 35 kilodalton and/or the 250 kilodalton proteins.
  • the CNS myelin associated inhibitory proteins may be isolated using immunoaffinity chroma ⁇ tography in which the proteins are applied to an antibody column in solubilized form.
  • Receptors for the CNS myelin associated inhibitory proteins can be isolated from cells whose attachment, spreading, growth and/or motility is inhibited by the CNS myelin associated inhibitory proteins.
  • Such cells include but are not limited to fibroblasts and neurons.
  • neurons are used as the source for isolation and purification of the receptors.
  • receptors to CNS myelin associated inhibitory proteins may be isolated by affinity chromatography of neuronal plasma membrane fractions, in which a myelin associated inhibitory protein or peptide fragment thereof is immobilized to a solid support.
  • receptor cDNA may be isolated by expressio cloning using purified 35 kD or 250 kD neurite growth inhibitory factor as a ligand for the selection of receptor-expressing clones.
  • the metalloproteases of the present invention may be isolated from cells of malignant tumors, in particular, glioblastomas.
  • a metalloprotease can be isolated from mammalian glioblastoma cells.
  • the metalloprotease is isolated from the plasma membrane fraction of such cells.
  • the cells may be obtained by dissociating and homogenizing tumors using procedures known in the art or from tumor cell lines.
  • Plasma membrane fractions may be obtained using procedures known in the art, e.g. , gradient centrifugation (Quigley, 1976, J. Cell Biol. 71:472-486).
  • the metalloprotease may be isolated from the membranes by solubilization with mild ionic or nonionic detergent (e.g...
  • an expression cloning system may be used by transfecting glioblastoma cDNA into, e.g., fibroblasts and screening for a membrane associated metalloproteolytic activity.
  • PROTEIN CHARACTERIZATION The neurite growth regulatory factors of the present invention can be analyzed by assays based on their physical, immunological, or functional properties. The half life of the neurite growth regulatory factors in cultured cells can be studied, for example, by use of cycloheximide, an inhibitor of protein synthesis (Vasquez, 1974, FEBS Lett. 40:563- 584).
  • a physiological receptor for a neurite growth regulatory factor could be identified by assays which detect complex formation with a neurite growth regulatory factor, e.g.. by use of affinity chromatography with immobilized neurite growth regulatory factor, binding to a labeled neurite growth regulatory factor followed by cross-linking and immunoprecipitation, etc.
  • Electrophoretic techniques such as SDS-polyacryl- amide gel electrophoresis and two-dimensional electro- phoresis can be used to study protein structure.
  • the amino acid sequences of primary myelin associated inhibitors or of the metalloprotease can be derived by deduction from the DNA sequence if such is available, or alternatively, by direct sequencing of the protein, e.g. , with an automated amino acid sequencer.
  • the protein sequences can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824- 3828) .
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the protein (and the corresponding regions of the gene sequence, if available, which encode such regions) .
  • Any mammalian cell can potentially serve as the nucleic acid source for the molecular cloning of the genes encoding the CNS myelin associated inhibitory proteins, including but not limited to the 35 kD and/or 250 kD myelin associated proteins (Caroni and Schwab, 1988 Neuron 1:85-96), or the glioblastoma associated metalloprotease, hereinafter referred to as neurite growth regulatory factor genes.
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g.. a DNA "library”) , by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired mammalian cell.
  • cloned DNA e.g.. a DNA "library”
  • chemical synthesis e.g., chemical synthesis
  • cDNA cloning e.g. a DNA "library”
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions, in addition to coding regions; clones derived from cDNA will contain only exon sequences. Whatever the source, a given neurite growth regulatory factor gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • DNA fragments are generated, some of which will encode the desired neurite growth regulatory factor gene.
  • the DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication.
  • the linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.
  • the DNA fragments are generated, identifica ⁇ tion of the specific DNA fragment containing a neurite g ->wth regulatory factor gene may be accomplished in a number of ways. For example, if an amount of a neurite growth regulatory factor gene or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated )NA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science
  • a portion of a neurite growth regulatory factor amino acid sequence can be used to deduce the DNA sequence, which DNA sequence can then be synthesized as an oligonucleotide for use as a hybridization probe.
  • nucleic acid fractions enriched in neurite growth regulatory factor may be used as a probe, as an initial selection procedure.
  • Such DNA fragments may represent available, purified neurite growth regulatory factor DNA, or DNA that has been enriched for neurite growth regulatory factor sequences.
  • Immunoprecipitation analysis or functional assays of the in vitro translation products of the isolated mRNAs identifies the mRNA and, therefore, the cDNA fragments that contain neurite growth regulatory factor sequences.
  • An example of such a functional assay involves an assay for nonpermissiveness in which the effect of the various translation products on the spreading of 3T3 cells on a polylysine coated tissue culture dish is observed (Caroni and Schwab, 1988 J. Cell Biol. 106:1281-1288).
  • isolating the neurite growth regulatory factor genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence or making cDNA to the mRNA which encodes the neurite growth regulatory factor gene. Other methods are possible and within the scope of the invention.
  • transformation of host cells with recombinant DNA molecules that incorporate an isolated neurite growth regulatory factor gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene.
  • the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.
  • the recombinant DNA molecule that incorporates a neurite growth regulatory factor gene can be modified so that the gene is flanked by virus sequences that allow for genetic recombination in cells infected with the virus so that the gene can be inserted into the viral genome.
  • neurite growth regulatory factor DNA- containing clone After the neurite growth regulatory factor DNA- containing clone has been identified, grown, and harvested, its DNA insert may be characterized as described in Section 5.3.4, infra.
  • a neurite growth regulatory factor gene When the genetic structure of a neurite growth regulatory factor gene is known, it is possible to manipulate the structure for optimal use in the present invention.
  • promoter DNA may be ligated 5' of a neurite growth regulatory factor coding sequence, in addition to or replacement of the native promoter to provide for increased expression of the protein. Many manipulations are possible, and within the scope of the present invention.
  • the nucleotide sequence coding for a neurite growth regulatory factor protein or a portion thereof can be inserted into an appropriate expression vector, i.e. , a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • the necessary transcrip- tional and translation signals can also be supplied by the native neurite growth regulatory factor gene and/or its flanking regions.
  • host-vector systems may be utilized to express the protein-coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g.. vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g..).
