US20110112280A1 - Use of rgm and its modulators - Google Patents

Use of rgm and its modulators Download PDF

Info

Publication number
US20110112280A1
US20110112280A1 US12/939,823 US93982310A US2011112280A1 US 20110112280 A1 US20110112280 A1 US 20110112280A1 US 93982310 A US93982310 A US 93982310A US 2011112280 A1 US2011112280 A1 US 2011112280A1
Authority
US
United States
Prior art keywords
rgm
derivative
polypeptide
disorders
fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US12/939,823
Inventor
Bernhard K. Mueller
Philippe P. Monnier
Paolo Macchi
Friedrich Bonhoeffer
Bernd Stahl
Matthias Mann
Jens S. Andersen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to US12/939,823 priority Critical patent/US20110112280A1/en
Publication of US20110112280A1 publication Critical patent/US20110112280A1/en
Priority to US13/493,005 priority patent/US8680239B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00Drugs for disorders of the urinary system
    • A61P13/12Drugs for disorders of the urinary system of the kidneys
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • A61P17/02Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/08Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/02Drugs for disorders of the nervous system for peripheral neuropathies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/08Antiepileptics; Anticonvulsants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/14Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/14Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16Anti-Parkinson drugs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/28Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/08Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04Antibacterial agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/08Antiallergic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00General protective or antinoxious agents
    • A61P39/02Antidotes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702Regulators; Modulating activity
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to the use of a modulator of a polypeptide having or comprising an amino acid sequence as disclosed herein or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with angiogenic disorders or disorders of the cardio-vascular system.
  • the invention provides for the use of a modulator of a polypeptide having or comprising said amino acid sequence or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with seizures, associated with angiogenic disorders or disorders of the cardio-vascular system.
  • the invention provides for the use of said polypeptide or said functional fragment or derivative thereof for the preparation of a pharmaceutical composition for preventing or treating tumor growth or formation of tumor metastases or as a marker of stem cells.
  • RGC axons Having arrived at the anterior pole of the optic tectum, RGC axons start to invade their tectal target to find their target neurons. Mapping occurs in such a way, that RGC axons from nasal retina project to posterior tectum and temporal axons to anterior tectum.
  • axons coming from dorsal retina terminate in ventral tectum, whereas those from ventral retina end up in dorsal tectum.
  • a precise topographic map is formed, where neighborhood relationships in the retina are preserved in the tectum, so that axons from neighboring ganglion cells in the retina synapse with neighboring tectal neurons.
  • RGM Repulsive Guidance Molecule
  • RGM Due to the abnormal biochemical behaviour of RGM, the precise amino acid sequence was not obtainable.
  • RGM was described as a molecule which is active during vertebrate development. Interestingly, RGM is down-regulated in the embryonic chick tectum after E12 and in the embryonic rat tectum after P2 and completely disappears after the embryonic stages (Müller (1992), Ph. D thesis University of Tübingen; Müller (1997) Japan Scientific Societies, 215-229) In 1996, Müller (loc. cit.) have shown that CALI (chromophore-assisted laser inactivation) of RGM eliminates the repulsive guidance activity of posterior tectal membranes/RGM.
  • CALI chromophore-assisted laser inactivation
  • RGM acts in concert with RAGS (now termed ephrin-A5) and ELF-1 (ephrin-A2). It was furthermore envisaged that RGM may be a co-factor potentiating the activity of RAGS and ELF-1 in embryonic guidance events.
  • Nogo-A Chon, Nature 403 (2000), 434-438.
  • the membrane-bound Nogo turned out to be a member of the reticulon family (GrandPré, Nature 403 (2000), 439-444).
  • semaphorin IV Further factors which mediate neuronal outgrowth inhibition have first been isolated in grasshoppers, and termed “fascidin IV” and later “collapsin” in chicken. These inhibitors belong to the so-called semaphorin family. Semaphorins have been reported in a wide range of species and described as transmembrane proteins (see, inter alia, Kolodkin Cell 75 (1993) 1389-99, POschel, Neuron 14 (1995), 941-948). Yet, it was also shown that not all semaphorins have inhibitory activity. Some members of said family, e.g. semaphorin E, act as an attractive guidance signal for cortical axons (Bagnard, Development 125 (1998), 5043-5053).
  • Ephrin-Eph A further system of repulsive guidance molecules is the ephrin-Eph system.
  • Ephrins are ligands of the Eph receptor kinases and are implicated as positional labels that may guide the development of neural topographic maps (Flanagan, Ann. Rev. Neurosc. 21 (1998), 309-345).
  • Ephrins are grouped in two classes, the A-ephrins which are linked to the membrane by a glycosylphosphatidylinositol-anchor (GPI-anchor) and the B-ephrins carrying a transmembrane domain (Eph nomenclature committee 1997).
  • GPI-anchor glycosylphosphatidylinositol-anchor
  • B-ephrins carrying a transmembrane domain
  • ephrin-A2 and ephrin-A5 Two members of the A-ephrins, ephrin-A2 and ephrin-A5, expressed in low anterior-high posterior gradients in the optic tectum, have recently been shown to be involved in repulsive guidance of retinal ganglion cell axons in vitro and in vivo (see, inter alia (Drescher, Cell 82 (1995), 359-70; Cheng, Cell 79 (1994), 157-168; Feldheim, Neuron 21 (1998), 563-74; Feldheim, Neuron 25 (2000), 563-74)
  • the technical problems underlying the present invention was to provide for means and methods for modifying altered developmental or cellular (migration) processes which lead to disease conditions.
  • the present invention relates to the use of an modulator of a polypeptide having or comprising the amino acid sequence of SEQ ID NOs.18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with angiogenic disorders or disorders of the cardio-vascular system and associated with tumor formation and tumor growth.
  • RGM repulsive guidance molecule
  • the present invention provides for the complete nucleotide sequence and/or amino acid of RGM (see, e.g. SEQ ID NO: 17 or 18 depicting the RGM sequence of chicken or SEQ ID NO: 20 to 25 depicting the human RGM homologues.)
  • RGM as pointed out herein above, is a glycoprotein, linked to membranes by a GPI-anchor.
  • Said GPI-anchor also carries a cross-reacting determinant (CRD) epitope and its carbohydrate part is able to bind peanut lectin.
  • CCD cross-reacting determinant
  • the RGM protein is a potent growth inhibitor and can assert neurite growth inhibition in picomolor concentrations.
  • modulator as employed herein relates to “inhibitors” as well as “activators” of RGM function. Most preferably said “modulation” is an inhibition, wherein said inhibition may be a partial or a complete inhibition.
  • amino acid sequence of SEQ ID NO: 18, 20, 23 or 25 as employed herein relates to the amino acid sequence of RGM (repulsive guidance molecule) and relates to the RGM polypeptide of chicken or human, respectively.
  • SEQ ID NOs: 20 and 21 depict human RGM1.
  • Human RGM1 has been localized on chromosome 15.
  • human RGMs comprise RGM2 and RGM3.
  • RGM2 is depicted in SEQ ID NO: 23 (amino acid sequence) and is encoded by a nucleotide sequence as shown in SEQ ID NO: 22. Human RGM2 has been localized on chromosome 5.
  • human RGM3 is shown in appended SEQ ID NO: 25 (amino acid sequence) and encoded by a nucleotide sequence as depicted in SEQ ID NO: 24. Human RGM3 is located on chromosome 1. Yet, as will be discussed herein below, said term relates also to further RGM homologues.
  • (poly)peptide means, in accordance with the present invention, a peptide, a protein, or a (poly)peptide which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds.
  • peptidomimetics of such RGM proteins/(poly)peptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention.
  • the present invention is not restricted to RGM from human, mouse or chicken and its inhibitors but also relates to the use of inhibitors of RGM or of RGM itself (or functional fragments or derivatives thereof) from other species. Since the present invention provides for the use of amino acid sequences/polypeptides of RGM and its corresponding inhibitors and since the amino acid sequences of human and chicken RGM are disclosed herein, the person skilled in the art is provided with the information to obtain RGM sequences from other species, like, inter alia, mouse, rat, pig, etc. The relevant methods are known in the art and may be carried out by standard methods, employing, inter alia, degenerate and non degenerate primers in PCR-techniques.
  • RGM RGM modulator
  • RGM-inhibitor also relates to RGM molecules (and their corresponding inhibitors) which are variants or homologs of the RGM molecules (and their inhibitors) as described herein.
  • Homology in this context is understood to refer in this context to a sequence identity of RGMs of at least 70%, preferably more than 80% and still more preferably more than 90% on the amino acid level.
  • the present invention comprises also (poly)peptides deviating from wildtype amino acid sequences of human or chicken RGMs described herein, wherein said deviation may be, for example, the result of amino acid and/or nucleotide substitution(s), deletion(s), addition(s), insertion(s), duplication(s), inversion(s) and/or recombination(s) either alone or in combination.
  • Those deviations may naturally occur or be produced via recombinant DNA techniques well known in the art.
  • the term “variation” as employed herein also comprises “allelic variants”. These allelic variations may be naturally occurring allelic variants, splice variants as well as synthetically produced or genetically engineered variants.
  • polynucleotide in accordance with the present invention comprises coding and, wherever applicable, non-coding sequences (like promotors, enhancers etc.). It comprises DNA, RNA as well as PNA.
  • polynucleotide/nucleic acid molecule comprises also any feasible derivative of a nucleic acid to which a nucleic acid probe may hybridize. Said nucleic acid probe itself may be a derivative of a nucleic acid molecule capable of hybridizing to said nucleic acid molecule or said derivative thereof.
  • nucleic acid molecule further comprises peptide nucleic acids (PNAs) containing DNA analogs with amide backbone linkages (Nielsen, Science 254 (1991), 1497-1500).
  • PNAs peptide nucleic acids
  • nucleic acid molecule which encodes a RGM (poly)peptide or a functional fragment/derivative thereof, in connection with the present invention, is defined either by (a) the specific nucleic acid sequences encoding said (poly)peptide specified in the present invention or (b) by nucleic acid sequences hybridizing under stringent conditions to the complementary strand of the nucleotide sequences of (a) and encoding a (poly)peptide deviating from the nucleic acid of (a) by one or more nucleotide substitutions, deletions, additions or inversions and wherein the nucleotide sequence shows at least 70%, more preferably at least 80% identity with the nucleotide sequence of said encoded RGM (poly)peptide
  • module as employed herein also comprises the term “inhibitor”, as mentioned herein above.
  • inhibitor of a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or a functional fragment or derivative thereof therefore, not only relates to the specific inhibitors of human or chicken RGM but also relates to inhibitors of RGM (or functional fragments or derivatives thereof) of other species.
  • Useful inhibitors are disclosed herein as well as described herein below and in the appended examples.
  • inhibitor also comprises “modulators” of the RGM polypeptides and/or the RGM encoding nucleic acid molecule/gene. In context of this invention it is also envisaged that said “modulation” leads, when desired, to an activation of RGM.
  • Functional fragments of the herein identified RGM molecules or RGM molecules of other species may be comprised in fusion and/or chimeric proteins.
  • “Functional fragments” comprise RGM fragments (or its encoding nucleic acid molecules) which are able to replace RGM full length molecules in corresponding assays (as disclosed herein in the appended examples, e.g.
  • polynucleotides encoding functional fragments of RGM and/or its derivatives have preferably at least 15, more preferably at least 30, more preferably at least 90, more preferably of at least 150, more preferably of at least 300 nucleotides.
  • derivative means in context of their invention derivatives of RGM molecules and/or their encoding nucleic acid molecules and refer to natural derivatives (like allelic variants) as well as recombinantly produced derivatives/variants which may differ from the herein described RGM molecules by at least one modification/mutation, e.g. at least one deletion, substitution, addition, inversion or duplication.
  • the term “derivative” also comprises chemical modifications.
  • derivative as employed herein in context of the RGM molecule also comprises soluble RGM molecules which do not comprise any membrane anchorage.
  • the present invention provides for the use of a modulator, preferably an inhibitor, of RGM molecules and/or their corresponding encoding polynucleotides/nucleic acid molecules for the preparation of a pharmaceutical composition for preventing, alleviating or treating various disorders of the nervous system, angiogenic disorders or disorders of the cardio-vascular system and malignancies of different etiology.
  • said disorders of the nervous system comprise degeneration or injury of vertebrate nervous tissue, in particular neurodegenerative diseases, nerve fiber injuries and disorders related to nerve fiber losses.
  • Said neurodegenerative diseases may be selected from the group consisting of motomeuronal diseases (MND), amyotrophic lateral sclerosis (ALS), Alzheimers disease, Parkinsons disease, progressive bulbar palsy, progressive muscular atrophy, HIV-related dementia and spinal muscular atrophy(ies), Down's Syndrome, Huntington's Disease, Creutzfeldt-Jacob Disease, Gerstmann-Straeussler Syndrome, kuru, Scrapie, transmissible mink encephalopathy, other unknown prion diseases, multiple system atrophy, Riley-Day familial dysautonomia
  • said nerve fiber injuries may be selected from the group consisting of spinal cord injury(ies), brain injuries related to raised intracranial pressure, trauma, secondary damage due to increased intracranial pressure, infection, infarction, exposure to toxic agents, malignancy and paraneoplastic syndromes and wherein said disorders related to nerve fiber losses may be selected from the group consisting of paresis of nervus facialis, nervus medianus, nervus ulnari
  • the above mentioned spinal cord and brain injuries not only comprise traumatic injuries but also relate to injuries caused by stroke, ischemia and the like. It is in particular envisaged that the inhibitors as defined herein below and comprising, inter alia, anti-RGM antibodies be employed in the medical art to stimulate nerve fiber growth in individuals, in particular in vertebrates, most preferably in humans.
  • the invention provides for the use of a modulator, preferably an inhibitor to RGM (or a functional fragment or derivative thereof) for the preparation of a pharmaceutical composition for the treatment of disorders of the cardio-vascular system, wherein these disorders, e.g., comprise disorders of the blood-brain barrier, brain oedema, secondary brain damages due to increased intracranial pressure, infection, infarction, ischemia, hypoxia, hypoglycemia, exposure to toxic agents, malignancy, paraneoplastic syndromes.
  • a modulator preferably an inhibitor to RGM (or a functional fragment or derivative thereof) for the preparation of a pharmaceutical composition for the treatment of disorders of the cardio-vascular system, wherein these disorders, e.g., comprise disorders of the blood-brain barrier, brain oedema, secondary brain damages due to increased intracranial pressure, infection, infarction, ischemia, hypoxia, hypoglycemia, exposure to toxic agents, malignancy, paraneoplastic syndromes.
  • RGM inhibitors may stimulate surviving neurons to project collateral fibers into the diseased tissue, e.g. the ischemic tissue.
  • RGM is expressed locally at the side of artificial transection of brain/spinal cord tissue in test animals (like rats), e.g., in the penumbra region surrounding an ischemic core of a human suffering focal ischemia in the temporal contex. Furthermore, it is documented in the appended examples that RGM is, surprisingly, expressed in tissue(s) having experienced from traumatic brain injuries.
  • the invention also relates to the use of a RGM polypeptide or a functional fragment or derivative thereof or the use of a polynucleotide encoding the same (polypeptides and polynucleotides as defined herein), wherein the above described disease or condition associated with seizures is epilepsy.
  • An epilepsy is thereby characterized by an epileptic seizure as a convulsion or transient abnormal event experienced by the subject, e.g. a human patient, due to a paroxysmal discharge of (cerebral) neurons.
  • the epileptic seizures comprise tonic seizures, tonic-clonic seizures (grand mal), myoclonic seizures, absence seizures as well as akinetic seizures.
  • Yet, also comprised are in context of this invention simple partial seizures, e.g. Jacksonian seizures and seizures due to perinatal trauma and/or fetal anoxia.
  • the uses described herein relate in particular to the preparation of pharmaceutical compositions for the treatment of diseases/conditions associated with aberrant sprouting of nerve fibres, like epilepsy, see also Routbort, Neuroscience 94 (1999), 755-765.
  • the modulator preferably the inhibitor of RGM (or of its functional fragment or derivative thereof or of its encoding nucleid acid molecule) is used for the preparation of a pharmaceutical composition for the modification of neovascularization.
  • Said modification may comprise activation as well as stimulation.
  • said neovascularisation be stimulated and/or activated in diseased tissue, like inter alia, ischemic and/or infarctious tissue.
  • the RGM-inhibitors described herein may be employed in the regulation of the blood-brain barrier permeability.
  • said modulators preferably said inhibitors for RGM be employed in the alleviation, prevention and/or inhibition of progression of vascular plaque formation (e.g. artherosclerosis) in cardio-vascular, cerebo-vascular and/or nephrovascular diseases/disorders.
  • vascular plaque formation e.g. artherosclerosis
  • the present invention provides for the use of a modulator, preferably an inhibitor of RGM as defined herein for the preparation of a pharmaceutical composition for remyelination. Therefore, the present invention provides for a pharmaceutical composition for the treatment of demyelinating diseases of the CNS, like multiple sclerosis or of demyelinating diseases like peripheral neuropathy caused by diphteria toxin, Landry-Guillain-Barré-Syndrom, Elsberg-Syndrom, Charcot-Marie-Tooth disease and other polyneuropatias.