  • baculovirus a virus containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
  • micro ⁇ organisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of these vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • any of the methods previously described for the insertion of DNA fragments into a vector may be used to. construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinations (genetic recombination) .
  • Expression vectors containing neurite growth regulatory factor gene inserts can be identified by three general approaches: (a) DNA-DNA hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences.
  • the presence of a foreign gene inserted in an expression vector can be detected by DNA-DNA hybridization using probes comprising sequences that are homologous to an inserted neurite growth regulatory factor gene.
  • the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g.. thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector.
  • recombinants containing the neurite growth regulatory factor insert can be identified by the absence of the marker gene function.
  • recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based on the physical, immunological, or functional properties of a given neurite growth regulatory factor gene product.
  • Expression in mammalian (e.g. COS) cells can be used to ensure "native" glycosylation of the heterologous neurite growth regulatory factor protein.
  • different vector/host expression systems may effect processing reactions such as proteolytic cleavages to different extents.
  • the gene product can be purified as described in Section 5.1, supra. and analyzed as described in Section 5.2, supra.
  • amino acid sequence of a given neurite growth regulatory factor protein can be deduced from the nucleotide sequence of the cloned gene, allowing the protein, or a fragment thereof, to be synthesized by standard chemical methods known in the art (e.g.. see Hunkapiller, et al., 1984, Nature 310:105-111).
  • such neurite growth regulatory factor proteins include but are not limited to those containing altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine, and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the invention are neurite growth regulatory factor proteins which are differen ⁇ tially modified during or after translation, e.g.. by glycosylation, proteolytic cleavage, etc.
  • the structure of a given neurite growth regulatory factor gene can be analyzed by various methods known in the art.
  • the cloned DNA or cDNA corresponding to a given neurite growth regulatory factor gene can be analyzed by methods including but not limited to Southern hybridization (Southern, 1975, J. Mol. Biol. 98:503- 517), Northern hybridization (Alwine, et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5350-5354; Wahl, et al., 1987, Meth. Enzy ol. 152:572-581), restriction endonuclease mapping (Maniatis, et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) , and DNA sequence analysis.
  • DNA sequence analysis can be performed by any techniques known in the art including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467), or use of an automated DNA sequenator (e.g.. Applied Biosystems, Foster City, CA) .
  • various host animals can be immunized by injection with a neurite growth regulatory factor protein, or a synthetic protein, or fragment thereof, including but not limited to rabbits, mice, rats, etc.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • a monoclonal antibody to an epitope of a neurite growth regulatory factor can be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), and the more recent human B cell hybridoma technique (Kozbor et al. , 1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et al. , 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In a particular embodiment, the hybridoma cell lines have been deposited with the European Collection of Animal Cell Cultures (ECACC) .
  • ECACC European Collection of Animal Cell Cultures
  • These cell lines have ECACC accession numbers 88102801 and 88102802. The production of these deposited hybridoma is detailed by Caroni and Schwab (1988, Neuron 1:85-96). These cell lines are used to express mouse monoclonal antibodies (IN-1 and IN-2) which recognize the 35 kD and 250 kD CNS myelin associated inhibitory proteins.
  • the monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g. , Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16).
  • Chimeric antibody molecules may be prepared containing a mouse antigen- binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci.
  • a molecular clone of an antibody to a neurite growth regulatory factor epitope can be prepared by known techniques. Recombinant DNA methodology (see e.g., Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab 1 fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment, and the 2 Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • the neurite growth regulatory factor-related derivatives, analogs, and peptides of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, a cloned neurite growth regulatory factor gene can be modified by any of numerous strategies known in the art (Maniatis, et al. , 1982, Molecular Cloning, A
  • a given neurite growth regulatory factor sequence can be cleaved at appropriate sites with restriction endonuclease(s) , subjected to enzymatic modifications if desired, isolated, and ligated in vitro.
  • a given neurite growth regulatory factor gene can be mutated .in vitro or jLn vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endo- nuclease sites or destroy preexisting ones, to facilitate further .in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site- directed mutagenesis (Hutchinson, et al., 1978, J. Biol. Chem. 253:6551), use of TAB® linkers (Pharmacia) , etc.
  • CNS myelin associated inhibitory proteins, analogs, derivatives, and subsequences thereof, and anti-inhibitory protein antibodies or peptides have uses in diagnostics.
  • Such molecules can be used in assays such as immunoassays to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting neurite growth extension, invasiveness, and regeneration. In one embodiment of the invention, these molecules may be used for the diagnosis of malignancies.
  • the CNS myelin associated inhibitory proteins, analogs, derivatives, and subsequences thereof and antibodies thereto may be used to monitor therapies for diseases and conditions which ultimately result in nerve damage; such diseases and conditions include but are not limited to CNS trauma, (e.g.
  • such molecules may be used to detect an increase in neurite outgrowth as an indicator of CNS fiber regeneration.
  • the absence of the CNS myelin associated inhibitory proteins in a patient sample containing CNS myelin can be a diagnostic marker for the presence of a malignancy, including but not limited to glioblastoma, neuro ⁇ blastoma, and melanoma, or a condition involving nerve growth, invasiveness, or regeneration in a patient.
  • the absence of the inhibitory proteins can be detected by means of an immunoassay in which the lack of any binding to anti- inhibitory protein antibodies (e.g., IN-1, IN-2) is observed.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as radioimmuno- assays, ELISA (enzyme linked immunosorbent assay) , "sandwich” immunoassays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, immunoelectro- phoresis assays, and immunohistochemistry on tissue sections, to name but a few.
  • ligands which bind to a CNS myelin associated inhibitory protein can be used in imaging techniques.
  • small peptides which bind to the inhibitory proteins, and which are able to penetrate through the blood-brain barrier, when labeled appropriately, can be used for imaging techniques such as PET (positron emission tomography) diagnosis or scintigraphy detection, under conditions n ⁇ ninvasive to the patient.
  • imaging techniques such as PET (positron emission tomography) diagnosis or scintigraphy detection, under conditions n ⁇ ninvasive to the patient.
  • Neurite growth inhibitory factor genes, DNA, cDNA, and RNA, and related nucleic acid sequences and subsequences, including complementary sequences can also be used in hybridization assays.