  • a particular preferred inhibitor of RGM in this context is an antibody directed against RGM, e.g. an IgM antibody.
  • IgM antibodies against RGM are known in the art and comprise e.g. the F3D4 described in the appended examples.
  • the invention provides for the use of a RGM polypeptide as defined herein or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the activity of autoreactive immune cells or with overactive inflammatory cells. Most preferably these cells are T-cells.
  • the present invention relates to the use of a modulator, preferably an inhibitor or another RGM binding molecule of a RGM polypeptide or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or of fragment/derivative thereof for modifying and/or altering the differentiation status of neuronal stem cells and/or their progenitors.
  • Said stem cells are normally found in the subventricular zones of many brain regions. It is known that factors in the microenvironment of the brain dramatically influence the differentiation of undifferentiated stem cells. It is assumed that due to the characteristic expression of RGM in the subventricular layers of many different brain regions, this molecule could be a marker for stem cells.
  • RGM inhibitors like antibodies could be useful markers for stem cells. Most important in stem cell biology is the understanding of factors influencing their differentiation. It is therefore assumed that RGM inhibitors change the developmental fate of these cells.
  • RGM is not only expressed in ischemic tissue but is also expressed in scar tissue surrounding (brain) lesions.
  • the modulator preferably the inhibitor of the RGM molecule (or its functional fragment or derivative) is an antibody or a fragment or a derivative thereof, is an aptamer, is a specific receptor molecule capable of interacting with a RGM polypeptide or with a functional fragment or derivative thereof, or is a specific nucleic acid molecule interacting with a polynucleotide encoding an RGM and/or the polypeptide as defined herein.
  • the antibody to be used in context of the present invention can be, for example, polyclonal or monoclonal antibodies. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. The production of specific anti-RGM antibodies is further known in the art (see, e.g. Müler (1996) loc.cit.) or described in the appended examples.
  • antibody as employed herein also comprises chimeric, single chain and humanized antibodies, as well as antibody fragments, like, inter alia, Fab fragments.
  • Antibody fragments or derivatives further comprise F(ab′) 2 , Fv or scFv fragments; see, for example, Harlow and Lane, loc.cit.
  • F(ab′) 2 , Fv or scFv fragments see, for example, Harlow and Lane, loc.cit.
  • the (antibody) derivatives can be produced by peptidomimetics.
  • techniques described for the production of single chain antibodies see, inter alia, U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to polypeptide(s) of this invention.
  • transgenic animals may be used to express humanized antibodies to polypeptides of this invention.
  • the antibody to be used in the invention is a monoclonal antibody, for example the F3D4 antibody described in the appended examples may be employed when an IgM is desired.
  • the general methodology for producing, monoclonal antibodies is well-known and has been described in, for example, Köhler and Milstein, Nature 256 (1975), 494-496 and reviewed in J. G. R. Hurrel, ed., “Monoclonal Hybridoma Antibodies Techniques and Applications”, CRC Press Inc., Boco Raron, Fla. (1982), as well as that taught by L. T. Mimms et al., Virology 176 (1990), 604-619.
  • said antibodies are directed against functional fragments of the RGM polypeptide.
  • functional fragments are easily deducible for the person skilled in the art and, correspondingly, relevant antibodies (or other inhibitors) may be produced.
  • the “modulator”, preferably the “inhibitor” as defined herein may also be an aptamer.
  • Aptamers are well known in the art and, inter alia, described in Famulok, Curr. Op. Chem. Biol. 2 (1998), 320-327.
  • the preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold, Ann. Rev. Biochem. 64 (1995), 763-797). Said other receptors may, for example, be derived from said antibody etc. by peptidomimetics.
  • Said specific “receptor” molecules which may function as inhibitors of the RGM polypeptides are also comprised in this invention.
  • Said specific receptors may be deduced by methods known in the art and comprise binding assays and/or interaction assays. These may, inter alia, involve assays in the ELISA-format or FRET-format.
  • Said “inhibitor” may also comprise specific peptides binding to and/or interfering with RGM.
  • the above recited “modulator”, preferably “inhibitor” may function at the level of RGM gene expression. Therefore, the inhibitor may be a (specific) nucleic acid molecule interacting with a polynucleotide encoding a RGM molecule (or a functional fragment or derivative thereof.) These inhibitors may, e.g., comprise antisense nucleic acid molecules or ribozymes.
  • the nucleic acid molecule encoding RGM may be employed to construct appropriate anti-sense oligonucleotides.
  • Said anti-sense oligonucleotides are able to inhibit the function of wild-type (or mutant) RGM genes and comprise, preferably, at least 15 nucleotides, more preferably at least 20 nucleotides, even more preferably 30 nucleotides and most preferably at least 40 nucleotides.
  • Ribozyme approaches are also envisaged for use in this invention. Ribozymes may specifically cleave the nucleic acid molecule encoding RGMs.
  • ribozymes comprise, inter alia, hammerhead ribozymes, hammerhead ribozymes with altered core sequences or deoxyribozymes (see, e.g., Santoro, Proc. Natl. Acad. Sci. USA 94 (1997), 4262) and may comprise natural and in vitro selected and/or synthesized ribozymes.
  • Nucleic acid molecules according to the present invention which are complementary to nucleic acid molecules coding for proteins/(poly)peptides regulating, causing or contributing to obesity and/or encoding a mammalian (poly)peptide involved in the regulation of body weight (see herein below) may be used for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) which specifically cleave nucleic acid molecules of the invention. Selection of the appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith, eds. Academic Press, Inc. (1995), 449-460.
  • Said “inhibitor” may also comprise double-stranded RNAs, which lead to RNA-mediated gene interference (see Sharp, Genes and Dev. 13 (1999), 139-141)
  • RGM RGM inhibitors
  • interaction assay and employing corresponding read-out systems.
  • These are known in the art and comprise, inter alia, two hybrid screenings (as, described, inter alia, in EP-0 963 376, WO 98/25947, WO 00/02911) GST-pull-down columns, co-precipitation assays from cell extracts as described, inter alia, in Kasus-Jacobi, Oncogene 19 (2000), 2052-2059, “interaction-trap” systems (as described, inter alia, in U.S. Pat. No. 6,004,746) expression cloning (e.g.
  • a further objective of the present invention is to provide for the use of a RGM polypeptide and/or of polypeptide having or comprising the amino acid sequence of SEQ ID NOs. 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with excessive collateral sprouting of nerve fibres.
  • the present invention therefore, provides for the medical use of RGM protein(s) and/or functional fragments/derivatives thereof or for the use of polynucleotides encoding said RGM protein(s) in conditions where excessive collateral sprouting occurs.
  • Said conditions comprise, but are not limited to, epilepsy, phantom pain and neuropathic pain.
  • epilepsy phantom pain and neuropathic pain.
  • McNamara Naat. Suppl. 399 (1999), A15-A22
  • the RGM molecule either naturally isolated or recombinantly produced, or its functional fragments/derivatives may therefore be employed as potent “stop” signals for growing nerve fibres. The feasibility of such an approach has been shown by Tanelian (Nat. Med. 3 (1997), 1398-1401) who employed a semaphorin for inhibition of nerve fiber growth.
  • the present invention provides for the use of RGM and/or of a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing or treating tumor growth or formation of tumor metastases.
  • RGM naturally isolated or recombinantly produced
  • RGM functional fragments thereof may be employed for the preparation of a pharmaceutical composition for the treatment of neoplastic disorders, in particular of disorders related to tumor (cell) migration, metastasis and/or tumor invasion.
  • RGM inhibits undesired neovascularisation. Said neovascularisation, as an angiogenic disorder during neoplastic events, should be prevented in order to limit, inter alia, tumor growth.
  • RGM and/or its functional fragments may be employed to actively stimulate withdrawal of lamellipodia of tumor cells and/or to induce their collapse. As demonstrated in the appended examples RGM also influences tumor growth behaviour, i.e. is capable of negatively influencing tumor growth.
  • the invention provides for the use of a RGM polypeptide as defined herein or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the activity of autoreactive immune cells or with overactive inflammatory cells. Most preferably these cells are T-cells.
  • the invention provides for the use of a RGM polypeptide having or comprising, inter alia, the amino acid sequence of SEQ ID NOs.18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for the treatment of inflammation processes and/or allergies, for wound healing or for the suppression/alleviation of scar formation.
  • Scar tissue is formed by invading cells, most importantly by fibroblasts and/or glial cells. Migration and adhesion of these cells are required to get to the lesion side.
  • RGM or an active fragment/derivative could prevent accumulation of these cells in the lesion side, thereby preventing or slowing down scar formation.
  • In inflammatory reactions cells migrate to the inflamed region and RGM or its active fragment/derivative prevent or reduce migration of these cells to the side of inflammation, thereby preventing overactive inflammatory reactions.
  • the term “pharmaceutical composition” also comprises optionally further comprising an acceptable carrier and/or diluent and/or excipient.
  • the pharmaceutical composition of the present invention may be particularly useful in preventing and/or treating pathological disorders in vertebrates, like humans.
  • Said pathological disorders comprise, but are not limited to, neurological, neurodegenerative and/or neoplastic disorders as well as disorders associated with seizures, e.g. epilepsy.
  • These disorders comprise, inter alia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (FALS/SALS), ischemia, stroke, epilepsy, AIDS dementia and cancer.
  • the pharmaceutical composition may also be used for prophylactic purposes.
  • Suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. However, it is also envisaged that the pharmaceutical compositions are directly applied to the nervous tissue. The dosage regimen will be determined by the attending physician and clinical factors.
  • dosages for any one patient depends upon many factors, including the patient's size, body surface area, general health, age, sex, the particular compound to be administered, time and route of administration, and other drugs being administered concurrently.
  • Pharmaceutically active matter may be present preferably, inter alia, in amounts between 1 ng and 1000 mg per dose, more preferably in amounts of 1 ng to 100 mg however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment.
  • the compositions of the invention may be administered locally or systemically.
  • compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents, depending on the intended use of the pharmaceutical composition.
  • agents may be drugs acting on the central nervous system as well as on small, unmyelinated sensory nerve terminals (like in the skin), neurons of the peripheral nervous system of the digestive tract., etc.
  • compositions as defined herein may comprise nucleic acid molecules encoding RGMs (and/or functional fragments or derivatives thereof) or corresponding RGM inhibitors or defined herein.
  • said inhibitors comprise, but are not limited to, antibodies, aptamer, RGM-interacting peptides as well as inhibitors interacting with the RGM-encoding polynucleotides.
  • the present invention also provides for a method of treating, preventing and/or alleviating pathological disorders and conditions as defined herein, whereby said method comprises administering to a subject in need of such a treatment a pharmaceutical composition/medicament as defined herein.
  • a pharmaceutical composition/medicament as defined herein.
  • said subject is a human.
  • the nucleic acid molecules may be particularly useful in gene therapy approaches and may comprise DNA, RNA as well as PNA. Said nucleic acid molecules may be comprised in suitable vectors, either inter alia, gene expression vectors.
  • suitable vectors may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the vectors may, in addition to the nucleic acid sequences encoding RGM or its corresponding inhibitors, comprise expression control elements, allowing proper expression of the coding regions in suitable host cells or tissues.
  • expression control elements are known to the artisan and may include a promoter, translation initiation codon, translation and insertion site for introducing an insert into the vector.
  • the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in (eukaryotic) cells. Particularly preferred are in this context control sequences which allow for correct expression in neuronal cells and/or cells derived from nervous tissue.
  • Control elements ensuring expression in eukaryotic cells are well known to those skilled in the art. As mentioned above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human elongation factor 1 ⁇ -promoter, CMV enhancer, CaM-kinase promoter or SV40-enhancer.
  • regulatory elements For the expression for example in nervous tissue and/or cells derived therefrom, several regulatory sequences are well known in the art, like the minimal promoter sequence of human neurofilament L (Charron, J. Biol. Chem 270 (1995), 25739-25745). Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (In-Vitrogene, as used, inter alia in the appended examples), pSPORTI (GIBCO BRL) or pGEMHE (Promega), Beside the nucleic acid molecules defined herein, the vector may further comprise nucleic acid sequences encoding for secretion signals. Such sequences are well known to the person skilled in the art.
  • leader sequences capable of directing the protein/(poly)peptide to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a part thereof,
  • said vector may also be, besides an expression vector, a gene transfer and/or gene targeting vector.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
  • Suitable vectors, vector systems and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res.
  • said vectors and/or gene delivery systems are also described in gene therapy approaches in neurological tissue/cells (see, inter alia Blömer, J. Virology 71 (1997) 6641-6649) or in the hypothalamus (see, inter alia, Geddes, Front Neuroendocrinol. 20 (1999), 296-316 or Geddes, Nat. Med. 3 (1997), 1402-1404).
  • Further suitable gene therapy constructs for use in neurological cells/tissues are known in the art, for example in Meier (1999), J. Neuropathol. Exp. Neurol. 58, 1099-1110.
  • the nucleic acid molecules and vectors of the invention may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g.
  • ballistic e.g. gene gun
  • a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules described herein.
  • treatment used herein to generally mean obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease.
  • treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.
  • the present invention provides for the use of a (RGM) polypeptide and/or a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative as a marker of stem cells. Since it is envisaged that stem cells as well as their undifferentiated progenitor cells express RGM, RGM and/or functional fragments or derivatives thereof may be employed to influence the differentiation/differentiation pattern of said stem cells.
  • antibodies directed against RGMs in particular directed against polypeptides disclosed herein or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 (or (a) functional fragment(s)/derivative(s) thereof) may be employed to influence the differentiation of (neuronal) stem cells and (neuronal) progenitor cells. It is particularly preferred that said antibodies (as well as other RGM-inhibitors and/or RGM-binding molecules) be employed to selectively label stem cells. Therefore these reagents may be employed as markers for stem cells. It is also envisaged that peptides or derivatives be employed in said purpose.
  • the polypeptide and/or fragment thereof which comprises or has an amino acid sequence as depicted in SEQ ID NOs 18, 20, 23 or 25 and/or is a RGM molecule to be used in accordance with their invention is a soluble, i.e. not membrane bound molecule.
  • ephrins As shown in Davis (1994), Science 266, 816-819 ephrins, in particular A-ephrins, are not active in soluble, monomeric form. In contrast, soluble RGMs are active and may function without any membrane-attachment RGM, in contrast to ephrins, is capable of self-formation of dimers and/or of the formation of higher aggregates.
  • the invention also provides for the use of a RGM molecule and/or a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or a fragment or a derivative for the preparation of a pharmaceutical composition for alleviating, preventing and/or treating homeostatic and/or bleeding disorders and/or vascular damage.
  • RGMs may, due to their structural homology to von-Willebrand factor (vWF), be employed in the treatment of said disorders/diseases. Furthermore, it is envisaged that RGM may interact with von-Willebrand factor and that said molecule, thereby, influences the activity of vWF. Furthermore, the inhibitors as defined herein should be employed in disorders where immune cells invade the brain, like multiple sclerosis, encephalomyelitis disseminata.
  • vWF von-Willebrand factor
  • the present invention also provides for the use of an antibody or a fragment or a derivative thereof, or an aptamer, or a binding molecule capable of interacting with a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or with functional fragment or derivative thereof or of an nucleid acid molecule capable of interacting with a polynucleotide encoding said polypeptide or a fragment thereof for the preparation of a diagnostic composition for detecting neurological and/or neurodegenerative disorders or dispositions thereto.
  • the diagnostic composition may be used, inter alia, for methods for determining the expression of the nucleic acids encoding RGM polypeptides by detecting, inter alia, the presence of the corresponding mRNA which comprises isolation of RNA from a cell, contacting the RNA so obtained with a nucleic acid probe as described above under hybridizing conditions, and detecting the presence of mRNAs hybridized to the probe.
  • RGM (poly)peptides can be detected with methods known in the art, which comprise, inter alia, immunological methods, like, ELISA or Western blotting.
  • the diagnostic composition of the invention may be useful, inter alia, in detecting the prevalence, the onset or the progress of a disease related to the aberant expression of a RGM polypeptide. Accordingly, the diagnostic composition of the invention may be used, inter alia, for assessing the prevalence, the onset and/or the disease status of neurological, neurodegenerative and/or inflammatory disorders, as defined herein above. It is also contemplated that anti-RGM antibodies, aptamers etc. and compositions comprising such antibodies, aptamers, etc. may be useful in discriminating (the) stage(s) of a disease.
  • the diagnostic composition optionally comprises suitable means for detection.
  • the nucleic acid molecule(s), vector(s), antibody(ies), (poly)peptide(s), described above are, for example, suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble or insoluble for the purposes of the invention.
  • Solid phase carriers are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes.
  • Suitable methods of immobilizing nucleic acid molecule(s), vector(s), host(s), antibody(ies), (poly)peptide(s), fusion protein(s) etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions and the like.