  • the neurite growth inhibitory factor nucleic acid sequences, or subsequences thereof comprising about at least 15 nucleotides, can be used as hybridization probes.
  • Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with changes in neurite growth inhibitory factor expression as described supra.
  • total RNA in myelin e.g., on biopsy tissue sections, from a patient can beassayed for the presence of neurite growth inhibitory factor mRNA, where the amount of neurite growth inhibitory factor mRNA is indicative of the level of inhibition of neurite outgrowth activity in a given patient.
  • CNS myelin associated inhibitory protein receptors as well as analogs, derivatives, and subsequences thereof, and anti-receptor antibodies have uses in diagnostics.
  • These molecules of the invention can be used in assays such as immunoassays or binding assays to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting neurite growth, extension, invasion, and regeneration. For example, it is possible that a lower level of expression of these receptors may be detected in various disorders associated with enhanced neurite sprouting and plasticity or regeneration such as those involving nerve damage, infarction, degenerative nerve diseases, or malignancies.
  • the metalloproteases of the invention may be used for diagnostic purposes. These molecules of the invention may be used in assays such as immuno ⁇ assays or inhibition type assays to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting neurite growth extension, inva- siveness, or regeneration. In a specific embodiment, the molecules of the present invention can be used to diagnose malignant tumors, in particular, glioblas- toma, by detecting the presence of or an increase in metalloprotease levels.
  • the molecules of the present invention may be used to monitor therapies for malignant tumors such as glioblastoma by detecting changes in metalloprotease levels.
  • decreases or the disappearance of metalloprotease levels should can be indicative of treatment efficacy.
  • metallo- protease levels can be relied upon as an indication of the malignant potential of a cell, e.g., a glial cell.
  • Malignant potential shall mean those properties associated with malignant tumors, e.g. invasiveness, lethality, and/or metastatic potential.
  • metalloprotease activity is measured by a competitive substrate assay, e.g., using the peptide carbobenzoxy- Phe-Ala-Phe-Tyr-amide (SEQ ID NO:l) (cbz-FAFY-NH2) as described in Section 7, infra.
  • the assays which can be used include but are not limited to those described supra in Section 5.6.1.1.
  • Metalloprotease genes and related nucleic acid sequences and subsequences, including complementary sequences, can also be used in hybridization assays, to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with changes in metalloprotease expression as described supra.
  • total RNA in a sample e.g., glial cells
  • metalloprotease mRNA e.g., glial cells
  • a malignancy that can be metastatic to the brain (e.g., glioblastoma) can be detected.
  • CNS myelin associated inhibitory proteins of the present invention can be therapeutically useful in the treatment of patients with malignant tumors including, but not limited to melanoma or tumors of nerve tissue (e.g. neuroblastoma) .
  • patients with neuroblastoma can be treated with the 35 kD and/or 250 kD proteins or analogs, derivatives, or subsequences thereof, and the human functional equivalents thereof, which are inhibitors of neurite extension.
  • a patient can be therapeutically administered both a CNS myelin- associated inhibitory protein and a metalloprotease inhibitor.
  • derivatives, analogs, or subsequences of CNS myelin inhibitory proteins which inhibit the native inhibitory protein function can be used in regimens where an increase in neurite extension, growth, or regeneration is desired, e.g., in patients with nervous system damage.
  • Patients suffering from traumatic disorders including but not limited to spinal cord injuries, spinal cord lesions, or other CNS pathway lesions
  • surgical nerve lesions damage secondary to infarction, infection, exposure to toxic agents, malignancy, paraneoplastic syndromes, or patients with various types of degenerative disorders of the central nervous system (Cutler, 1987, In: Scientific American Medicines v. 2, Scientific American Inc., NY, pp. 11-1-11-13) can be treated with such inhibitory protein antagonists.
  • antibodies directed to the 35 kD and/or 250 kD myelin associated inhibitory protein may be used to promote the regeneration of nerve fibers over long distances following spinal cord damage; in a specific example, monoclonal antibody IN-1 may be used.
  • Various delivery systems are known and can be used for delivery of CNS myelin inhibitory proteins, related molecules, or antibodies thereto, e.g.. encapsulation in liposomes or semipermeable membranes, expression by bacteria, etc.
  • Linkage to ligands such as antibodies can be used to target myelin associated protein-related molecules to therapeutically desirable sites in vivo.
  • Methods of introduction include but are not limited to intradermal, intramuscular, mtraperitoneal, intravenous, subcutaneous, oral, and intranasal routes, and infusion into ventricles or a site of operation (e.g. for spinal cord lesions) or tumor removal.
  • cells secreting CNS myelin inhibitory protein antagonist activity for example, and not by way of limitation, hybridoma cells, encapsulated in a suitable biological membrane may be implanted in a patient so as to provide a continuous source of anti-CNS myelin inhibiting protein antibodies.
  • any method which results in decreased synthesis of CNS myelin inhibitory proteins may be used to diminish their biological function.
  • agents toxic to the cells which synthesize CNS myelin inhibitory proteins e.g. oligodendrocytes
  • CNS myelin associated inhibitory protein receptors or fragments thereof, and antibodies thereto can be therapeutically useful in the treatment of patients with nervous system damage including but not limited to that resulting from CNS trauma (e.g., spinal cord injuries), infarction, or degenerative disorders of the central nervous system which include but are not limited to Alzheimer's disease, Parkinson's disease, Huntington's Chorea, amyotrophic lateral sclerosis, or progressive supranuclear palsy.
  • CNS trauma e.g., spinal cord injuries
  • infarction e.g., infarction
  • degenerative disorders of the central nervous system which include but are not limited to Alzheimer's disease, Parkinson's disease, Huntington's Chorea, amyotrophic lateral sclerosis, or progressive supranuclear palsy.
  • CNS myelin associated inhibitory protein receptors can be administered to a patient to "compete out” binding of the inhibi ⁇ tory proteins to their natural receptor, and to thus promote nerve growth or regeneration in the patient.
  • antibodies to the inhibitory protein receptor can be administered to a patient in order to prevent receptor function and thus promote nerve growth or regeneration in the patient.
  • Patients in whom such a therapy may be desired include but are not limited to those with nerve damage, stroke, or degenerative disorders of the central nervous system as described supra.