  • immunoassays which can utilize said compounds of the invention are competitive and non-competitive immunoassays in either a direct or indirect format.
  • Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods.
  • immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Northern or Southern blot assay.
  • these detection methods comprise, inter alia, IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay).
  • the diagnostic compounds of the present invention may be are employed in techniques like FRET (Fluorescence Resonance Energy Transfer) assays.
  • nucleic acid sequences encoding RGMs of other species as well as variants of RGMs are easily deducible from the information provided herein. These nucleic acid sequences are particularly useful, as pointed out herein above, in medical and/or diagnostic setting, but they also provide for important research tools. These tools may be employed, inter alia, for the generation of transgenic animals which overexpress or suppress RGMs or wherein the RGM gene is silenced and/or deleted. Furthermore, said sequences may be employed to detect and/or ellucidate RGM interaction partners and/or molecules binding to and/or interfering with RGMs.
  • FIG. 1 RGM protein fractions induce collapse of RGC growth cones.
  • FIG. 2 Comparative two dimensional gel analysis of tectal proteins and RGM sequences.
  • FIG. 3 Nucleotide and amino acid sequence of RGM.
  • FIG. 4 The polyclonal and the monoclonal RGM antibody recognize the same 33 kDa protein.
  • FIG. 5 The RGM anti-sense probe hybridizes to an mRNA with graded expression along the anterio-posterior axis.
  • FIG. 6 Recombinant RGM induces collapse of retinal growth cones.
  • FIG. 7 Recombinant RGM guides temporal retinal axons in the stripe assay.
  • FIG. 8 RGM staining in endothel of (human) brain.
  • FIG. 9 RGM expression in a lesion of a human being deceased due to severe brain injury (1-2 hours after his death). RGM expression on infiltrating cells from the immune system.
  • FIG. 10 RGM expression in a brain lesion (human).
  • RGM in contrast to the A-ephrins, bound to a strong cation exchanger and was eluted at a salt concentration of 200-400 mM NaCl.
  • RGM-fractions fractions 4+5, FIG. 1
  • RGM-free fractions fraction 6, FIG. 1 .
  • ephrin-A5 nor ephrin-A2 was present in these fractions, proving thereby that RGM function does not require presence of the A-ephrins.
  • Tecta from 100 chick embryos were isolated and were divided into three parts equal in length along the anterior-posterior axis. The middle tectal parts were discarded and the anterior and posterior parts were worked up separately. Membranes were washed with PBS containing protease inhibitors and were centrifuged. Tectal membrane pellets were resuspended in triethanolamin buffer and were treated with the enzyme PI-PLC (50 mU Boehringer Mannheim/Roche Diagnostics GmbH), to remove glycosylphosphatidylinositol-anchored (GPI-anchored) proteins from the membranes. No PI-PLC was added to the other anterior and posterior tectal membrane fractions, the control-fractions (C).
  • PI-PLC 50 mU Boehringer Mannheim/Roche Diagnostics GmbH
  • Enzyme (E) and control (C) fractions were incubated at 37° C. for 1.5 hours and membrane suspensions were centrifuged at 400.000 xg in a Beckmann TLA 100.3 rotor. Supernatants were collected and their protein concentrations were determined (Bradford 1976, modified by Zor and Selinger, 1996). Supernatants were precipitated with ice cold 10% trichloroacetic acid, were centrifuged and protein pellets were washed in ethanol-ether (1:1 v/v) and solubilized in sample buffer (8.5 M urea, 5% ⁇ -mercaptoethanol, 2.5% ampholytes pH 3-10, 2% NP 40).
  • E fractions and C fractions were loaded onto two dimensional gels, and after silver staining candidate proteins in the E-fractions ( FIG. 2A , arrows) were cut out and subject to in gel tryptic digestion and nanoelectrospray ionization (Wilm, Nature 379 (1996), 466-9).
  • Tectal proteins resuspended in sample buffer were separated using two dimensional gel electrophoresis. 20 ⁇ g of tectal protein was loaded on each gel. Non-equilibrium pH gradient electrophoresis (NEPHGE) followed by SDS-PAGE in the second dimension was performed as described by Boxberg (1988). After the SDS PAGE, gels were stained by a modified silver staining protocol from Heukeshoven and Demick (Heukeshoven & Demick, Electrophoresis 9, (1988), 372-375).
  • Silver-stained proteins in the 2D gels with a basic isoelectric point and a molecular weight of 33/35 kDa, present in the PI-PLC treated E fraction but not in the C fraction, were cut out using a sharp and sterile scalpel.
  • Microsequencing was done using the technique of nano-electrospray tandem mass spectrometry as previously described (Wilm et al., 1996).
  • the protein spots were digested in gel by trypsin and the resulting peptides were adsorbed and stepwise eluted into the electrospray source for mass spectral analysis.
  • Nanoelectrospray was performed on an API III (Perkin-Elmer) mass spectrometer as described by Wilm and Mann (Wilm & Mann, Anal. Chem. 68 (1996), 1-8). After selecting an ionized peptide from the peptide mixture, the peptide was fragmented and the peptide fragments were analysed.
  • Mass spectrometric microsequencing of ionized peptides from the spot marked by arrows in FIG. 2 yielded ten different peptides, with lengths of 5-14 amino acids as shown in FIG. 2B ; (SEQ ID NOs 1-10).
  • the selected spot was present in anterior and posterior PI-PLC supernatantes at similar levels.
  • RGM is however more abundant in posterior than in anterior tectal membranes and the disappearance of the ap-difference in the 2D-gels was most likely caused by two different proteins unrelated to RGM and present in the selected spot.
  • P1F (SEQ ID NO: 11) 5′-ATGCC(AGCT)GA(AG)GA(AG)GT(AGCT)GT(AGCT)-3′ P1R: (SEQ ID NO: 12) 5′-TT(AGCT)AC(AGCT)AC(CT)TC(CT)TC(AGCT)GGCAT-3′ P2F: (SEQ ID NO: 13) 5′-GA(CT)AC(AGCT)TT(CT)CA(AG)AC(AGCT)TG(CT)AA-3′ P2R: (SEQ ID NO: 14) 5′-TT(AG)CA(AGCT)GT(CT)TG(AG)AA(AGCT)GT(AG)TC-3′ P3F: (SEQ ID NO: 15) 5′-AA(CT)CA(AG)CA(AG)(CT)T(AGCT)GA(CT)TT(CT)CA-3′ P3R: (SEQ ID NO: 16) 5′-TG(AG)AA(AG)TC(AGCT)A(AG)(CT)TG(
  • Moloney murine leukemia virus reverse transcriptase and random hexamer primers were used to synthesize single-stranded cDNA from E9 chick tectum total RNA. Combinations of forward (F) and reverse (R) primers were added to the cDNA and PCR amplification was done using Taq polymerase. The following PCR conditions were used: an initial denaturation step at 95° C. for 5 min followed by 30 cycles of 95° C. for 40 s, 50° C. for 1 min, 72° C. for 2 min. The PCR products were cloned into the pGEM T vector (Promega) and four positive clones were sequenced using the ALF express sequencer (Pharmacia).
  • the 459 by fragment was used for screening a cDNA library to obtain the full length sequence and for further analysis such as Northern blotting and in situ hybridization.
  • PCR products were loaded onto agarose gels stained with ethidium bromide and a PCR product of 459 by in length, was obtained and cloned into the pGEM T vector. After sequencing, most of the peptide sequences were found in the PCR product, confirming that the correct candidate was amplified.
  • the 459 by fragment was used for screening an E14 chicken brain cDNA library. Positive clones contained an insert of approximately 4 kb and sequencing confirmed the presence of the 459 by fragment and additional downstream sequences, including a stop codon. Upstream sequences were obtained by performing 5′-RACE.
  • the 459 by probe was used to screen 500.000 plaques of an E14 chicken brain library, cloned in the ⁇ Zap vector. After two screening rounds, eight single plaques were isolated and the related inserts were cloned into the Bluescript vector using the rapid excision kit (Stratagene). The positive clones, analysed by restriction digestions, contained an insert of approximately 4 kb and sequencing confirmed the presence of the 459 by fragment and additional downstream sequences, including a stop codon. To get the sequence of the region upstream of the 459 by fragment, a 5′-RACE was performed according to the manufacturer's protocol using the RACE kit from Boehringer Mannheim and total RNA from E9 chick tecta.
  • a 700 by band was amplified, purified, cloned into pGEM T vector, and 5 positive clones were sequenced.
  • the sequence had an ORF with two methionines which could act as potential start sites.
  • the full length sequence of RGM was confirmed independently several times.
  • the 459 by fragment was cloned into the Bluescript KS vector (Stratagene) and anti-sense and sense probes were produced by using the SP6 and T7 polymerases, respectively.
  • This 5′-RACE yielded an ORF with two methionines, potential start sites.
  • the complete ORF of RGM is 1302 nucleotides in length and encodes a protein consisting of 434 amino acids ( FIG. 3A ; SEQ ID NO:17).
  • Two hydrophobic domains are present at the N-terminus and C-terminus, respectively ( FIG. 3B ; SEQ ID NO:18), and two different algorithms suggested that the N-terminal hydrophobic domain encodes a signal peptide (best cleavage site predicted: at aa 29), the C-terminal domain, a GPI-anchor domain (best cleavage site predicted: at aa 406).
  • RGM has no significant homology to any other protein, present in the databases and does not carry any specific domain or motif, except an triamino acid motif, the RGD site, a potential cell attachment site (Ruoshlahti, Annu. Rev. Cell Dev. Biol. 12 (1996), 697-715). Preliminary results suggest that this site is dispensable for RGM function.
  • Polyclonal antibodies named anti-RGM1 (against aas: 276-293) and anti-RGM2 (against aas: 110-130), raised against two peptides of the recombinant RGM molecule, recognize a GPI-anchored 33 kDa molecule, which is present at higher levels in posterior than in anterior tectal membrane PI-PLC supernatants ( FIG. 4A ). In membrane fractions at least three protein bands appear, a double band at 33 kDa and a single band at 35 kDa. These protein bands are recognized by the polyclonal anti-RGM1 antibody and the monoclonal F3D4 antibody (Müller (1996), loc. cit) ( FIG. 4B ).
  • RGM is the first member of a new class of axon guidance molecules, sharing no sequence homology with ephrins, netrins, slits, semaphorins and any other axon guidance molecules.
  • the corresponding human RGM sequence (SEQ ID NO:20) could be deduced by screening the human genome database with the deduced chicken RGM sequence.
  • RGM-mRNA in the tectum opticum was used in in situ hybridization experiments on cryostat sections from E9 chick tecta. Strongest staining is observed in the periventricular layer, surrounding the tectal ventricle and staining intensity is much stronger in posterior tectum than in anterior tectum ( FIG. 5A , B). Cell bodies of radial glial cells are located in the periventricular layer and the staining pattern confirms previous data using the monoclonal F3D4 antibody, where staining of glial endfeet and of glial cell bodies was observed (Mueller; (1996), loc. cit.; Mueller, (1997), loc. cit.).
  • tectal neurons are RGM-positive. This is in line with the expression of RGM by a subpopulation of tectal neurons.
  • the staining pattern with the RGM anti-sense probe looks very similar to the expression pattern of ephrin-A5 with both messages being found in a periventricular and in a more superficial tectal layer. No staining is detectable with the RGM-sense probe.
  • the RGM anti-sense probe marked two transcripts at 5.5 and 6.1 kb. Both messages are down-regulated at E14 with the smaller message being no longer detectable and the larger transcript being clearly present, albeit at lower levels.
  • RGM is active in in vitro assays and shows a graded expression in the tectum opticum of vertebrates. Based on Southern blot data it is assumed that there are least two additional family members which might have similar guidance activity. (see FIG. 11 )
  • Recombinant RGM is Active in Collapse and Stripe Assay
  • the full length RGM cDNA was used to transfect cos-7 cells with a lipofection procedure.
  • the full length RGM cDNA was cloned into the Kpnl site of the expression vector pTriEx-1 (Novagen).
  • Cos-7 cells were transfected with the pTriEx-1 plasmid containing RGM cDNA or with the empty plasmid using the Superfect transfection reagent (Qiagen) according to the manufacturer's protocol.
  • the DNA-Superfect mixture was added to Cos-7 cells growing in 10 cm dishes. 2 hours later medium was removed, cells were washed with PBS and grown for an additional 48 hours in fresh medium.
  • Conditioned medium was collected, run over an RGM-affinity column and RGM-containing fractions and RGM-free control fractions were directly used in collapse assay experiments.
  • RGM-transfected Cos-7 cells and empty plasmid transfected cells were washed with PBS and harvested using a rubber policeman in the presence of homogenization buffer containing protease inhibitors.
  • Conditioned medium of cos-7 cells transfected with the RGM-pTriEx-1 plasmid was collected and run over an anti-RGM1 antibody column. Eluted fractions were evaluated with a sensitive and rapid dot blot assay and RGM-positive fractions were added to retinal axons growing on a laminin substratum.
  • soluble RGM induced collapse of 90% of temporal RGC growth cones ( FIG. 6A ).
  • Recombinant RGM is active in soluble form, is a strong difference between RGM and the A-ephrins and suggests a role for a chemotropic mechanism, in etablishing the retinotectal map.
  • Membrane carpets consisted of lanes of anterior tectal membranes mixed with membranes from RGM-transfected cos cells (ratio: 1:1), alternating with lanes consisting of anterior membranes mixed with membranes from empty plasmid transfected cos cells (ratio 1:1).
  • membrane carpets consisting of alternating lanes of membranes from RGM-transfected cos cells and of control cos membranes, were incubated for 2 hours at 37° C. with 20 ⁇ g/ml laminin (Becton-Dickinson). Before use, the carpets were washed with Hank's buffer (2 ⁇ ).
  • RGM shares with the A-ephrins A2 and A5 the GPI-anchor, the graded expression and functional activity in two different in vitro systems. Its activity is however different from the two A-ephrins in other respects.
  • the specificity of its activity is not restricted to temporal axons and growth cones. Nasal axons and growth cones also react, albeit at higher RGM concentrations. This is in line with the previous observations, that temporal retinal axons react more strongly to RGM than nasal retinal axons (Stahl, (1990), loc.cit).
  • RGM functional relationship of RGM with ephrin-A2 and ephrin-A5 and the in vivo role of RGM need to be addressed, especially since both ephrins have been shown to be important molecular determinants for topographic map formation in vertebrates (Nakamoto, Cell 86 (1996), 755-66; Frisen, Neuron 20 (1998), 235-43; Feldheim, Neuron 21 (1998), 563-74; Picker, Development 126 (1999), 2967-78; Feldheim, Neuron 25 (2000), 563-74; Brown, Cell 102 (2000), 77-88).
  • the zebrafish mutant acerebella is mutant in fgf8 and lacks the midbrain-hindbrain boundary region and the cerebellum (Reifers, Development 125 (1998), 2381-95; Picker, (1999), loc. cit.).
  • the tectum is much smaller in ace mutants than in wildtype and the expression levels of all three zebrafish A-ephrins are changed with ephrin-A2 and ephrin-A5a being still expressed at low and anterior levels in ace tecta and with ephrin-A5b being completely eliminated (Picker et al., 1999).
  • RGM with its graded expression along the anterior-posterior axis of the tectum and its ability to function in a secreted and membrane-coupled way, is an important player for topographic map formation.
  • Infarctioned brain tissue was derived from an updated stroke and trauma brain-bank (Table 1,2) reported previously (Postler et al., 1997, Beschomer et al., 2000). Tissue specimen procurement was performed according to the ethical guidelines of the University of Tuebingen. Patients with altered immune status because of immunosuppressive therapy or meningitis/encephalitis were excluded from this study. As controls, the results were compared to tissue from corresponding areas of 4 normal non-ischemic brains described previously (Schwab et al., 2000).
  • haematoxyline-eosine (HE), luxol fast blue (LFB) and iron (Fe) staining was used for evaluation of the typical histological features defined as standard indication of infarct (Kalimo et al., 1996) and trauma age (Graham and Gennarelli, 1996).
  • Monospecific polyclonal antibodies directed against RGM were diluted (1:10) in 1% BSA (bovine serum albumin) TBS (Tris-balanced salt solution, containing 0.025 M Tris, 0.15 M NaCl) and incubated over night at room temperature. Specific binding of the antibodies were detected with a secondary biotinylated swine anti-rabbit IgG F(ab) 2 antibody fragment 1:400 for 30 min (DAKO, Hamburg, FRG), followed by incubation with a peroxidase conjugated streptavidin-biotin complex (DAKO, Hamburg, FRG). The enzyme was visualized with diaminobenzidine as a chromogen (Fluka, Neu-Ulm, FRG).
  • BSA bovine serum albumin
  • TBS Tris-balanced salt solution, containing 0.025 M Tris, 0.15 M NaCl
  • Sections were counterstained with Mayer's Hemalaun. Negative controls consisted of sections incubated in the absence of the primary antibody. Specificity of polyclonal RGM antibody was confirmed by inhibition of staining using human ischemic brain tissue after pre-incubation for 3 h on ice with access of the cognate RGM peptide.
  • a cell-type or activation specific antigen was first labelled using the ABC procedure in combination with alkaline phosphatase conjugates.
  • Specific antigens were labelled with antibodies against GFAP (glial fibrillary acidic protein, monoclonal, Boehringer Mannheim, Germany, 1:100) to detect astrocytes, MBP (myelin basic protein, polyclonal, oligodendrocytes, Dako, 1:500) and CD68 (Dako, 1:100) for microglia/macrophage identification.
  • GFAP glial fibrillary acidic protein, monoclonal, Boehringer Mannheim, Germany, 1:100
  • MBP myelin basic protein, polyclonal, oligodendrocytes, Dako, 1:500
  • CD68 Dako, 1:100
  • Activated microglia/macrophages were detected with antibodies directed against HLA-DR, -DP, -DQ (MHC class II, DAKO, Glostrup, Denmark, 1:100) or MRP-8 (8-5C2, BMA, Augst, Switzerland, 1:100) (Postler et al., 1997). Lymphocytic subpopulations were classified with monoclonal antibodies against CD4 (T-helper lymphocytes, 1:10, Dako) and CD8 (T cytotoxic/suppressor lymphocytes, 1:500, Dako) and CD20 (pan B cell marker, 1:200, Dako).
  • CD4 T-helper lymphocytes, 1:10, Dako
  • CD8 T cytotoxic/suppressor lymphocytes, 1:500, Dako
  • CD20 pan B cell marker, 1:200, Dako
  • mice laminin (1:500, Chemicon) antibodies were used and rabbit fibronectin (1:100, Dako) antibodies were used to detect matrix deposition.
  • S phase specific PCNA proliferating cell nuclear antigen, 1:100, Dako
  • slices were deparaffinized, irradiated in a microwave oven for antigen retrieval and incubated with non specific porcine serum as described above. Visualization was achieved by adding biotinylated secondary antibodies (1:400) for 30 min and alkaline phosphatase conjugated ABC complex diluted 1:400 in TBS-BSA for 30 min.
  • slices were developed with Fast-Blue BB salt chromogen-substrate solution yielding a blue reaction product. Between double labelling experiments, slices were irradiated in a microwave for 5 min in citrate buffer. Then RGM was immunodetected as described above.
  • Border zones were defined as peri-lesional areas adjacent to the developing necrotic core demarcating the region of major damage.
  • RGM + cells were counted in ten high power fields (HPF, ⁇ 200 magnification with an eye-piece-grid representing 0.25 mm 2 ).
  • FCI focal cerebral infarctions
  • TBI traumatic brain injury
  • RGM immunoreactivity was detected on white matter fibres, oliogodendrocytes, the perikarya of some neurons and RGM + cells were also detected in the choroid plexus ( FIG. 8 ) and ependyma. Only single cells were detected in peri-vascular spaces. Further, some smooth muscle cells and few endothelial cells but no astrocytes were labelled.
  • FCI Focal Cerebral Ischemia
  • RGM expression was lesion-associated.
  • Cellular RGM expression was confined to neurons, few reactive astrocytes and invading leukocytes. With the ageing of the lesions, RGM-positive extracellular laminae components were found in the constituting scar.
  • RGM-positive cells accumulated in infarctioned white matter, hemorrhagic areas, infarction core and peri-infarctional areas, respectively.
  • the morphological described peri-infarctional areas were part of the physiologically defined penumbra.
  • RGM-positive cells formed clusters in the Virchow-Robin spaces from day 1-7, which subsided later. These peri-vascular cells, also referred to as adventitial or perithelial cells are characteristically alert immune cells (Kato and Walz, 2000; Streit et al., 1999). With lesion aging, from day 3 onwards, lesional RGM expression by few reactive astrocytes, was observed. At later stages, arising 1 week after infarction, extracellular RGM deposits were detected constituting neo-laminae localized to areas of ongoing scar formation. These RGM-positive laminae increased in magnitude and regional extend over time. With tissue reorganisation of the lesion, also “foamy”, lipid loaded RGM-positive phagocytic RGM-positive microglia/macrophages were observed.
  • Upregulation of cellular RGM expression correlated with the time course and appearance of infiltrating leukocytes and activation of microglia/macrophages after injury (Stoll et al., 1998). Whereas upregulation of extracellular RGM expression correlated with the time course and the appearance of the scar after injury. In few cases ( ⁇ 5% of counterstained nuclei) some reactive astrocytes restricted to the demarcating lesion core also expressed RGM.
  • RGM immunoreactivity was also detected in endothelial and vascular smooth muscle cells (SMC) but no significant differences were observed between injured and control brains.
  • Hybridoma cells secreting the RGM-specific F3D4 monoclonal antibody were injected into the peritoneum of mice, primed with mineral oil. Normally the hybridoma cells continue to divide in the peritoneum and the hybridoma cells secreted large amounts of antibody, resulting in formation of large ascites tumors. Mice receiving the F3D4-producing hybridoma cells did not develop ascites tumors in the peritoneal cavity, but developed solid, adherent tumors. The F3D4 monoclonal antibody resulted in a change of phenotype of the tumorigenic hybridoma cells from a less invasive, non-adherent state to an invasive adherent state. Masking of endogeneous RGM by the antibodies secreted from the hybridoma cells, enabled adhesion and invasion of these tumor cells and was responsible for this outcome.
  • RGM was cloned into the pTriEx vector and the vector was cut inside the polylinker side and inside the RGM sequence using Sad in the first step. After ligation of both ends the RGM containing vector was cut in the second step with Stul inside the RGM sequence and in the polylinker with Pa. After ligation of both ends, the vector with the shorter RGM-fragments was transfected into COS7 cells. Cell lysates of these COS cells were purified using an anti-RGM1 affinity column and RGM-containing fractions were used in collapse assay experiments. A fragment as described in SEQ ID NO:19 was active in said assays.
  • NCBI National Center for Biotechnology Information

Abstract

The present invention relates to the use of a modulator of a polypeptide having or comprising an amino acid sequence as disclosed herein or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with seizures or associated with angiogenic disorders or disorders of the cardiovascular system. Furthermore, the invention provides for the use of a modulator of a polypeptide having or comprising said amino acid sequence of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with angiogenic disorders or disorders of the cardiovascular system. In addition, the invention provides for the use of said polypeptide or said functional fragment or derivative thereof for the preparation of a pharmaceutical compositions for preventing or treating tumor growth or formation of tumor metastases or as a marker of stem cells.

Description

  • The present invention relates to the use of a modulator of a polypeptide having or comprising an amino acid sequence as disclosed herein or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with angiogenic disorders or disorders of the cardio-vascular system. Furthermore, the invention provides for the use of a modulator of a polypeptide having or comprising said amino acid sequence or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with seizures, associated with angiogenic disorders or disorders of the cardio-vascular system. In addition the invention provides for the use of said polypeptide or said functional fragment or derivative thereof for the preparation of a pharmaceutical composition for preventing or treating tumor growth or formation of tumor metastases or as a marker of stem cells.
  • Several documents are cited throughout the text of this specification. The disclosure content of each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated by reference.
  • The most important mechanism in formation of embryonic nervous systems is the guidance of axons and growth cones by directional guidance cues (Goodman, Annu. Rev. Neurosci. 19 (1996), 341-77; Mueller, Annu. Rev. Neurosci 22, (1999), 351-88). A suitable model system for studying this guidance process is the retinotectal system of vertebrates. In the chick embryo approximately 2 million retinal ganglion cell (RGC) axons leave each eye and grow towards the contralateral tectum opticum to form a precise map (Mey & Thanos, (1992); J. Himforschung 33, 673-702). Having arrived at the anterior pole of the optic tectum, RGC axons start to invade their tectal target to find their target neurons. Mapping occurs in such a way, that RGC axons from nasal retina project to posterior tectum and temporal axons to anterior tectum. Along the dorso-ventral axis, axons coming from dorsal retina terminate in ventral tectum, whereas those from ventral retina end up in dorsal tectum. In the end a precise topographic map is formed, where neighborhood relationships in the retina are preserved in the tectum, so that axons from neighboring ganglion cells in the retina synapse with neighboring tectal neurons. Most important for formation of this map, are graded tectal guidance cues, read by retinal growth cones carrying corresponding receptors which also show a graded distribution (Sperry, Proc. Natl. Acad. Sci. USA 50 (1963), 703-710; Bonhoeffer & Gierer, Trends Neurosci. 7 (1984) 378-381). Position of each retinal growth cone in the tectal field is therefore determined by two sets of gradients: receptor gradients on ingrowing retinal axons and growth cones and ligand gradients on tectal cells (Gierer, Development 101 (1987), 479-489). The existence of the graded tectal ligands has been postulated from anatomical work, their identification however proved to be extremely difficult and was only made possible with the development of simple in vitro systems (Walter, Development 101 (1987), 685-96; Cox, Neuron 4 (1990), 31-7). In the stripe assay RGC axons grow on a membrane carpet, consisting of alternating lanes of anterior (a) and posterior (p) tectal membranes. On these carpets, temporal retinal axons grow on anterior tectal membranes and are repelled by the posterior lanes, whereas nasal axons do not distinguish between a and p membranes (Walter, Development 101 (1987), 685-96). The same specificity is also observed in the growth cone collapse assay (Raper & Kapfhammer, Neuron 4 (1990), 21-29), where temporal retinal growth cones collapse after addition of posterior tectal membrane vesicles but do not react to anterior tectal vesicles and where nasal growth cones are insensitive to either type of vesicles (Cox, (1990), loc. cit.). In both assay systems, treatment of posterior tectal membranes with the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC) which cleaves the lipid anchor of glycosylphosphatidylinositol (GPI)-linked proteins, removed their repellent and collapse-inducing activity (Walter, J. Physiol 84 (1990), 104-10).
  • One of the first repulsive guidance molecules identified in the retinotectal system of chick embryos was a GPI-anchored glycoprotein with a molecular weight of 33/35 kDa (Stahl, Neuron 5 (1990), 735-43). This 33/35 kDa molecule, later termed RGM (Repulsive Guidance Molecule), was active in both stripe and collapse-assays and was shown to be expressed in a low-anterior high-posterior gradient in the embryonic tecta of chick and rat (Mueller, Curr. Biol. 6 (1996), 1497-502; Mueller, Japan Scientific Societies Press (1997), 215-229). Due to the abnormal biochemical behaviour of RGM, the precise amino acid sequence was not obtainable. RGM was described as a molecule which is active during vertebrate development. Interestingly, RGM is down-regulated in the embryonic chick tectum after E12 and in the embryonic rat tectum after P2 and completely disappears after the embryonic stages (Müller (1992), Ph. D thesis University of Tübingen; Müller (1997) Japan Scientific Societies, 215-229) In 1996, Müller (loc. cit.) have shown that CALI (chromophore-assisted laser inactivation) of RGM eliminates the repulsive guidance activity of posterior tectal membranes/RGM. However, due to the presence of other guidance molecules, in particular of RAGS (repulsive axon guidance signal) and ELF-1 (Eph ligand family 1), a complete elimination of guidance was not always detected and it was speculated that RGM acts in concert with RAGS (now termed ephrin-A5) and ELF-1 (ephrin-A2). It was furthermore envisaged that RGM may be a co-factor potentiating the activity of RAGS and ELF-1 in embryonic guidance events.
  • In 1980/81 the group of Aguayo found that, when peripheral neurons are transplanted/grafted into injured CNS of adult, axon growth of CNS neurons is induced (David, Science 214 (1981), 931-933). Therefore, it was speculated that CNS neurons have still the ability and capacity of neurite-outgrowth and/or regeneration, if a suitable environment would be provided. Furthermore, it was speculated that “CNS-neuron regeneration inhibitors” may exist.
  • In 1988, Caroni and Schwab (Neuron 1, 85-96) described two inhibitiors of 35 kDa and 250 kDa, isolated from rat CNS myelin (NI-35 and NI-250; see also Schnell, Nature 343 (1990) 269-272; Caroni, J. Cell Biol. 106 (1988), 1291-1288).
  • In 2000, the DNA encoding for NI-220/250 was deduced and the corresponding potent inhibitor of neurite growth was termed Nogo-A (Chen, Nature 403 (2000), 434-438. The membrane-bound Nogo turned out to be a member of the reticulon family (GrandPré, Nature 403 (2000), 439-444).
  • Further factors which mediate neuronal outgrowth inhibition have first been isolated in grasshoppers, and termed “fascidin IV” and later “collapsin” in chicken. These inhibitors belong to the so-called semaphorin family. Semaphorins have been reported in a wide range of species and described as transmembrane proteins (see, inter alia, Kolodkin Cell 75 (1993) 1389-99, POschel, Neuron 14 (1995), 941-948). Yet, it was also shown that not all semaphorins have inhibitory activity. Some members of said family, e.g. semaphorin E, act as an attractive guidance signal for cortical axons (Bagnard, Development 125 (1998), 5043-5053).
  • A further system of repulsive guidance molecules is the ephrin-Eph system. Ephrins are ligands of the Eph receptor kinases and are implicated as positional labels that may guide the development of neural topographic maps (Flanagan, Ann. Rev. Neurosc. 21 (1998), 309-345). Ephrins are grouped in two classes, the A-ephrins which are linked to the membrane by a glycosylphosphatidylinositol-anchor (GPI-anchor) and the B-ephrins carrying a transmembrane domain (Eph nomenclature committee 1997). Two members of the A-ephrins, ephrin-A2 and ephrin-A5, expressed in low anterior-high posterior gradients in the optic tectum, have recently been shown to be involved in repulsive guidance of retinal ganglion cell axons in vitro and in vivo (see, inter alia (Drescher, Cell 82 (1995), 359-70; Cheng, Cell 79 (1994), 157-168; Feldheim, Neuron 21 (1998), 563-74; Feldheim, Neuron 25 (2000), 563-74)
  • Considering the fact that a plurality of physiological disorders or injuries are related to altered cellular migration processes, the technical problems underlying the present invention was to provide for means and methods for modifying altered developmental or cellular (migration) processes which lead to disease conditions.
  • Accordingly, the present invention relates to the use of an modulator of a polypeptide having or comprising the amino acid sequence of SEQ ID NOs.18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with angiogenic disorders or disorders of the cardio-vascular system and associated with tumor formation and tumor growth.
  • In context of the present invention, and as documented in the appended examples, it was surprisingly found that the repulsive guidance molecule (RGM) is not only expressed during vertebrate development but is re-expressed in adult tissue, in particular in damaged adult tissues. It was, inter alia, surprisingly found that RGM is re-expressed after damage of the nervous tissue, after traumatic events or focal ischemias. The present invention provides for the complete nucleotide sequence and/or amino acid of RGM (see, e.g. SEQ ID NO: 17 or 18 depicting the RGM sequence of chicken or SEQ ID NO: 20 to 25 depicting the human RGM homologues.) RGM, as pointed out herein above, is a glycoprotein, linked to membranes by a GPI-anchor. Said GPI-anchor also carries a cross-reacting determinant (CRD) epitope and its carbohydrate part is able to bind peanut lectin. As documented herein, the RGM protein is a potent growth inhibitor and can assert neurite growth inhibition in picomolor concentrations.
  • The term “modulator” as employed herein relates to “inhibitors” as well as “activators” of RGM function. Most preferably said “modulation” is an inhibition, wherein said inhibition may be a partial or a complete inhibition.
  • The term “amino acid sequence of SEQ ID NO: 18, 20, 23 or 25 as employed herein relates to the amino acid sequence of RGM (repulsive guidance molecule) and relates to the RGM polypeptide of chicken or human, respectively. In particular, SEQ ID NOs: 20 and 21 depict human RGM1. Human RGM1 has been localized on chromosome 15. Further, human RGMs comprise RGM2 and RGM3. RGM2 is depicted in SEQ ID NO: 23 (amino acid sequence) and is encoded by a nucleotide sequence as shown in SEQ ID NO: 22. Human RGM2 has been localized on chromosome 5. Furthermore, human RGM3 is shown in appended SEQ ID NO: 25 (amino acid sequence) and encoded by a nucleotide sequence as depicted in SEQ ID NO: 24. Human RGM3 is located on chromosome 1. Yet, as will be discussed herein below, said term relates also to further RGM homologues.
  • The term “(poly)peptide” means, in accordance with the present invention, a peptide, a protein, or a (poly)peptide which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds. However, peptidomimetics of such RGM proteins/(poly)peptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention.
  • The present invention is not restricted to RGM from human, mouse or chicken and its inhibitors but also relates to the use of inhibitors of RGM or of RGM itself (or functional fragments or derivatives thereof) from other species. Since the present invention provides for the use of amino acid sequences/polypeptides of RGM and its corresponding inhibitors and since the amino acid sequences of human and chicken RGM are disclosed herein, the person skilled in the art is provided with the information to obtain RGM sequences from other species, like, inter alia, mouse, rat, pig, etc. The relevant methods are known in the art and may be carried out by standard methods, employing, inter alia, degenerate and non degenerate primers in PCR-techniques. Such molecular biology methods are well known in the art and, e.g., described in Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y. (1989)) and Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates; and Wiley Interscience, N.Y. (1989).