  • Various delivery systems are known and can be used for delivery of CNS myelin associated inhibitory protein receptors, related molecules, or antibodies thereto, e.g.. encapsulation in liposomes, expression by bacteria, etc.
  • Linkage to ligands such as anti ⁇ bodies can be used to target myelin associated protein receptor-related molecules to therapeutically desirable sites in vivo.
  • Methods of introduction include but are not limited to intradermal, intra ⁇ muscular, intraperitoneal, intravenous, subcutaneous, oral, intranasal routes ⁇ nd infusion into ventricles or a site of tumor remo.-i.
  • the metalloproteases of the present invention can be therapeutically useful in the treatment of various types of nervous system damage or degenerative disorders of the central nervous system (such as those described supra. Section 5.6.2.2)
  • patients su f fering from nervous system damage resulting from trauma, stroke, or neurodegenerative disorders can be treated with the metalloprotease or proteolytically active analogs, derivatives, or subsequences thereof which stimulate neurite extension or regeneration of CNS fiber.
  • a competitive inhibitor such as carbobenzoxy-Phe-Ala-Phe-Tyr-amide (SEQ ID NO:l) (cbz- FAFY-NH2)
  • o-phenanthroline, EDTA, and EGTA may be obtained from commercial vendors (e.g. Sigma Chemical Co.).
  • Cbz-tyr-tyr, cbz-gly-phe-NH2, cbz-phe-phe-NH2, and cbz-gly-phe-phe-NH2 may also be obtained from commercial vendors (e.g. Vega Biotechnologies) , or may be chemically synthesized.
  • patients with various types of malignant tumors in particular, those metastatic to the brain, can be treated with such inhibitors.
  • a patient with a glioblastoma can be treated with such inhibitors.
  • administration of antibodies directed to an epitope of the metalloprotease can also be used to inhibit metalloprotease function in patients.
  • metalloprotease inhibitors and a CNS myelin associated inhibitory protein can both be administered to a patient for the treatment of a malignant tumor, examples of which include but are not limited to glioblastoma, neuroblastoma, or a melanoma.
  • Various delivery systems are known and can be used for the delivery of metalloproteases and related molecules, e.g.. encapsulation in liposomes or semi ⁇ permeable membranes, expression by bacteria, etc.
  • Linkage to ligands such as antibodies can be used to target molecules to therapeutically desirable sites in vivo.
  • Methods of introduction include but are not limited to intradermal, intramuscular, mtraperi ⁇ toneal, intravenous, subcutaneous, oral, and intra- nasal routes, and infusion into ventricles or a site of tumor removal.
  • C6 cells infiltrate optic nerve explants, attach and spread on white and grey matter of cerebellar frozen sections or on CNS myelin.
  • the metal ions chelator 1,10- phenanthroline and the dipeptide cbz-tyr-tyr, but not inhibitors for three other classes of proteases, blocked up to 67% of C6 cell spreading on CNS myelin.
  • the same inhibitors of metalloprotease also impaired infiltration of CNS nerve explants and spreading on the CNS white matter of cerebellar frozen sections.
  • Rat C6, mouse NIH 3T3 and B16 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supple ⁇ mented with 10% fetal calf serum (FCS) , usually to maximally 70-80% confluency. Cells were harvested with a short trypsin treatment (0.1% in Ca 2+ /Mg 2+ -free Hank's medium for 90 seconds) stopped by addition of FCS in excess, collected by centrifugation. Cells were resuspended in either DMEM/FCS or defined serum- free medium (MEM) and used for experiments. Disso- ciated rat CNS glial cells were prepared starting from optic nerves of 6-7 days old Lewis rats as described below.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • Optic nerves were dissected from 6-7 day old Wistar rats and collected in plating medium (air- buffered enriched L 1S with 5% rat serum; Mains and Patterson, 1973, J. Cell Biol. 59:329-345). The meninges and blood vessels were carefully removed under a microscope and the nerves were cut into small pieces. Dissociation of 10 day old nerves was done for 25 minutes twice in 0.25% trypsin (Sigma) and 0.02% collagenase (Worthington) (Raff et al. , 1979, Brain Res. 174:283-318) in CMF-PBS (Ca ++ /Mg ++ - free phosphate buffered saline) at 37°C.
  • CMF-PBS Ca ++ /Mg ++ - free phosphate buffered saline
  • Inhibitory oligodendrocytes were identified by double labelling using the specific mouse monoclonal antibodies 0 * and 0 4 (Sommer and Schachner, 1981, Dev. Biol. 83:311-327) as described below.
  • the specific antibodies were visualized by the corresponding anti-mouse, anti-rabbit or anti-goat - fluorescein isothiocyanate (FITC) or - rhodamine isothiocyanate (RITC) linked secondary antibodies (Cappel, NC) .
  • FITC fluorescein isothiocyanate
  • RITC - rhodamine isothiocyanate
  • Cappel NC
  • the cultures Prior to staining, the cultures were washed twice with PBS containing 5% sucrose and 0.1% bovine serum albumin (BSA) .
  • the antibodies were directed against surface antigens and were therefore incubated on the living cultures at room temperature for 30 minutes at a dilution of 1:20 in PBS/s
  • Double-labeled cultures were evaluated by systematically screening in the fluorescence microscope for the presence of one antigen (usually 0 4 ) , and every labeled cell was examined for the presence of the other antigen, e.g. 0,.
  • Optic nerve and sciatic nerve explants were prepared as described (Schwab and Thoenen, 1985, J. Neurosci. 5:2415-2423). Briefly, the nerves were rapidly dissected from about 8 week old male rats, cleaned from the meninges, 3 times frozen and thawed using liquid nitrogen, and placed under a teflon ring (diameter 13 mm, thickness 1 mm) sealed to a culture dish with silicon grease. Two chambers connected only by the explants were in this way obtained. 300,000 C6, 3T3 or B16 cells were plated in the inner chamber in DMEM/FCS and incubated for 5 to 20 days. The medium was changed every other day. Cultures were fixed overnight with 4% formalin.
  • the nerve explants were mounted with Tissue-Tek, 10 to 15 ⁇ m sections were cut in a cryostate and collected on gelatine coated cover slips. After drying at room temperature 5 overnight, the sections were stained in 0.75% cresyl violet, and evaluated. The infiltrated cells were counted for each 0.1 mm of the explants, starting from the tip where cells were added. Due to the 15 day incubation, the explants were often different in 0 diameter. Therefore, only the central part of the nerves (0.25 mm) were considered, since only this part of the explants presented a good histological quality. Inhibition experiments were performed with nerve explants previously injected from both sides with 2 ⁇ l 5 of 3 mM cbz-tyr-tyr or cbz-ala-phe solutions.
  • Membrane fractions in sucrose solutions containing no more than 50 mM ionic species were adsorbed for several hours onto the wells of PLYS-coated tissue culture dishes (about 0.1 g of protein per cm 2 of tissue culture dish) . Unbound membranes were removed by three washes with CMF-Hank's solution. Coated dishes were then immediately used in substrate testing experiments. In experiments with sympathetic or sensory neurons small droplets of central or peripheral myelin were deposited in defined patterns over 35 mm culture dishes.
  • the myelin was then dried overnight onto PLYS coated wells (20 ⁇ g protein/well of 100 mm 2 surface) . Unbound membranes were removed by three washes with Ca 2+ /Mg 2+ -free Hank's solution. Myelin coated wells were immediately used in substrate testing assays by the addition of 9,000 cells (C6, 3T3, or B16) per cm 2 . Alternatively, we used extracted CNS myelin protein, or SDS-PAGE purified 35 and 250 kD inhibitory proteins reconstituted in liposomes (Caroni and Schwab, 1988, J. Cell Biol. 106:1281-1288). Experiments were scored at different time points using a phase contrast micro ⁇ scope equipped with a photocamera.
  • Quantifications were done using a surface integration program; three arbitrary fields were photographed for each well at a magnitude of 8OX, at least 25 cells per picture were measured. Each point represents the mean of at least 3 wells + SEM. Results are expressed as ⁇ 2 of projected cell surface, or as degree, which was calculated by subtracting from the projected surface value of a spreading cell, the surface value of a completely spheric cell.
  • sucrose interphase C6 plasma membranes fraction
  • 40-60% sucrose interphase C6 mitochondrial fraction
  • CNS myelin coated PLYS wells were prepared as described in the previous section, but instead of being immediately tested as substrate, they were first incubated with 50 ⁇ l of C6 plasma membranes (contain ⁇ ing 0.8 mg protein/ml MEM) at 37° for 30 minutes. Dishes were then rinsed twice with Hank's medium and immediately used as substrates for 3T3 cells. In some experiments, protease blockers were added to the membranes using 10 times concentrated solutions.
  • C6 cells were confronted with the SDS-PAGE purified inhibitors (35 kD and 250 kD) reconstituted in liposomes, and also with living, cultured oligoden ⁇ drocytes. Again, 35 kD and 250 kD liposomes strongly inhibited 3T3 cell spreading, but they did not impair C6 cell spreading; C6 cells adhered and rapidly assumed the well spread characteristic "fried egg" appearance also on these reconstituted CNS myelin fractions. 6.2.3. SPECIFIC BLOCKERS OF METALLOPROTEASES INHIBIT C6 CELL SPREADING ON CNS MYELIN
  • proteases involved in C6 behavior was investigated by determining the effect of inhibitors of proteases on C6 cell spreading on either CNS myelin or PLYS. Cys-, Ser- and Asp-protease blockers at the adequate concentrations had no discernible effect on
  • TIMP Tissue inhibitor of metalloproteases.
  • Cocktail - trasylol, 200 U/ml; leuptine, 0.3 mM; pepstatine, 0.3 mM.
  • Cocktail + same as cocktail -, but with 0.3 mM 1,10-phenantroline. nq: not quantified, only qualitative
  • 1,10-phenanthroline inhibited C6 spreading on myelin up to 67% after 2 hours in culture (Table I) . None of the blockers tested showed a significant effect on C6 cell spreading on PLYS. 1,10-phenanthroline is a general metalloprotease inhibitor due to its property of metal ion chelation. However, inhibition by this substance is not sufficient to define a proteolytic activity, since other metallodependent enzymes are also inhibited. Many other inhibitors of metallo ⁇ proteases have been found, but they usually turned out not to be as general as 1,10-phenanthroline.
  • TIMP also does not inhibit a brain membrane associated metalloprotease degrading enkephaline.
  • Carboxymethyl-phe-leu (Fournie-Zaluski, M.C. et al., 1983, J. Med. Chem. 26:60-65), a modified peptide with high affinity for enkephalinase (Almenoff, J. and M. Orlowski, 1983, Biochemistry 22:590-599), did not inhibit C6 cell spreading (Table I) .
  • C6 cells were plated on cerebellar frozen sections or added to optic nerve explants in the presence of two metalloprotease inhibitors (1,10-phenanthroline and cbz-tyr-tyr).
  • Parallel cultures contained inhibitors for the three other classes of proteases (leupeptine, pepstatine or trasylol) , or a control dipeptide (cbz-ala-phe) .
  • Rat optic nerves were injected with 4 ⁇ l of 3 mM solutions of either cbz-ala-phe or cbz-tyr-tyr. Cells were incubated with medium containing 0.5 mM peptide. In the outer chamber, where no cells were present, the peptide concentration was 1 mM. After 14 days, the immigration of C6 cells into the explants differed greatly (Fig. 7) . Cbz-ala-phe-injected nerves contained more cells, and C6 cell infiltration was not affected, as compared to explants injected with culture medium only. On the other hand, cbz-tyr-tyr inhibited C6 cell infiltration in all the 8 nerves examined (2 experiments) . C6 cells were found mainly at the cut end of these nerve explants, and deep infiltration, which occurred massively in control explants, was strongly reduced by cbz-tyr-tyr.
  • Metalloproteases form an increasingly numerous gr.oup, the members of which differ in their sensitivity to various blockers.
  • the most general blocker is 1,10- phenanthroline which impaired C6 cell spreading on CNS myelin up to 67%, whereas most inhibitors of the other classes of proteases had no detectable effects.
  • an effect of trypsin-like serine-protease inhibitors was also observed.
  • the effect of 1,10-phenanthroline was dose- dependent, with an IC 50 of 200 ⁇ M.
  • CNS myelin as a substrate, since normal, rapid spreading of C6 cells was observed on other substrates such as CNS grey matter, PNS myelin, glass or PLYS in the presence of 1,10-phenanthroline.