  • Furthermore, as employed in the context of the present invention, the term “RGM”, “RGM modulator” and “RGM-inhibitor” also relates to RGM molecules (and their corresponding inhibitors) which are variants or homologs of the RGM molecules (and their inhibitors) as described herein. “Homology” in this context is understood to refer in this context to a sequence identity of RGMs of at least 70%, preferably more than 80% and still more preferably more than 90% on the amino acid level. The present invention, however, comprises also (poly)peptides deviating from wildtype amino acid sequences of human or chicken RGMs described herein, wherein said deviation may be, for example, the result of amino acid and/or nucleotide substitution(s), deletion(s), addition(s), insertion(s), duplication(s), inversion(s) and/or recombination(s) either alone or in combination. Those deviations may naturally occur or be produced via recombinant DNA techniques well known in the art. The term “variation” as employed herein also comprises “allelic variants”. These allelic variations may be naturally occurring allelic variants, splice variants as well as synthetically produced or genetically engineered variants.
  • The term “polynucleotide” in accordance with the present invention comprises coding and, wherever applicable, non-coding sequences (like promotors, enhancers etc.). It comprises DNA, RNA as well as PNA. In accordance with the present invention, the term “polynucleotide/nucleic acid molecule” comprises also any feasible derivative of a nucleic acid to which a nucleic acid probe may hybridize. Said nucleic acid probe itself may be a derivative of a nucleic acid molecule capable of hybridizing to said nucleic acid molecule or said derivative thereof. The term “nucleic acid molecule” further comprises peptide nucleic acids (PNAs) containing DNA analogs with amide backbone linkages (Nielsen, Science 254 (1991), 1497-1500). The term “nucleic acid molecule” which encodes a RGM (poly)peptide or a functional fragment/derivative thereof, in connection with the present invention, is defined either by (a) the specific nucleic acid sequences encoding said (poly)peptide specified in the present invention or (b) by nucleic acid sequences hybridizing under stringent conditions to the complementary strand of the nucleotide sequences of (a) and encoding a (poly)peptide deviating from the nucleic acid of (a) by one or more nucleotide substitutions, deletions, additions or inversions and wherein the nucleotide sequence shows at least 70%, more preferably at least 80% identity with the nucleotide sequence of said encoded RGM (poly)peptide having an amino acid sequence as defined herein above and functions as a RGM (or a functional fragment/derivative thereof).
  • The term “modulator” as employed herein also comprises the term “inhibitor”, as mentioned herein above.
  • The term “inhibitor of a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or a functional fragment or derivative thereof”, therefore, not only relates to the specific inhibitors of human or chicken RGM but also relates to inhibitors of RGM (or functional fragments or derivatives thereof) of other species. Useful inhibitors are disclosed herein as well as described herein below and in the appended examples.
  • The term “inhibitor” also comprises “modulators” of the RGM polypeptides and/or the RGM encoding nucleic acid molecule/gene. In context of this invention it is also envisaged that said “modulation” leads, when desired, to an activation of RGM.
  • The term “functional fragment or derivative thereof” in context of the present invention and in relation to the herein described RGM molecules comprises fragments of the RGM molecules defined herein having a length of at least 25, more preferably at least 50, more preferably at least 75, even more preferably at least 100 amino acids. Functional fragments of the herein identified RGM molecules or RGM molecules of other species (homologous RGMs) may be comprised in fusion and/or chimeric proteins. “Functional fragments” comprise RGM fragments (or its encoding nucleic acid molecules) which are able to replace RGM full length molecules in corresponding assays (as disclosed herein in the appended examples, e.g. collapse and/or stripe assays) or may elucidate an anti-RGM specific immune-response and/or lead to specific anti-RGM antibodies. An example of such a “functional fragment” is, inter alia, the functional fragment of chicken RGM depicted in SEQ ID NO: 19. In context of the present invention, polynucleotides encoding functional fragments of RGM and/or its derivatives have preferably at least 15, more preferably at least 30, more preferably at least 90, more preferably of at least 150, more preferably of at least 300 nucleotides.
  • The term “derivative” means in context of their invention derivatives of RGM molecules and/or their encoding nucleic acid molecules and refer to natural derivatives (like allelic variants) as well as recombinantly produced derivatives/variants which may differ from the herein described RGM molecules by at least one modification/mutation, e.g. at least one deletion, substitution, addition, inversion or duplication. The term “derivative” also comprises chemical modifications. The term “derivative” as employed herein in context of the RGM molecule also comprises soluble RGM molecules which do not comprise any membrane anchorage.
  • As mentioned herein above, the present invention provides for the use of a modulator, preferably an inhibitor, of RGM molecules and/or their corresponding encoding polynucleotides/nucleic acid molecules for the preparation of a pharmaceutical composition for preventing, alleviating or treating various disorders of the nervous system, angiogenic disorders or disorders of the cardio-vascular system and malignancies of different etiology.
  • In a preferred embodiment, said disorders of the nervous system comprise degeneration or injury of vertebrate nervous tissue, in particular neurodegenerative diseases, nerve fiber injuries and disorders related to nerve fiber losses.
  • Said neurodegenerative diseases may be selected from the group consisting of motomeuronal diseases (MND), amyotrophic lateral sclerosis (ALS), Alzheimers disease, Parkinsons disease, progressive bulbar palsy, progressive muscular atrophy, HIV-related dementia and spinal muscular atrophy(ies), Down's Syndrome, Huntington's Disease, Creutzfeldt-Jacob Disease, Gerstmann-Straeussler Syndrome, kuru, Scrapie, transmissible mink encephalopathy, other unknown prion diseases, multiple system atrophy, Riley-Day familial dysautonomia said nerve fiber injuries may be selected from the group consisting of spinal cord injury(ies), brain injuries related to raised intracranial pressure, trauma, secondary damage due to increased intracranial pressure, infection, infarction, exposure to toxic agents, malignancy and paraneoplastic syndromes and wherein said disorders related to nerve fiber losses may be selected from the group consisting of paresis of nervus facialis, nervus medianus, nervus ulnaris, nervus axillaris, nervus thoracicus longus, nervus radialis and for of other peripheral nerves, and other aquired and non-aquired diseases of the (human) central and peripheral nervous system.
  • The above mentioned spinal cord and brain injuries not only comprise traumatic injuries but also relate to injuries caused by stroke, ischemia and the like. It is in particular envisaged that the inhibitors as defined herein below and comprising, inter alia, anti-RGM antibodies be employed in the medical art to stimulate nerve fiber growth in individuals, in particular in vertebrates, most preferably in humans.
  • In a more preferred embodiment of the present invention, the invention provides for the use of a modulator, preferably an inhibitor to RGM (or a functional fragment or derivative thereof) for the preparation of a pharmaceutical composition for the treatment of disorders of the cardio-vascular system, wherein these disorders, e.g., comprise disorders of the blood-brain barrier, brain oedema, secondary brain damages due to increased intracranial pressure, infection, infarction, ischemia, hypoxia, hypoglycemia, exposure to toxic agents, malignancy, paraneoplastic syndromes.
  • It is envisaged, without being bound by theory, that RGM inhibitors may stimulate surviving neurons to project collateral fibers into the diseased tissue, e.g. the ischemic tissue.
  • As illustrated in the appended examples, RGM is expressed locally at the side of artificial transection of brain/spinal cord tissue in test animals (like rats), e.g., in the penumbra region surrounding an ischemic core of a human suffering focal ischemia in the temporal contex. Furthermore, it is documented in the appended examples that RGM is, surprisingly, expressed in tissue(s) having experienced from traumatic brain injuries. The invention also relates to the use of a RGM polypeptide or a functional fragment or derivative thereof or the use of a polynucleotide encoding the same (polypeptides and polynucleotides as defined herein), wherein the above described disease or condition associated with seizures is epilepsy. An epilepsy is thereby characterized by an epileptic seizure as a convulsion or transient abnormal event experienced by the subject, e.g. a human patient, due to a paroxysmal discharge of (cerebral) neurons. The epileptic seizures comprise tonic seizures, tonic-clonic seizures (grand mal), myoclonic seizures, absence seizures as well as akinetic seizures. Yet, also comprised are in context of this invention simple partial seizures, e.g. Jacksonian seizures and seizures due to perinatal trauma and/or fetal anoxia. As mentioned herein below, the uses described herein relate in particular to the preparation of pharmaceutical compositions for the treatment of diseases/conditions associated with aberrant sprouting of nerve fibres, like epilepsy, see also Routbort, Neuroscience 94 (1999), 755-765.
  • In a even more preferred embodiment of the invention, the modulator, preferably the inhibitor of RGM (or of its functional fragment or derivative thereof or of its encoding nucleid acid molecule) is used for the preparation of a pharmaceutical composition for the modification of neovascularization. Said modification may comprise activation as well as stimulation. It is in particular envisaged that said neovascularisation be stimulated and/or activated in diseased tissue, like inter alia, ischemic and/or infarctious tissue. Furthermore, it is envisaged that the RGM-inhibitors described herein may be employed in the regulation of the blood-brain barrier permeability.
  • It is furthermore envisaged that said modulators, preferably said inhibitors for RGM be employed in the alleviation, prevention and/or inhibition of progression of vascular plaque formation (e.g. artherosclerosis) in cardio-vascular, cerebo-vascular and/or nephrovascular diseases/disorders.
  • Furthermore, the present invention provides for the use of a modulator, preferably an inhibitor of RGM as defined herein for the preparation of a pharmaceutical composition for remyelination. Therefore, the present invention provides for a pharmaceutical composition for the treatment of demyelinating diseases of the CNS, like multiple sclerosis or of demyelinating diseases like peripheral neuropathy caused by diphteria toxin, Landry-Guillain-Barré-Syndrom, Elsberg-Syndrom, Charcot-Marie-Tooth disease and other polyneuropatias. A particular preferred inhibitor of RGM in this context is an antibody directed against RGM, e.g. an IgM antibody. It has previously be shown that certain IgMs bind to oligodendrocytes and thereby induce remyelination. IgM antibodies against RGM are known in the art and comprise e.g. the F3D4 described in the appended examples.
  • In addition the invention provides for the use of a RGM polypeptide as defined herein or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the activity of autoreactive immune cells or with overactive inflammatory cells. Most preferably these cells are T-cells.
  • Furthermore, the present invention relates to the use of a modulator, preferably an inhibitor or another RGM binding molecule of a RGM polypeptide or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or of fragment/derivative thereof for modifying and/or altering the differentiation status of neuronal stem cells and/or their progenitors. Said stem cells are normally found in the subventricular zones of many brain regions. It is known that factors in the microenvironment of the brain dramatically influence the differentiation of undifferentiated stem cells. It is assumed that due to the characteristic expression of RGM in the subventricular layers of many different brain regions, this molecule could be a marker for stem cells. Furthermore, RGM inhibitors, like antibodies could be useful markers for stem cells. Most important in stem cell biology is the understanding of factors influencing their differentiation. It is therefore assumed that RGM inhibitors change the developmental fate of these cells.
  • As documented in the appended examples, RGM is not only expressed in ischemic tissue but is also expressed in scar tissue surrounding (brain) lesions.
  • It is particularly preferred that the modulator, preferably the inhibitor of the RGM molecule (or its functional fragment or derivative) is an antibody or a fragment or a derivative thereof, is an aptamer, is a specific receptor molecule capable of interacting with a RGM polypeptide or with a functional fragment or derivative thereof, or is a specific nucleic acid molecule interacting with a polynucleotide encoding an RGM and/or the polypeptide as defined herein.
  • The antibody to be used in context of the present invention can be, for example, polyclonal or monoclonal antibodies. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. The production of specific anti-RGM antibodies is further known in the art (see, e.g. Müler (1996) loc.cit.) or described in the appended examples.
  • The term “antibody” as employed herein also comprises chimeric, single chain and humanized antibodies, as well as antibody fragments, like, inter alia, Fab fragments. Antibody fragments or derivatives further comprise F(ab′)2, Fv or scFv fragments; see, for example, Harlow and Lane, loc.cit. Various procedures are known in the art and may be used for the production of such antibodies and/or fragments, see also appended examples. Thus, the (antibody) derivatives can be produced by peptidomimetics. Further, techniques described for the production of single chain antibodies (see, inter alia, U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to polypeptide(s) of this invention. Also, transgenic animals may be used to express humanized antibodies to polypeptides of this invention. Most preferably, the antibody to be used in the invention is a monoclonal antibody, for example the F3D4 antibody described in the appended examples may be employed when an IgM is desired. The general methodology for producing, monoclonal antibodies is well-known and has been described in, for example, Köhler and Milstein, Nature 256 (1975), 494-496 and reviewed in J. G. R. Hurrel, ed., “Monoclonal Hybridoma Antibodies Techniques and Applications”, CRC Press Inc., Boco Raron, Fla. (1982), as well as that taught by L. T. Mimms et al., Virology 176 (1990), 604-619.
  • Preferably, said antibodies (or inhibitors) are directed against functional fragments of the RGM polypeptide. As pointed out herein above and as documented in the appended examples, such functional fragments are easily deducible for the person skilled in the art and, correspondingly, relevant antibodies (or other inhibitors) may be produced.
  • The “modulator”, preferably the “inhibitor” as defined herein may also be an aptamer. In the context of the present invention, the term “aptamer” comprises nucleic acids such as RNA, ssDNA (ss=single stranded), modified RNA, modified ssDNA or PNAs which bind a plurality of target sequences having a high specificity and affinity. Aptamers are well known in the art and, inter alia, described in Famulok, Curr. Op. Chem. Biol. 2 (1998), 320-327. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold, Ann. Rev. Biochem. 64 (1995), 763-797). Said other receptors may, for example, be derived from said antibody etc. by peptidomimetics.
  • Other specific “receptor” molecules which may function as inhibitors of the RGM polypeptides are also comprised in this invention. Said specific receptors may be deduced by methods known in the art and comprise binding assays and/or interaction assays. These may, inter alia, involve assays in the ELISA-format or FRET-format. Said “inhibitor” may also comprise specific peptides binding to and/or interfering with RGM.
  • Furthermore, the above recited “modulator”, preferably “inhibitor” may function at the level of RGM gene expression. Therefore, the inhibitor may be a (specific) nucleic acid molecule interacting with a polynucleotide encoding a RGM molecule (or a functional fragment or derivative thereof.) These inhibitors may, e.g., comprise antisense nucleic acid molecules or ribozymes.
  • The nucleic acid molecule encoding RGM (and as disclosed herein, e.g., SEQ ID NO: 17) may be employed to construct appropriate anti-sense oligonucleotides. Said anti-sense oligonucleotides are able to inhibit the function of wild-type (or mutant) RGM genes and comprise, preferably, at least 15 nucleotides, more preferably at least 20 nucleotides, even more preferably 30 nucleotides and most preferably at least 40 nucleotides.
  • In addition, ribozyme approaches are also envisaged for use in this invention. Ribozymes may specifically cleave the nucleic acid molecule encoding RGMs.
  • In the context of the present invention ribozymes comprise, inter alia, hammerhead ribozymes, hammerhead ribozymes with altered core sequences or deoxyribozymes (see, e.g., Santoro, Proc. Natl. Acad. Sci. USA 94 (1997), 4262) and may comprise natural and in vitro selected and/or synthesized ribozymes. Nucleic acid molecules according to the present invention which are complementary to nucleic acid molecules coding for proteins/(poly)peptides regulating, causing or contributing to obesity and/or encoding a mammalian (poly)peptide involved in the regulation of body weight (see herein below) may be used for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) which specifically cleave nucleic acid molecules of the invention. Selection of the appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith, eds. Academic Press, Inc. (1995), 449-460.
  • Said “inhibitor” may also comprise double-stranded RNAs, which lead to RNA-mediated gene interference (see Sharp, Genes and Dev. 13 (1999), 139-141)
  • Further potential inhibitors of RGM may be found and/or deduced by interaction assay and employing corresponding read-out systems. These are known in the art and comprise, inter alia, two hybrid screenings (as, described, inter alia, in EP-0 963 376, WO 98/25947, WO 00/02911) GST-pull-down columns, co-precipitation assays from cell extracts as described, inter alia, in Kasus-Jacobi, Oncogene 19 (2000), 2052-2059, “interaction-trap” systems (as described, inter alia, in U.S. Pat. No. 6,004,746) expression cloning (e.g. lambda gtll), phage display (as described, inter alia, in U.S. Pat. No. 5,541,109), in vitro binding assays and the like. Further interaction assay methods and corresponding read out systems are, inter alia, described in U.S. Pat. No. 5,525,490, WO 99/51741, WO 00/17221, WO 00/14271 or WO 00/05410.
  • A further objective of the present invention is to provide for the use of a RGM polypeptide and/or of polypeptide having or comprising the amino acid sequence of SEQ ID NOs. 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with excessive collateral sprouting of nerve fibres.