  • Other known metalloprotease blockers like bestatine (inhibitor of aminopeptidases; Umezawa, et al., 1976, J. Antibiot. 29:857-859), phosphoramidone (inhibitor of thermolysin-like metalloproteases; Komiyama, et al., 1975, Biochem. Biophys. Res. Commun.
  • B16 cells did not migrate into optic nerve explants, but responded to the myelin-associated inhibitors in a way very similar to 3T3 cells or neurons.
  • B16 cells upon intraventricular injection, form mainly meningiomas or intraventricular tumors without significant infiltration of the brain parenchyma.
  • the mechanisms providing metastatic behavior to B16 cells in the periphery are different from those conferring high mobility to C6 cells in the CNS tissue.
  • C6-associated metalloprotease not only inhibited C6 spreading on CNS myelin, but also abolished C6 cell attachment, spreading, and migration on CNS white matter, and the dipeptide, cbz-tyr-tyr strongly impaired the migration of C6 cells into optic nerve explants.
  • This metalloprotease activity(ies) may, therefore, be crucially involved in the infiltrative behavior of C6 glioblastoma cells in CNS tissue, also in vivo.
  • C6 glioblastoma cell spreading on CNS myelin as a model, we describe here a tetrapeptide, carbo- benzoxy-Phe-Ala-Phe-Tyr-amide (SEQ ID NO:l) (cbz-FAFY- NH 2 ) , that acted as a competitive substrate to block C6 cell spreading on CNS myelin.
  • the specific cleavage of this radio-iodinated substrate could be used as a sensitive, specific enzymatic assay to further charac ⁇ terize the metalloprotease involved in C6 invasive behavior in CNS tissue.
  • Rat C6 glioblastoma cells were a kind gift of Prof. D. Monard (Friedrich Miescher Institute, Basel, Switzerland) .
  • Culture media were purchased from GIBCO BRL (Basel, Switzerland) ; cell culture plastic- wares were from Falcon (Becton Dickinson, Oxnard, CA) . All chemicals were purchased from SIGMA (St. Louis, MO) .
  • Tri-and tetrapeptide protease blockers were synthesized by BACHEM AG (Basel, Switzerland).
  • Carbobenzoxy-Gly-Phe-Phe-amide (cbz-GFF-NH 2 ) was synthesized from cbz-G-OH (I) and H 2 N-FF-amide (II) as follows: 1 mmole of I was activated at -20°C with 1 mmole N-methylmorpholine and 1 mmole isobutylchloro- formate in 10 ml tetrahydrofuran (THF) . To start the reaction, 1 mmole of II in 5 ml THF was added. The reaction was continued by warming the reaction vessel to room temperature. The mixture was then stirred overnight. The product was crystallized by addition of a few drops of water, and was analysed by ['HJNMR- spectrometry. The reaction profile was monitored on thin layer chromatography.
  • Rat C6 glioblastoma cells (Benda et al., 1968, Science 161:370-371) were cultured in Dulbecco's modified Eagle's medium (DMEM) supple ⁇ mented with 10% fetal calf serum (FCS) , usually to maximally 70-80% confluency (Paganetti et al., 1988, J. Cell Biol. 107:2281-2291). All cell culture media were routinely supplemented with penicillin and strep ⁇ tomycin. Cells were harvested by a short trypsin treatment (0.1% in Ca 2+ /Mg 2+ -free [CMF] PBS/EDTA solution for 90 seconds) followed by addition of
  • DMEM/10% FCS DMEM/10% FCS and centrifugation (600 X g, 6 min) .
  • the cell pellet was rinsed with 5 ml DMEM/10% FCS and centrifuged.
  • cells were resuspended at 10 6 /ml in defined serum-free medium (MEM ⁇ ) supplemented with 0.2 ⁇ g/ml insulin, 0.5 ⁇ g/ml . transferrin and 0.12 ⁇ g/ml bovine serum albumin (BSA).
  • Protease blockers or peptides were added directly to the cell suspension from 10X solutions 10 min before plating.
  • Spinal cord myelin from young adult Lewis rats was purified on a discontinuous sucrose gradient (Colman et al., 1982, J. Cell Biol.
  • a crude vesicular fraction was obtained by centrifugating the nuclear supernatant in a Sorvall S22 rotor (20,000 X g, 30 min), and was resuspended in 10 ml CMF-Hank's. After adding 20 ml 2.25 M sucrose in CMF-Hank's, this suspension was loaded on 4X 8 ml 2.25 M sucrose in CMF-Hank's and centrifuged at 80,000 X g for 1 h in a Beckman SW28 motor. Plasma membranes were harvested at the top and the mitochondria1 fraction at the interphase of this 2 step gradient.
  • Conditioned medium was obtained as described (Paganetti et al., 1988, J. Cell Biol. 107:2281-2291) by collecting MEM ⁇ incubated for 1 day on a C6 cell layer.
  • Radio-iodination of cbz-FAFY-NH 2 Radio-iodination was performed using Iodo-Beads (PIERCE, IL, USA) .
  • the probe was then diluted to 10 ml with 10% acetonitrile (ACN)/0.1% trifluor-acetic acid (TFA), loaded on a SEP-PAK cartridge (Waters/Millipore, MA, USA), rinsed with 10 ml 20% ACN/0.1% TFA and eluted with 1 ml 80% ACN, 0.1% TFA.
  • This partially purified probe was then lyophilized, resuspended in 2 ml 10% ACN, 0.1% TFA (buffer A) and loaded on a reverse-phase column (300-5 C I8 , Macherey-Nagel, Switzerland; HPLC System Gold, Beckman CA, USA) .
  • cell layers or plasma membrane suspensions were preincubated for 30 min with protease blockers or peptides.
  • NEM or iodacetamide incubations of plasma membranes were performed at pH 8.5. The incubation was stopped by loading a 10 ⁇ l aliquot of the incubation medium on a thin layer chromatography plate (10 cm height, Kieselgel 60 F ⁇ , MERCK, Germany) and air dried. Thin layer chromatography was routinely run with CHCl 3 :CH 3 OH:CH 3 COOH 32% (5:3:1). Plates were then air dried and exposed on Kodak X-OMAT films (NY, USA) with an intensifying screen.
  • C6 glioblastoma cell spreading on CNS mvelin- coated wells is impaired by short oligopeptides.
  • C6 glioblastoma cells plated on CNS myelin as a culture substrate rapidly spread and overcome the inhibitory effect of the myelin-associated neurite growth inhibitors of 35 kD (NI-35) and 250 kD (NI-250) (most other cell types remain round and are inhibited from spreading on this substrate) .
  • cbz-FAFY-NH 2 The tetrapeptide cbz-FAFY-NH 2 (SEQ ID NO:l) was the longest peptide tested that clearly impaired C6 cell spreading on CNS myelin. Control experiments were performed with peptides containing unrelated amino acid sequences (e.g. cbz-GGG-NH 2 ) or with partially unpro- tected peptides (e.g. AFY-NH 2 ) . cbz-FAFY-NH 2 had no effect on PLYS-coated wells.
  • the dipeptides cbz-GF-NH 2 and cbz-AF-NH 2 required a concentration of 600 ⁇ M for half-maximal inhibition of spreading on CNS myelin, whereas on PLYS concentrations up to 2 mM (saturation limit!) had no effect.
  • the tripeptides cbz-GFF-OCH 3 and cbz-GLF-NH 2 impaired C6 cell spreading on CNS myelin with significantly lower IC 50 (5 ⁇ M) .
  • the most potent blocker of C6 cell spreading on CNS myelin was the tetrapeptide cbz-FAFY-NH 2 (SEQ ID NO:l) with an half-maximal inhibition at 3 ⁇ M. Protection of both amino- and carboxy- terminals, seems to be a prerequisite for activity, since unprotected, charged peptides had no effect. This property suggests the involvement of an endoprotease rather than of an exopeptidase.
  • Cbz-FAFY-NH ? is degraded by a plasma membrane associated metalloprotease.
  • cbz-FAF[ 125 I]Y-NH 2 degradation by different subcellular fractions Maximal activity was associated with the plasma membrane (2.7 nMol/min) .
  • crude homogenate (0.08 nMol/min) and mitochondrial fraction (0.27 nMol/min) were clearly less active.
  • the substrate cbz-FAF[ I25 I]Y-H, (SEQ ID NO:2) was incubated initially with 100 ⁇ g for each protease. The amount of protease was then reduced stepwise by a factor of 3 for each further point. Activity was measured after 30 min (37°C, pH 7.4) . Shown are the amounts of protease necessary to obtain degradation of 50% of the substrate.
  • cbz-FAFY-NH 2 SEQ ID NO:l
  • uPA urokinase
  • tPA tissue-type plasminogen activator
  • Half maximal activity for Cathepsin D was obtained with 5 ng at pH 4.0, but no activity was detected at pH 7.4 with up to 100 ⁇ g protein.
  • 1.5 ⁇ g C6 plasma membranes resulted in 50% substrate cleavage (Table V) . Total degradation of the substrate by plasma membranes was completed after 2h (not shown) .
  • the rate of C6 metalloendoprotease activity was measured at different pH values. We found activity in the pH range of 4.0-8.5 (Fig. 12a). This broad range could be due to the presence of a contamination of acidic proteases in the plasma membrane fraction. To test for this, we studied the effect of the general acidic protease blocker pepstatin and of o-phenanthroline at the different pH values. In fact, a pepstatin sensitive activity was found at pH 4.0, but was absent at neutral pH (Fig. 12b). In contrast, the o-phenanthroline sensitive activity was maximal in the pH range of 5.5-7.0 (Fig. 12c), with peak activity between pH 5.5 and 6.0 (Fig. 12d) .
  • HPLC fractions containing protein peaks were lyophilized, resuspended in sample buffer and subjected to ran on a SDS- PAGE under reducing conditions.
  • the HPLC chromatographs from bovine 33 kd, 35 kd and 250 kd gel eluted proteins show two common identical peaks, which elute at -62% (peak I) and -73% (peak II) aceto- nitrile. Aliquots of these peak fractions migrate at 33 kD on a SDS-PAGE gel.
  • the amino acid composi ⁇ tions (Fig. 15) (determined by the orthophtaldialdehyde method) of peaks I and II derived from 33 kD or 35 kD or 250 kD. material are very similar, which implies that they represent two subforms of one heterogenous protein.
  • N-terminal sequence data of peak II derived from 33 kD or 35 kD protein (bovine) indicate that both fractions are identical and therefore must be derivatives of the same protein.
  • FIG. 16 Also shown in Figure 16 are the partial sequences of its predicted mRNA (SEQ ID NO:5), and a complementary DNA (SEQ ID NO:6) which can be used as a hybridization probe for cloning the NI-35 (or NI-250, see infra) gene.
  • the N-terminus of peak II derived from rat 35 kD or 250 kD protein fractions was determined to be identical to each other. This result therefore shows that the 250 kD protein is a complex containing the 35 kD protein. Endoproteinase Lys-C digestion of the rat 35 kD HPLC- purified protein resulted in several fragments which were separated by HPLC. A partial sequence of 17 amino acids (SEQ ID NO:7) was determined and is shown in Figure 17, along with its predicted partial mRNA sequence (SEQ ID NO:8), and a DNA (SEQ ID NO:9) which can be used as a hybridization probe in cloning.
  • Human CNS myelin was prepared by the same procedure described by Caroni and Schwab (1988, J. Cell Biol.
  • the glioblastoma cell lines were also tested for in vivo invasiveness by intracerebral implantation in nude mice, and survival time was measured. A clear correlation was found between malignant potential in vivo (first animals died in 6 weeks with cell line SKI E; longest survival time was about 5-6 months with cell lines SKI H and I) , and infiltrative behavior in vitro (spreading on CNS myelin, sensitivity to metalloprotease blockers) . In contrast, the production of plasminogen activator was not at all related to high malignancy.