  • The present invention, therefore, provides for the medical use of RGM protein(s) and/or functional fragments/derivatives thereof or for the use of polynucleotides encoding said RGM protein(s) in conditions where excessive collateral sprouting occurs. Said conditions comprise, but are not limited to, epilepsy, phantom pain and neuropathic pain. For example, McNamara (Nat. Suppl. 399 (1999), A15-A22) has described that said sprouting occurs in certain types of epilepsy. The RGM molecule, either naturally isolated or recombinantly produced, or its functional fragments/derivatives may therefore be employed as potent “stop” signals for growing nerve fibres. The feasibility of such an approach has been shown by Tanelian (Nat. Med. 3 (1997), 1398-1401) who employed a semaphorin for inhibition of nerve fiber growth.
  • In yet another embodiment, the present invention provides for the use of RGM and/or of a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing or treating tumor growth or formation of tumor metastases.
  • RGM (naturally isolated or recombinantly produced) and/or functional fragments thereof may be employed for the preparation of a pharmaceutical composition for the treatment of neoplastic disorders, in particular of disorders related to tumor (cell) migration, metastasis and/or tumor invasion. Furthermore, it is envisaged that RGM inhibits undesired neovascularisation. Said neovascularisation, as an angiogenic disorder during neoplastic events, should be prevented in order to limit, inter alia, tumor growth.
  • Growth cones of neurons and (invasive) tumor cells secrets a cocktail of proteases (uPA, tPA, MNPs, etc.) in order to degrade extracellular matrix. Furthermore, similar mechanisms for adhesion and (cell) migration are employed by these cellular systems.
  • RGM and/or its functional fragments may be employed to actively stimulate withdrawal of lamellipodia of tumor cells and/or to induce their collapse. As demonstrated in the appended examples RGM also influences tumor growth behaviour, i.e. is capable of negatively influencing tumor growth.
  • In addition the invention provides for the use of a RGM polypeptide as defined herein or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the activity of autoreactive immune cells or with overactive inflammatory cells. Most preferably these cells are T-cells.
  • In yet another embodiment, the invention provides for the use of a RGM polypeptide having or comprising, inter alia, the amino acid sequence of SEQ ID NOs.18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for the treatment of inflammation processes and/or allergies, for wound healing or for the suppression/alleviation of scar formation. Scar tissue is formed by invading cells, most importantly by fibroblasts and/or glial cells. Migration and adhesion of these cells are required to get to the lesion side. RGM or an active fragment/derivative could prevent accumulation of these cells in the lesion side, thereby preventing or slowing down scar formation. In inflammatory reactions cells migrate to the inflamed region and RGM or its active fragment/derivative prevent or reduce migration of these cells to the side of inflammation, thereby preventing overactive inflammatory reactions.
  • In context of the present invention, the term “pharmaceutical composition” also comprises optionally further comprising an acceptable carrier and/or diluent and/or excipient. The pharmaceutical composition of the present invention may be particularly useful in preventing and/or treating pathological disorders in vertebrates, like humans. Said pathological disorders comprise, but are not limited to, neurological, neurodegenerative and/or neoplastic disorders as well as disorders associated with seizures, e.g. epilepsy. These disorders comprise, inter alia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (FALS/SALS), ischemia, stroke, epilepsy, AIDS dementia and cancer. The pharmaceutical composition may also be used for prophylactic purposes. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. However, it is also envisaged that the pharmaceutical compositions are directly applied to the nervous tissue. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, general health, age, sex, the particular compound to be administered, time and route of administration, and other drugs being administered concurrently. Pharmaceutically active matter may be present preferably, inter alia, in amounts between 1 ng and 1000 mg per dose, more preferably in amounts of 1 ng to 100 mg however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously. The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents, depending on the intended use of the pharmaceutical composition. Such agents may be drugs acting on the central nervous system as well as on small, unmyelinated sensory nerve terminals (like in the skin), neurons of the peripheral nervous system of the digestive tract., etc.
  • It is also understood that the pharmaceutical composition as defined herein may comprise nucleic acid molecules encoding RGMs (and/or functional fragments or derivatives thereof) or corresponding RGM inhibitors or defined herein. As mentioned herein-above, said inhibitors comprise, but are not limited to, antibodies, aptamer, RGM-interacting peptides as well as inhibitors interacting with the RGM-encoding polynucleotides.
  • Accordingly, the present invention also provides for a method of treating, preventing and/or alleviating pathological disorders and conditions as defined herein, whereby said method comprises administering to a subject in need of such a treatment a pharmaceutical composition/medicament as defined herein. Preferably, said subject is a human.
  • The nucleic acid molecules may be particularly useful in gene therapy approaches and may comprise DNA, RNA as well as PNA. Said nucleic acid molecules may be comprised in suitable vectors, either inter alia, gene expression vectors. Such a vector may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • Furthermore, the vectors may, in addition to the nucleic acid sequences encoding RGM or its corresponding inhibitors, comprise expression control elements, allowing proper expression of the coding regions in suitable host cells or tissues. Such control elements are known to the artisan and may include a promoter, translation initiation codon, translation and insertion site for introducing an insert into the vector. Preferably, the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in (eukaryotic) cells. Particularly preferred are in this context control sequences which allow for correct expression in neuronal cells and/or cells derived from nervous tissue.
  • Control elements ensuring expression in eukaryotic cells are well known to those skilled in the art. As mentioned above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human elongation factor 1α-promoter, CMV enhancer, CaM-kinase promoter or SV40-enhancer. For the expression for example in nervous tissue and/or cells derived therefrom, several regulatory sequences are well known in the art, like the minimal promoter sequence of human neurofilament L (Charron, J. Biol. Chem 270 (1995), 25739-25745). Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (In-Vitrogene, as used, inter alia in the appended examples), pSPORTI (GIBCO BRL) or pGEMHE (Promega), Beside the nucleic acid molecules defined herein, the vector may further comprise nucleic acid sequences encoding for secretion signals. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used leader sequences capable of directing the protein/(poly)peptide to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a part thereof,
  • As mentioned herein above, said vector may also be, besides an expression vector, a gene transfer and/or gene targeting vector. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, vector systems and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, Schaper, Current Opinion in Biotechnology 7 (1996), 635-640 Verma, Nature 389 (1997), 239-242 WO 94/29469, WO 97/00957, U.S. Pat. No. 5,580,859, US 589,66 or U.S. Pat. No. 4,394,448 and references cited therein.
  • In particular, said vectors and/or gene delivery systems are also described in gene therapy approaches in neurological tissue/cells (see, inter alia Blömer, J. Virology 71 (1997) 6641-6649) or in the hypothalamus (see, inter alia, Geddes, Front Neuroendocrinol. 20 (1999), 296-316 or Geddes, Nat. Med. 3 (1997), 1402-1404). Further suitable gene therapy constructs for use in neurological cells/tissues are known in the art, for example in Meier (1999), J. Neuropathol. Exp. Neurol. 58, 1099-1110. The nucleic acid molecules and vectors of the invention may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules described herein.
  • The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term “treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.
  • In yet another embodiment, the present invention provides for the use of a (RGM) polypeptide and/or a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative as a marker of stem cells. Since it is envisaged that stem cells as well as their undifferentiated progenitor cells express RGM, RGM and/or functional fragments or derivatives thereof may be employed to influence the differentiation/differentiation pattern of said stem cells.
  • It is furthermore envisaged that antibodies directed against RGMs, in particular directed against polypeptides disclosed herein or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 (or (a) functional fragment(s)/derivative(s) thereof) may be employed to influence the differentiation of (neuronal) stem cells and (neuronal) progenitor cells. It is particularly preferred that said antibodies (as well as other RGM-inhibitors and/or RGM-binding molecules) be employed to selectively label stem cells. Therefore these reagents may be employed as markers for stem cells. It is also envisaged that peptides or derivatives be employed in said purpose.
  • In a particularly preferred embodiment of the present invention, the polypeptide and/or fragment thereof which comprises or has an amino acid sequence as depicted in SEQ ID NOs 18, 20, 23 or 25 and/or is a RGM molecule to be used in accordance with their invention is a soluble, i.e. not membrane bound molecule.
  • As shown in Davis (1994), Science 266, 816-819 ephrins, in particular A-ephrins, are not active in soluble, monomeric form. In contrast, soluble RGMs are active and may function without any membrane-attachment RGM, in contrast to ephrins, is capable of self-formation of dimers and/or of the formation of higher aggregates. The invention also provides for the use of a RGM molecule and/or a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or a fragment or a derivative for the preparation of a pharmaceutical composition for alleviating, preventing and/or treating homeostatic and/or bleeding disorders and/or vascular damage.
  • It is envisaged, without being bound by theory, that RGMs may, due to their structural homology to von-Willebrand factor (vWF), be employed in the treatment of said disorders/diseases. Furthermore, it is envisaged that RGM may interact with von-Willebrand factor and that said molecule, thereby, influences the activity of vWF. Furthermore, the inhibitors as defined herein should be employed in disorders where immune cells invade the brain, like multiple sclerosis, encephalomyelitis disseminata.
  • The present invention also provides for the use of an antibody or a fragment or a derivative thereof, or an aptamer, or a binding molecule capable of interacting with a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or with functional fragment or derivative thereof or of an nucleid acid molecule capable of interacting with a polynucleotide encoding said polypeptide or a fragment thereof for the preparation of a diagnostic composition for detecting neurological and/or neurodegenerative disorders or dispositions thereto.
  • The diagnostic composition may be used, inter alia, for methods for determining the expression of the nucleic acids encoding RGM polypeptides by detecting, inter alia, the presence of the corresponding mRNA which comprises isolation of RNA from a cell, contacting the RNA so obtained with a nucleic acid probe as described above under hybridizing conditions, and detecting the presence of mRNAs hybridized to the probe.
  • Furthermore, corresponding mutations and/or alterations may be detected. Furthermore, RGM (poly)peptides can be detected with methods known in the art, which comprise, inter alia, immunological methods, like, ELISA or Western blotting.
  • The diagnostic composition of the invention may be useful, inter alia, in detecting the prevalence, the onset or the progress of a disease related to the aberant expression of a RGM polypeptide. Accordingly, the diagnostic composition of the invention may be used, inter alia, for assessing the prevalence, the onset and/or the disease status of neurological, neurodegenerative and/or inflammatory disorders, as defined herein above. It is also contemplated that anti-RGM antibodies, aptamers etc. and compositions comprising such antibodies, aptamers, etc. may be useful in discriminating (the) stage(s) of a disease.
  • The diagnostic composition optionally comprises suitable means for detection. The nucleic acid molecule(s), vector(s), antibody(ies), (poly)peptide(s), described above are, for example, suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention.
  • Solid phase carriers are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes. Suitable methods of immobilizing nucleic acid molecule(s), vector(s), host(s), antibody(ies), (poly)peptide(s), fusion protein(s) etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions and the like. Examples of immunoassays which can utilize said compounds of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Northern or Southern blot assay. Furthermore, these detection methods comprise, inter alia, IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay). Furthermore, the diagnostic compounds of the present invention may be are employed in techniques like FRET (Fluorescence Resonance Energy Transfer) assays.
  • The nucleic acid sequences encoding RGMs of other species as well as variants of RGMs are easily deducible from the information provided herein. These nucleic acid sequences are particularly useful, as pointed out herein above, in medical and/or diagnostic setting, but they also provide for important research tools. These tools may be employed, inter alia, for the generation of transgenic animals which overexpress or suppress RGMs or wherein the RGM gene is silenced and/or deleted. Furthermore, said sequences may be employed to detect and/or ellucidate RGM interaction partners and/or molecules binding to and/or interfering with RGMs.
  • THE FIGURES SHOW
  • FIG. 1: RGM protein fractions induce collapse of RGC growth cones.
      • Solubilized membrane proteins from E9/E10 chick brains were loaded on two different ion exchange columns, a DEAE anion exchange column and a cation exchange column. RGM was eluted from the cation exchange column at a NaCl concentration of 200-400 mM in two 1 ml fractions (4+5) was incorporated into lecithin vesicles and lecithin vesicles were used in collapse experiments with RGC growth cones. RGM-containing fractions (4+5, arrows), but not RGM-free fractions induced extensive collapse (>90%) of RGC growth cones. Neither ephrin-A5 nor ephrin-A2 could be detected with specific antibodies, in RGM-fractions. RGC axons and growth cones on laminin were stained with Alexa-Phalloidin. Western blots from two dimensional gels were incubated with the F3D4 monoclonal antibody, and were subsequently stained by a whole protein, india ink stain.
  • FIG. 2: Comparative two dimensional gel analysis of tectal proteins and RGM sequences.
      • A: Membranes from E9/10 anterior and posterior chick tecta were enriched and treated with buffer (C) or with PI-PLC (E), to remove GPI-anchored proteins. The putative RGM (arrow in Anterior-E+Posterior-E), a PI-PLC cleavable basic protein with a molecular weight of 33 kDa, was cut out and was used for nanoelectrospray tandem mass spectrometry. Two dimensional gels were stained with silver. No anterior-posterior difference of the RGM candidate is observed in these gels, this is probably due to the presence of two other proteins in the selected spot. B: Deduced RGM peptide sequences
  • FIG. 3: Nucleotide and amino acid sequence of RGM.
      • A. Nucleotide sequence of RGM.
      • B. Amino acid sequence of RGM.Peptides derived from microsequencing are highlighted in bold and peptides used for making polyclonal antibodies are underlined. Potential N-glycosylation sites and an RGD tripeptide, potential cell attachment site are underlined by asterics.
      • C. Schematic view of the RGM protein. Hydrophobic domains are present a the N- and C-termini of the protein. Epitopes of the two polyclonal anti-RGM antibodies are demarcated.
  • FIG. 4: The polyclonal and the monoclonal RGM antibody recognize the same 33 kDa protein.
      • A. The anti-RGM1 antibody binds to a GPI-anchored CRD- (cross reacting determinant) positive 33 kDa protein. Left blot: An anti-CRD antibody binds to a low abundant, 33 kDa protein (arrow), present in the E (PI-PLC supernatant) but not the C fraction (control supernatant). Right blot: Anti-RGM1 staining of a GPI-anchored 33 kDa protein on a western blot with supernatant from E9/E10 chick brain membranes.
      • B. The GPI-anchored 33 kDa antigen of the anti-RGM1 antibody is more abundant in posterior (pos.) than in anterior (ant.) tectal membranes. Left blot: rabbit preimmune serum did not bind to any protein on western blots with PI-PLC supernatant protein from anterior and posterior tectum. Right blot: Anti-RGM1 binding to a 33 kDa protein. E=PI-PLC supernatant from tectal membranes, C=control supernatants from tectal membranes.
      • C. Anti-RGM1 and F3D4 recognize the same antigens in tectal membranes. Left blot: F3D4 staining of tectal membrane proteins. A double band at 33 kDa (lower arrow) and a hardly visible band at 35 kDa (upper arrow) are recognized.
        • Right blot: Anti-RGM1 staining reveals the same staining pattern with 33 and 35 kDa antigens (arrows). Contrary to the membrane fraction, where 3 different protein bands are observed, only one band is detected in most western blots with PI-PLC supernatants.
      • For detection on western blots, a secondary, alkaline phosphatase-conjugated antibody was used and NBT (nitro blue tetrazolium) and BCIP (bromochloroindolyl phosphat) was used for the colour reaction.
  • FIG. 5: The RGM anti-sense probe hybridizes to an mRNA with graded expression along the anterio-posterior axis.
      • A, B: RGM-mRNA is expressed in a periventricular gradient in the tectum of an E9 chick embryo. In a more superficial layer (arrows), RGM is also expressed but at much lower level. The anterior tectal pole is to the right, the posterior to the left.
      • C, D: No staining is detected with the RGM sense probe, on parallel cryostat sections from E9 chick tecta. The anterior tectal pole is to the right, the posterior to the left.
  • FIG. 6: Recombinant RGM induces collapse of retinal growth cones.
      • A: RGC axons were grown on laminin-coated coverslips and affinity purified recombinant RGM was added at a final concentration of 10 ng/ml. More than 90% of temporal retinal growth cones are collapsed.
      • B: Neighboring, RGM-free fractions from affinity purification did not induce collapse of temporal growth cones. Supernatants from cos-7 cells transfected with an empty plasmid, did not possess any collapse-inducing activity (data not shown).
      • In A and B, retinal axons and growth cones were stained with the F-actin stain Alexa-Phalloidin.
  • FIG. 7: Recombinant RGM guides temporal retinal axons in the stripe assay.
      • A, B: Temporal retinal axons avoid the RGM-containing stripes (demarcated with red fluorescent beads). Membranes from RGM-transfected cos-7 cells (marked with beads) and anterior tectal membranes were used to prepare striped carpets.
      • C, D: Temporal retinal axons do not show any avoidance recation, when membranes from cos-7 cells, transfected with an empty plasmid (red beads) were used.