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Abstract

Gènes et protéines codées de ceux-ci servant à réguler la croissance des neurites, et utilisations diagnostique et thérapeutique de ces protéines (ci-après: facteurs régulateurs de la croissance des neurites). Lesdites protéines incluent des protéines associées à la myéline du système nerveux central (CNS) et des métalloprotéases associées aux cellules de glioblastomes et aux autres tumeurs malignes aptes à métastaser dans le cerveau. Les protéines associées à la myéline du CNS inhibent l'excroissance de neurites dans les cellules nerveuses et les cellules de neuroblastomes, et peuvent également inhiber la propagation fibroblastique. Ces protéines d'inhibition incluent des protéines dont les poids moléculaires sont de 35 000 et 250 000 daltons respectivement, leurs analogues, leurs dérivés et leurs fragments. Lesdites protéines d'inhibition associées à la myéline du CNS peuvent être utilisées dans le traitement des tumeurs malignes. Lesdites métalloprotéases sont utiles dans le traitement des lésions nerveuses et des troubles dégénératifs du système nerveux. On a également prévu des inhibiteurs des métalloprotéases pouvant s'utiliser, en association avec lesdites protéines d'inhibition associées à la myéline du CNS, dans le traitement des tumeurs malignes.
PCT/EP1992/001384 1991-06-24 1992-06-19 Facteurs regulateurs de la croissance des neurites WO1993000427A2 (fr)

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Cited By (11)

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WO1994017831A1 (fr) * 1993-02-11 1994-08-18 Erziehungsdirektion Of The Canton Zurich Association de la neurotrophine et d'un anticorps contre la proteine inhibitrice de la croissance de neurites associee a la myeline stimulant la regeneration du systeme nerveux central
WO1996032959A1 (fr) * 1995-04-19 1996-10-24 Acorda Therapeutics Modulateurs de la croissance de l'axone et des dendrites du systeme nerveux central, compositions, cellules et procedes dans lesquels ils sont mis en ×uvre et utilises
US5717092A (en) * 1996-03-29 1998-02-10 Vertex Pharmaceuticals Inc. Compounds with improved multi-drug resistance activity
US6037370A (en) * 1995-06-08 2000-03-14 Vertex Pharmaceuticals Incorporated Methods and compositions for stimulating neurite growth
WO2000060064A1 (fr) * 1999-04-01 2000-10-12 The Procter & Gamble Company Processus de purification d'une enzyme du type fixant le calcium, par exemple une métalloprotéase, à l'aide d'un tampon exempt de calcium
WO2000060083A1 (fr) * 1999-04-08 2000-10-12 Chiron Corporation Nouvelle proteine associee a la reponse de la cellule a un stress
EP1124846A4 (fr) * 1998-11-06 2002-06-12 Martin E Schwab Sequences nucleotidiques et proteiques de gene nogo et procedes reposant sur ces sequences
US6576607B1 (en) 1995-04-19 2003-06-10 Acorda Therapeutics Methods using CNS neurite outgrowth modulators
US6858598B1 (en) 1998-12-23 2005-02-22 G. D. Searle & Co. Method of using a matrix metalloproteinase inhibitor and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia
US7186530B1 (en) 2000-04-07 2007-03-06 Chiron Corporation Protein associated with cell stress response
US7560102B2 (en) 1998-05-19 2009-07-14 Yeda Research And Development Co., Ltd Method for reducing neuronal degeneration so as to ameliorate the effects of injury or disease

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CA1173823A (fr) * 1980-08-26 1984-09-04 Leonard M. Hjelmeland Detersif zwitterionique non denaturant pour la biochimie des membranes
AU652537B2 (en) * 1988-11-04 1994-09-01 Erziehungsdirektion Of The Canton Zurich Neurite growth regulatory factors

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994017831A1 (fr) * 1993-02-11 1994-08-18 Erziehungsdirektion Of The Canton Zurich Association de la neurotrophine et d'un anticorps contre la proteine inhibitrice de la croissance de neurites associee a la myeline stimulant la regeneration du systeme nerveux central
US6576607B1 (en) 1995-04-19 2003-06-10 Acorda Therapeutics Methods using CNS neurite outgrowth modulators
WO1996032959A1 (fr) * 1995-04-19 1996-10-24 Acorda Therapeutics Modulateurs de la croissance de l'axone et des dendrites du systeme nerveux central, compositions, cellules et procedes dans lesquels ils sont mis en ×uvre et utilises
US7951373B2 (en) 1995-04-19 2011-05-31 Melitta Schachner Methods using agonist antibodies to CNS neurite outgrowth modulators
EP1666037A3 (fr) * 1995-06-08 2006-06-21 Vertex Pharmaceuticals Incorporated Compositions pour la stimulation de la croissance des neurites
US6326387B1 (en) 1995-06-08 2001-12-04 Vertex Pharmaceuticals Incorporated Methods and compositions for stimulating neurite growth
US6037370A (en) * 1995-06-08 2000-03-14 Vertex Pharmaceuticals Incorporated Methods and compositions for stimulating neurite growth
US5935954A (en) * 1996-03-29 1999-08-10 Vertex Pharmaceuticals Incorporated Compounds with improved multi-drug resistance activity
US5717092A (en) * 1996-03-29 1998-02-10 Vertex Pharmaceuticals Inc. Compounds with improved multi-drug resistance activity
US7560102B2 (en) 1998-05-19 2009-07-14 Yeda Research And Development Co., Ltd Method for reducing neuronal degeneration so as to ameliorate the effects of injury or disease
US7425334B2 (en) 1998-11-06 2008-09-16 The University Of Zurich Methods of making antibodies that bind Nogo
JP2003531566A (ja) * 1998-11-06 2003-10-28 シュヴァブ，マーティン，イー． Ｎｏｇｏ遺伝子のヌクレオチド配列およびタンパク質配列、ならびにそれに基づく方法
EP1124846A4 (fr) * 1998-11-06 2002-06-12 Martin E Schwab Sequences nucleotidiques et proteiques de gene nogo et procedes reposant sur ces sequences
US7781188B1 (en) 1998-11-06 2010-08-24 University Of Zurich Nucleotide and protein sequences of Nogo genes and methods based thereon
US6858598B1 (en) 1998-12-23 2005-02-22 G. D. Searle & Co. Method of using a matrix metalloproteinase inhibitor and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia
WO2000060064A1 (fr) * 1999-04-01 2000-10-12 The Procter & Gamble Company Processus de purification d'une enzyme du type fixant le calcium, par exemple une métalloprotéase, à l'aide d'un tampon exempt de calcium
WO2000060083A1 (fr) * 1999-04-08 2000-10-12 Chiron Corporation Nouvelle proteine associee a la reponse de la cellule a un stress
US7186530B1 (en) 2000-04-07 2007-03-06 Chiron Corporation Protein associated with cell stress response

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