      • In A-D, striped membrane carpets were in addition coated with laminin to enhance retinal axon growth in accordance with a previous protocol (Monschau et al. 1997).
  • FIG. 8: RGM staining in endothel of (human) brain.
      • RGM immunoreactivity was detected in endothelial and vascular smooth muscle cells (SMC), both, in healthy, neuropathological unaltered control brains and injured brains, suggesting a constitutive, physiological role in vascular homeostasis.
  • FIG. 9: RGM expression in a lesion of a human being deceased due to severe brain injury (1-2 hours after his death). RGM expression on infiltrating cells from the immune system.
      • Upregulation of cellular RGM expression correlated with the time course and appearance of infiltrating leukocytes and activation of microglia/macrophages after injury (Stoll et al., 1998).
      • Early after injury (up to 2.5 days), RGM immunoreactivity was found on leukocytes of granulocytic, monocytic and lymphocytic origin in vessels within ischemic tissue.
      • Paralleled by edema formation, up to 1-7 days, RGM-positive cells were found extravasating outside the vascular walls into the focal ischemic lesioned parenchyma. In perivascular regions, RGM-positive cells formed clusters in the Virchow-Robin spaces from day 1-7, which subsided later. These peri-vascular cells, also referred to as adventitial or perithelial cells are characteristically alert immune cells (Kato and Walz, 2000; Streit et al., 1999).
  • FIG. 10: RGM expression in a brain lesion (human).
      • With increasing time after brain injury, most remarkable changes corresponded to areas of ongoing scar formation. In these areas, well defined extracellular RGM-positive laminae and RGM-positive fibroblastoid and reactive astrocytic cells were visible condensing adjacent to the border zone. These RGM-positive laminae increased in magnitude and regional extend over time.
  • The Examples illustrate the invention.
  • EXAMPLE I Microsequencing of an RGM Candidate
  • To separate RGM from the A-ephrins, a combination of two different ionexchange columns was employed. RGM, in contrast to the A-ephrins, bound to a strong cation exchanger and was eluted at a salt concentration of 200-400 mM NaCl. After incorporation of RGM into lecithin vesicles, strong collapse-inducing activity was observed in RGM-fractions (fractions 4+5, FIG. 1) but not in neighboring RGM-free fractions (fraction 6, FIG. 1). Neither ephrin-A5 nor ephrin-A2 was present in these fractions, proving thereby that RGM function does not require presence of the A-ephrins.
  • To get peptide sequences from RGM, microsequencing of all proteins, (cleaved from the membrane by treatment with the enzyme PI-PLC and having a molecular weight of 30-35 kDa and an isoelectric point between 7 and 9 was carried out). To this aim, anterior and posterior membranes from embryonic chick tecta (E9/10) were prepared with some modifications as described previously (Walter, Development 101, (1987), 685-96) and membrane pellets were subject to treatment with enzyme PI-PLC (E fraction) or buffer alone (C fraction). In particular, Membranes from embryonic chick tecta (E9/10) were prepared with some modifications as described previously (Walter et al., 1987). All steps were performed at 4° C. Tecta from 100 chick embryos were isolated and were divided into three parts equal in length along the anterior-posterior axis. The middle tectal parts were discarded and the anterior and posterior parts were worked up separately. Membranes were washed with PBS containing protease inhibitors and were centrifuged. Tectal membrane pellets were resuspended in triethanolamin buffer and were treated with the enzyme PI-PLC (50 mU Boehringer Mannheim/Roche Diagnostics GmbH), to remove glycosylphosphatidylinositol-anchored (GPI-anchored) proteins from the membranes. No PI-PLC was added to the other anterior and posterior tectal membrane fractions, the control-fractions (C). Enzyme (E) and control (C) fractions were incubated at 37° C. for 1.5 hours and membrane suspensions were centrifuged at 400.000 xg in a Beckmann TLA 100.3 rotor. Supernatants were collected and their protein concentrations were determined (Bradford 1976, modified by Zor and Selinger, 1996). Supernatants were precipitated with ice cold 10% trichloroacetic acid, were centrifuged and protein pellets were washed in ethanol-ether (1:1 v/v) and solubilized in sample buffer (8.5 M urea, 5% β-mercaptoethanol, 2.5% ampholytes pH 3-10, 2% NP 40).
  • E fractions and C fractions were loaded onto two dimensional gels, and after silver staining candidate proteins in the E-fractions (FIG. 2A, arrows) were cut out and subject to in gel tryptic digestion and nanoelectrospray ionization (Wilm, Nature 379 (1996), 466-9).
  • In detail, said 2D gelelectrophoresis and the protein sequence analysis was carried out as outlined herein below:
  • Tectal proteins resuspended in sample buffer, were separated using two dimensional gel electrophoresis. 20 μg of tectal protein was loaded on each gel. Non-equilibrium pH gradient electrophoresis (NEPHGE) followed by SDS-PAGE in the second dimension was performed as described by Boxberg (1988). After the SDS PAGE, gels were stained by a modified silver staining protocol from Heukeshoven and Demick (Heukeshoven & Demick, Electrophoresis 9, (1988), 372-375).
  • Silver-stained proteins in the 2D gels, with a basic isoelectric point and a molecular weight of 33/35 kDa, present in the PI-PLC treated E fraction but not in the C fraction, were cut out using a sharp and sterile scalpel.
  • Microsequencing was done using the technique of nano-electrospray tandem mass spectrometry as previously described (Wilm et al., 1996). The protein spots were digested in gel by trypsin and the resulting peptides were adsorbed and stepwise eluted into the electrospray source for mass spectral analysis. Nanoelectrospray was performed on an API III (Perkin-Elmer) mass spectrometer as described by Wilm and Mann (Wilm & Mann, Anal. Chem. 68 (1996), 1-8). After selecting an ionized peptide from the peptide mixture, the peptide was fragmented and the peptide fragments were analysed.
  • Mass spectrometric microsequencing of ionized peptides from the spot marked by arrows in FIG. 2, yielded ten different peptides, with lengths of 5-14 amino acids as shown in FIG. 2B; (SEQ ID NOs 1-10). The selected spot, was present in anterior and posterior PI-PLC supernatantes at similar levels. RGM is however more abundant in posterior than in anterior tectal membranes and the disappearance of the ap-difference in the 2D-gels was most likely caused by two different proteins unrelated to RGM and present in the selected spot.
  • EXAMPLE II Cloning of the RGM Gene
  • Three out of the ten peptide sequences (SEQ ID NOs 1 to 10) obtained by nanoelectrospray tandem mass spectrometry were used for synthesis of degenerate oligonucleotide primers and PCR experiments were performed as follows: Three out of the ten peptide sequences obtained by nanoelectrospray tandem mass spectrometry were used for synthesis of degenerate oligonucleotide primers and their complementary sequences.
  • P1F:
    (SEQ ID NO: 11)
    5′-ATGCC(AGCT)GA(AG)GA(AG)GT(AGCT)GT(AGCT)-3′
    P1R:
    (SEQ ID NO: 12)
    5′-TT(AGCT)AC(AGCT)AC(CT)TC(CT)TC(AGCT)GGCAT-3′
    P2F:
    (SEQ ID NO: 13)
    5′-GA(CT)AC(AGCT)TT(CT)CA(AG)AC(AGCT)TG(CT)AA-3′
    P2R:
    (SEQ ID NO: 14)
    5′-TT(AG)CA(AGCT)GT(CT)TG(AG)AA(AGCT)GT(AG)TC-3′
    P3F:
    (SEQ ID NO: 15)
    5′-AA(CT)CA(AG)CA(AG)(CT)T(AGCT)GA(CT)TT(CT)CA-3′
    P3R:
    (SEQ ID NO: 16)
    5′-TG(AG)AA(AG)TC(AGCT)A(AG)(CT)TG(CT)TG(AG)TT-3′
  • Moloney murine leukemia virus reverse transcriptase and random hexamer primers were used to synthesize single-stranded cDNA from E9 chick tectum total RNA. Combinations of forward (F) and reverse (R) primers were added to the cDNA and PCR amplification was done using Taq polymerase. The following PCR conditions were used: an initial denaturation step at 95° C. for 5 min followed by 30 cycles of 95° C. for 40 s, 50° C. for 1 min, 72° C. for 2 min. The PCR products were cloned into the pGEM T vector (Promega) and four positive clones were sequenced using the ALF express sequencer (Pharmacia). The sequence yielded an ORF, containing most of the peptide sequences obtained by microsequencing. The 459 by fragment was used for screening a cDNA library to obtain the full length sequence and for further analysis such as Northern blotting and in situ hybridization.
  • The PCR products were loaded onto agarose gels stained with ethidium bromide and a PCR product of 459 by in length, was obtained and cloned into the pGEM T vector. After sequencing, most of the peptide sequences were found in the PCR product, confirming that the correct candidate was amplified. The 459 by fragment was used for screening an E14 chicken brain cDNA library. Positive clones contained an insert of approximately 4 kb and sequencing confirmed the presence of the 459 by fragment and additional downstream sequences, including a stop codon. Upstream sequences were obtained by performing 5′-RACE.
  • In detail, the 459 by probe was used to screen 500.000 plaques of an E14 chicken brain library, cloned in the λ Zap vector. After two screening rounds, eight single plaques were isolated and the related inserts were cloned into the Bluescript vector using the rapid excision kit (Stratagene). The positive clones, analysed by restriction digestions, contained an insert of approximately 4 kb and sequencing confirmed the presence of the 459 by fragment and additional downstream sequences, including a stop codon. To get the sequence of the region upstream of the 459 by fragment, a 5′-RACE was performed according to the manufacturer's protocol using the RACE kit from Boehringer Mannheim and total RNA from E9 chick tecta. A 700 by band was amplified, purified, cloned into pGEM T vector, and 5 positive clones were sequenced. The sequence had an ORF with two methionines which could act as potential start sites. The full length sequence of RGM was confirmed independently several times.
  • For in situ and Northern blot experiments, the 459 by fragment was cloned into the Bluescript KS vector (Stratagene) and anti-sense and sense probes were produced by using the SP6 and T7 polymerases, respectively.
  • This 5′-RACE yielded an ORF with two methionines, potential start sites. The complete ORF of RGM is 1302 nucleotides in length and encodes a protein consisting of 434 amino acids (FIG. 3A; SEQ ID NO:17). Two hydrophobic domains are present at the N-terminus and C-terminus, respectively (FIG. 3B; SEQ ID NO:18), and two different algorithms suggested that the N-terminal hydrophobic domain encodes a signal peptide (best cleavage site predicted: at aa 29), the C-terminal domain, a GPI-anchor domain (best cleavage site predicted: at aa 406). RGM has no significant homology to any other protein, present in the databases and does not carry any specific domain or motif, except an triamino acid motif, the RGD site, a potential cell attachment site (Ruoshlahti, Annu. Rev. Cell Dev. Biol. 12 (1996), 697-715). Preliminary results suggest that this site is dispensable for RGM function. Polyclonal antibodies, named anti-RGM1 (against aas: 276-293) and anti-RGM2 (against aas: 110-130), raised against two peptides of the recombinant RGM molecule, recognize a GPI-anchored 33 kDa molecule, which is present at higher levels in posterior than in anterior tectal membrane PI-PLC supernatants (FIG. 4A). In membrane fractions at least three protein bands appear, a double band at 33 kDa and a single band at 35 kDa. These protein bands are recognized by the polyclonal anti-RGM1 antibody and the monoclonal F3D4 antibody (Müller (1996), loc. cit) (FIG. 4B). Both antibodies show identical staining patterns on western blots and immunoprecipitation experiments with anti-RGM1 resulted in pull down of a GPI-anchored, F3D4-positive protein. These results prove, that the antigens of the F3D4 monoclonal antibody and of the anti-RGM1 polyclonal antibody are identical.
  • RGM is the first member of a new class of axon guidance molecules, sharing no sequence homology with ephrins, netrins, slits, semaphorins and any other axon guidance molecules.
  • The corresponding human RGM sequence (SEQ ID NO:20) could be deduced by screening the human genome database with the deduced chicken RGM sequence.
  • EXAMPLE III RGM mRNA is Expressed in a Gradient in the Optic Tectum
  • To analyse expression of RGM-mRNA in the tectum opticum, an RGM anti-sense probe was used in in situ hybridization experiments on cryostat sections from E9 chick tecta. Strongest staining is observed in the periventricular layer, surrounding the tectal ventricle and staining intensity is much stronger in posterior tectum than in anterior tectum (FIG. 5A, B). Cell bodies of radial glial cells are located in the periventricular layer and the staining pattern confirms previous data using the monoclonal F3D4 antibody, where staining of glial endfeet and of glial cell bodies was observed (Mueller; (1996), loc. cit.; Mueller, (1997), loc. cit.). In a more superficial layer, a much weaker staining is detected with the RGM anti-sense probe but a differential expression between anterior and posterior tectal poles is hard to detect in this layer. In this layer tectal neurons are RGM-positive. This is in line with the expression of RGM by a subpopulation of tectal neurons. Overall, the staining pattern with the RGM anti-sense probe looks very similar to the expression pattern of ephrin-A5 with both messages being found in a periventricular and in a more superficial tectal layer. No staining is detectable with the RGM-sense probe.
  • On northern blots with tectal RNA, the RGM anti-sense probe marked two transcripts at 5.5 and 6.1 kb. Both messages are down-regulated at E14 with the smaller message being no longer detectable and the larger transcript being clearly present, albeit at lower levels.
  • RGM is active in in vitro assays and shows a graded expression in the tectum opticum of vertebrates. Based on Southern blot data it is assumed that there are least two additional family members which might have similar guidance activity. (see FIG. 11)
  • EXAMPLE IV Recombinant RGM is Active in Collapse and Stripe Assay
  • To analyse the function of recombinant RGM, the full length RGM cDNA was used to transfect cos-7 cells with a lipofection procedure. The full length RGM cDNA was cloned into the Kpnl site of the expression vector pTriEx-1 (Novagen). Cos-7 cells were transfected with the pTriEx-1 plasmid containing RGM cDNA or with the empty plasmid using the Superfect transfection reagent (Qiagen) according to the manufacturer's protocol. The DNA-Superfect mixture was added to Cos-7 cells growing in 10 cm dishes. 2 hours later medium was removed, cells were washed with PBS and grown for an additional 48 hours in fresh medium. Conditioned medium was collected, run over an RGM-affinity column and RGM-containing fractions and RGM-free control fractions were directly used in collapse assay experiments. For stripe assay experiments, RGM-transfected Cos-7 cells and empty plasmid transfected cells were washed with PBS and harvested using a rubber policeman in the presence of homogenization buffer containing protease inhibitors. Conditioned medium of cos-7 cells transfected with the RGM-pTriEx-1 plasmid was collected and run over an anti-RGM1 antibody column. Eluted fractions were evaluated with a sensitive and rapid dot blot assay and RGM-positive fractions were added to retinal axons growing on a laminin substratum. At a final concentration of 10 ng/ml, soluble RGM induced collapse of 90% of temporal RGC growth cones (FIG. 6A). Neighboring, RGM-free fractions or conditioned and concentrated supernatants from cos-7 cells transfected with the empty plasmid did not possess any collapse-inducing activity (FIG. 6B). Recombinant RGM is active in soluble form, is a strong difference between RGM and the A-ephrins and suggests a role for a chemotropic mechanism, in etablishing the retinotectal map.
  • For preparation of striped membrane carpets, membranes from RGM- or mock-transfected cells were used. Carpets consisting of alternate lanes of membranes from mock-transfected cos-7 cells and from RGM-transfected cells were offered to temporal and nasal RGC axons. To enhance the poor outgrowth-stimulating activity of cos-7 membranes, anterior tectal membranes or laminin were added. Collapse assay and stripe assays were prepared and employed as follows: The collapse assay was performed as descibed (Cox, (1990), loc. cit.; Wahl, J. Cell Biol. 149(2) (2000), 263-70). 5 μl of the RGM-positive fraction from the RGM-cos supernatant or supernatant from control cos cells or RGM-free fractions, was added to the retinal cultures. One hour later cultures were fixed by carefully adding 1 ml of fixative (4% paraformaldehyde, 0.33 M sucrose, pH 7.4). 4-12 hours later, cultures were washed and stained by Alexa-Phalloidin (Molecular Probes), following the recommendations of the manufacturer. Stained cultures were stored on a computer using a CCD camera and the images were analysed with the SIS analysis imaging software.
  • Stripe assay experiments were performed as previously described by Walter et al. (1987). Membrane carpets consisted of lanes of anterior tectal membranes mixed with membranes from RGM-transfected cos cells (ratio: 1:1), alternating with lanes consisting of anterior membranes mixed with membranes from empty plasmid transfected cos cells (ratio 1:1). In an alternative protocol, membrane carpets consisting of alternating lanes of membranes from RGM-transfected cos cells and of control cos membranes, were incubated for 2 hours at 37° C. with 20 μg/ml laminin (Becton-Dickinson). Before use, the carpets were washed with Hank's buffer (2×).
  • On these carpets, temporal RGC axons, but not nasal axons, showed a clear repulsive avoidance behaviour, growing on the RGM-free membrane stripes (FIG. 7 A-D). These results demonstrate, that the recombinant RGM protein is not only active in collapse but also in stripe assays.
  • RGM shares with the A-ephrins A2 and A5 the GPI-anchor, the graded expression and functional activity in two different in vitro systems. Its activity is however different from the two A-ephrins in other respects. The specificity of its activity is not restricted to temporal axons and growth cones. Nasal axons and growth cones also react, albeit at higher RGM concentrations. This is in line with the previous observations, that temporal retinal axons react more strongly to RGM than nasal retinal axons (Stahl, (1990), loc.cit). For ephrin-A5, a slight difference in sensitivity of temporal and nasal retinal axons has been observed, this difference is however not as pronounced as with RGM (Drescher, Cell 82 (1995), 359-70). Besides the stronger concentration dependancy of RGM function, another crucial difference is that RGM, in contrast to both ephrin-A5 and ephrin-A2, seems to be active in soluble form and apparently does not require aggregation to stimulate its currently unknown retinal receptor. These in vitro results underscore the difference between ephrins and RGM. In the stripe assay, inactivation of RGM using the F3D4 monoclonal antibody and the chromophore-assisted laser inactivation (CALI) method, resulted in complete neutralization of repulsive guidance activities of posterior tectal membranes in more than 50% of the experiments (Mueller, (1996), loc. cit.) F3D4 however neither binds ephrin-A2 nor ephrin-A5 (Mueller, (1997), loc. cit.) and it was therefore suggested that the A-ephrins and RGM somehow interact in special membrane domains to which they are recruited by their GPI-anchors. Such a colocalization could explain the result, that inactivation of RGM lead in addition to inactivation of ephrin-A2 and ephrin-A5 and could explain the complete neutralization observed in the stripe assay experiments (Mueller, (1996), loc. cit.). The functional relationship of RGM with ephrin-A2 and ephrin-A5 and the in vivo role of RGM need to be addressed, especially since both ephrins have been shown to be important molecular determinants for topographic map formation in vertebrates (Nakamoto, Cell 86 (1996), 755-66; Frisen, Neuron 20 (1998), 235-43; Feldheim, Neuron 21 (1998), 563-74; Picker, Development 126 (1999), 2967-78; Feldheim, Neuron 25 (2000), 563-74; Brown, Cell 102 (2000), 77-88). There are however evidences from two vertebrates, which suggest that others factors, besides the ephrins, are required for formation of the retinotectal map. Deletion of either the ephrin-A2 or the ephrin-A5 gene in mice, resulted in mapping phenotypes with some retinal axons forming ectopic termination zones in the superior colliculus (SC), the mammalian homologue of the optic tectum, and with nasal retinal axons overshooting the SC and terminating in the inferior colliculus. In ephrin-A2−/− mice, temporal axons showed mapping errors with ectopic termination zones, but nasal axons did not show any mapping errors in contrast to the ephrin-A5−/− mice which had defects in topographic mapping of nasal but not temporal axons (Frisen, (1998), loc.cit.; Feldheim, (2000), loc. cit.). Deletion of both genes should therefore result in a much more disturbed mapping of both nasal and temporal retinal axons along the anterior-posterior axis of the SC. This is actually observed in double mutant ephrin-A2−/− A5−/− homozygotes but a topographic bias of both nasal and temporal axons was still present, with the majority of temporal and nasal retinal axons being confined to their anterior and posterior tectal halfs, respectively (Feldheim, (2000), loc. cit.; Goodhill, Neuron 25 (2000), 501-3). These results point to a role of RGM as one of the additional factors required for mapping along the anterior-posterior axis. Such a role is supported by the graded anterior-low posterior-high expression of this molecule in the SC of mammals (Mueller, (1997), loc. cit.).
  • The zebrafish mutant acerebella (ace) is mutant in fgf8 and lacks the midbrain-hindbrain boundary region and the cerebellum (Reifers, Development 125 (1998), 2381-95; Picker, (1999), loc. cit.). As a result the tectum is much smaller in ace mutants than in wildtype and the expression levels of all three zebrafish A-ephrins are changed with ephrin-A2 and ephrin-A5a being still expressed at low and anterior levels in ace tecta and with ephrin-A5b being completely eliminated (Picker et al., 1999). In ace mutant tecta, mapping of retinal axons along the anterior-posterior axis is normal in dorsal tectum and is not completely lost in ventral tectum, suggesting the involvement of other graded guidance cues, not seriously affected by the fgf8 mutation in the ace zebrafish mutants (Picker et al., 1999). Dorsoventral patterning in both zebrafish ace mutants and ephrin-A2−/− A5−/− double knock out mice is affected.
  • RGM, with its graded expression along the anterior-posterior axis of the tectum and its ability to function in a secreted and membrane-coupled way, is an important player for topographic map formation.
  • EXAMPLE V Materials and Methods 1. Patients
  • 21 brains of patients with clinical history and neuropathologically confirmed diagnosis of focal cerebral infarctions and 25 brains of patients with traumatic brain injury were included in this study. Infarctioned brain tissue was derived from an updated stroke and trauma brain-bank (Table 1,2) reported previously (Postler et al., 1997, Beschomer et al., 2000). Tissue specimen procurement was performed according to the ethical guidelines of the University of Tuebingen. Patients with altered immune status because of immunosuppressive therapy or meningitis/encephalitis were excluded from this study. As controls, the results were compared to tissue from corresponding areas of 4 normal non-ischemic brains described previously (Schwab et al., 2000). In addition to patient data, haematoxyline-eosine (HE), luxol fast blue (LFB) and iron (Fe) staining was used for evaluation of the typical histological features defined as standard indication of infarct (Kalimo et al., 1996) and trauma age (Graham and Gennarelli, 1996).
  • 2. Immunohistochemistry
  • After formaldehyde fixation and paraffin-embedding, rehydrated 2 μm sections were boiled (in an 600 W microwave oven) seven times for 5 min in citrate buffer (2.1 g sodium citrate/liter, pH 7.4). Endogenous peroxsidase was inhibited with 1% H2O2 in methanol (1:10; 15 min). Sections were incubated with 10% normal porcine serum (Biochrom, Berlin, FRG) to block non-specific binding of immunoglobulins. Monospecific polyclonal antibodies directed against RGM were diluted (1:10) in 1% BSA (bovine serum albumin) TBS (Tris-balanced salt solution, containing 0.025 M Tris, 0.15 M NaCl) and incubated over night at room temperature. Specific binding of the antibodies were detected with a secondary biotinylated swine anti-rabbit IgG F(ab)2 antibody fragment 1:400 for 30 min (DAKO, Hamburg, FRG), followed by incubation with a peroxidase conjugated streptavidin-biotin complex (DAKO, Hamburg, FRG). The enzyme was visualized with diaminobenzidine as a chromogen (Fluka, Neu-Ulm, FRG). Sections were counterstained with Mayer's Hemalaun. Negative controls consisted of sections incubated in the absence of the primary antibody. Specificity of polyclonal RGM antibody was confirmed by inhibition of staining using human ischemic brain tissue after pre-incubation for 3 h on ice with access of the cognate RGM peptide.
  • 3. Double Labeling Experiments
  • In double labelling experiments, a cell-type or activation specific antigen was first labelled using the ABC procedure in combination with alkaline phosphatase conjugates. Specific antigens were labelled with antibodies against GFAP (glial fibrillary acidic protein, monoclonal, Boehringer Mannheim, Germany, 1:100) to detect astrocytes, MBP (myelin basic protein, polyclonal, oligodendrocytes, Dako, 1:500) and CD68 (Dako, 1:100) for microglia/macrophage identification. Activated microglia/macrophages were detected with antibodies directed against HLA-DR, -DP, -DQ (MHC class II, DAKO, Glostrup, Denmark, 1:100) or MRP-8 (8-5C2, BMA, Augst, Switzerland, 1:100) (Postler et al., 1997). Lymphocytic subpopulations were classified with monoclonal antibodies against CD4 (T-helper lymphocytes, 1:10, Dako) and CD8 (T cytotoxic/suppressor lymphocytes, 1:500, Dako) and CD20 (pan B cell marker, 1:200, Dako). In order to detect extracellular basal lamina structures in vessels and during scar formation mouse laminin (1:500, Chemicon) antibodies were used and rabbit fibronectin (1:100, Dako) antibodies were used to detect matrix deposition. Furthermore, in order to characterize the cellular proliferation response, sections were incubated with the S phase specific PCNA (proliferating cell nuclear antigen, 1:100, Dako) monoclonal antibodies. Briefly, slices were deparaffinized, irradiated in a microwave oven for antigen retrieval and incubated with non specific porcine serum as described above. Visualization was achieved by adding biotinylated secondary antibodies (1:400) for 30 min and alkaline phosphatase conjugated ABC complex diluted 1:400 in TBS-BSA for 30 min. Consecutively, slices were developed with Fast-Blue BB salt chromogen-substrate solution yielding a blue reaction product. Between double labelling experiments, slices were irradiated in a microwave for 5 min in citrate buffer. Then RGM was immunodetected as described above.
  • 4. Evaluation and Statistical Analysis
  • Data were calculated as means of labelled cells (MLC, ±SEM) from border zones or remote areas of the same tissue section and were compared to normal control brains using the two-tailed unpaired student's t-test. Border zones were defined as peri-lesional areas adjacent to the developing necrotic core demarcating the region of major damage. RGM+ cells were counted in ten high power fields (HPF, ×200 magnification with an eye-piece-grid representing 0.25 mm2).
  • Results
  • 21 brains of patients with focal cerebral infarctions (FCI), 25 brains with traumatic brain injury (TBI) and 4 control brains were evaluated for RGM protein expression by immunohistochemistry.
  • 1. Healthy, Neuropathological Unaltered Control Brains
  • In control brains without neuropathologically alterations, RGM immunoreactivity was detected on white matter fibres, oliogodendrocytes, the perikarya of some neurons and RGM+ cells were also detected in the choroid plexus (FIG. 8) and ependyma. Only single cells were detected in peri-vascular spaces. Further, some smooth muscle cells and few endothelial cells but no astrocytes were labelled.
  • 2. Focal Cerebral Ischemia (FCI)
  • It was analysed whether number and distribution of RGM expressing cells is altered after cerebral infarctions. Results suggested, that RGM expression is lesion-associated. Cellular RGM expression was confined to neurons, few reactive astrocytes and invading leukocytes. With the ageing of the lesions, RGM-positive extracellular laminae components were found in the constituting scar.
  • RGM-positive cells accumulated in infarctioned white matter, hemorrhagic areas, infarction core and peri-infarctional areas, respectively. Using the student's t-test, a significantly (P<0.0001) higher number of RGM+ cells was detected in peri-infarcional areas (MLC=24, SEM=1.1) than in remote areas (MLC=2, SEM=0.2) or control tissue (MLC=6, SEM=0.8). The morphological described peri-infarctional areas were part of the physiologically defined penumbra. In these areas the number of RGM-positive cells accumulated already up to day 1 (p<0.0001, MLC=31.93, SEM=2.3) reached their maximum 1.5-2.5 days (MLC=34, SEM=3.2) after infarction and remained elevated up to several weeks and months of survival (MLC=11, SEM=1.4). Early after ischemic damage (up to 2.5 days), RGM immunoreactivity was predominantly found on neurons and leukocytes of granulocytic, monocytic and lymphocytic origin in vessels within ischemic tissue. Paralleled by edema formation, up to 1-7 days, RGM-positive cells were found extravasating outside the vascular walls into the focal ischemic lesioned parenchyma. In perivascular regions, RGM-positive cells formed clusters in the Virchow-Robin spaces from day 1-7, which subsided later. These peri-vascular cells, also referred to as adventitial or perithelial cells are characteristically alert immune cells (Kato and Walz, 2000; Streit et al., 1999). With lesion aging, from day 3 onwards, lesional RGM expression by few reactive astrocytes, was observed. At later stages, arising 1 week after infarction, extracellular RGM deposits were detected constituting neo-laminae localized to areas of ongoing scar formation. These RGM-positive laminae increased in magnitude and regional extend over time. With tissue reorganisation of the lesion, also “foamy”, lipid loaded RGM-positive phagocytic RGM-positive microglia/macrophages were observed.
  • Upregulation of cellular RGM expression correlated with the time course and appearance of infiltrating leukocytes and activation of microglia/macrophages after injury (Stoll et al., 1998). Whereas upregulation of extracellular RGM expression correlated with the time course and the appearance of the scar after injury. In few cases (<5% of counterstained nuclei) some reactive astrocytes restricted to the demarcating lesion core also expressed RGM.
  • 3. Traumatic Brain Injury
  • In patients who died after TBI, in accordance to cerebral infarction (FCI) the immunohistological evaluation revealed early cellular membranous, cytoplasmatic and nuclear RGM expression by leukocytes, few reactive astrocytes and neurons with strong staining of their perikarya, dendrites and axons (FIG. 9). During the observed time post TBI, within the necrotic core and the bordering peri-necrotic parenchyma accumulation of RGM-positive cells (p<0.0001) was detected in border zones (MLC=22, SEM=0.7) compared to remote areas (MLC=1, SEM=0.1) and normal brain controls (MLC=5.8, SEM=0.8). Following TBI, RGM-positive cell numbers arose already during the first 24 hours (p<0.0001) where RGM-positive cell numbers reached maximum levels (MLC=29, SEM=0.9) and decreased subsequently. With increasing time after TBI, most remarkable changes corresponded to areas of ongoing scar formation (FIG. 10). In these areas, well defined extracellular RGM-positive laminae were visible condensing adjacent to the border zone. RGM immunoreactivity was also detected in endothelial and vascular smooth muscle cells (SMC) but no significant differences were observed between injured and control brains.
  • References as mentioned in the example herein above:
    • Beschomer, Acta Neuropathol. 100 (2000), 377-384
    • Graham, “Greenfield's Neuropathology.” D. I. Graham and P. L. Lantos (eds), 6th. Edn., Edward Arnold, London (1996), pps. 197-248
    • Kalimo, Greenfield's Neuropathology 6th. Edn. Arnold, London Sydney Auckland (1996), pp 315-381.
    • Kato, Brain Pathol., 10 (2000), 137-143.
    • Postler, Glia 19 (1997), 27-34.
    • Schwab, Acta Neuropathol. 99 (2000), 609-614.
    • Stoll, Prog. Neurobiol. 56 (1998), 149-171.
    • Streit, Prog. Neurobiol. 57 (1999), 563-581.
    EXAMPLE VI Change of Tumor Growth Behaviour in Mice
  • Hybridoma cells secreting the RGM-specific F3D4 monoclonal antibody were injected into the peritoneum of mice, primed with mineral oil. Normally the hybridoma cells continue to divide in the peritoneum and the hybridoma cells secreted large amounts of antibody, resulting in formation of large ascites tumors. Mice receiving the F3D4-producing hybridoma cells did not develop ascites tumors in the peritoneal cavity, but developed solid, adherent tumors. The F3D4 monoclonal antibody resulted in a change of phenotype of the tumorigenic hybridoma cells from a less invasive, non-adherent state to an invasive adherent state. Masking of endogeneous RGM by the antibodies secreted from the hybridoma cells, enabled adhesion and invasion of these tumor cells and was responsible for this outcome.
  • EXAMPLE VII Detection of a Functional RGM Fragment
  • RGM was cloned into the pTriEx vector and the vector was cut inside the polylinker side and inside the RGM sequence using Sad in the first step. After ligation of both ends the RGM containing vector was cut in the second step with Stul inside the RGM sequence and in the polylinker with Pa. After ligation of both ends, the vector with the shorter RGM-fragments was transfected into COS7 cells. Cell lysates of these COS cells were purified using an anti-RGM1 affinity column and RGM-containing fractions were used in collapse assay experiments. A fragment as described in SEQ ID NO:19 was active in said assays.
  • EXAMPLE VIII Detection of Further RGMs
  • A publicly available computer database at the National Center for Biotechnology Information (NCBI, USA) was used to identify human genes homologous to chicken RGM, employing the information and data illustrated in the examples herein above. A search strategy based on the Blast algorithm (NCBI) resulted in three human genes located on chromosomes 1, 5 and 15. The corresponding contigs are NT021932.5 (RGM3), NT029283.2 (RGM2) and NT010370.5 (RGM1), respectively. cDNA sequences for RGM 1, 2 and 3 were derived from these genomic sequences by omitting introns and fusing the remaining exons.
  • Corresponding amino acid and nucleotide sequences for human RGM2 are illustrated in appended SEQ ID NOs: 22 and 23. Human RGM3-sequences are shown in SEQ ID NOs: 24 and 25.

Claims (13)

1. The method of a modulator of a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for preventing, alleviating or treating diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue, associated with seizures associated with angiogenic disorders or disorders of the cardio-vascular system.
2. The method of claim 1 wherein said diseases or conditions associated with the degeneration or injury of vertebrate nervous tissue are selected from the group consisting of neurodegenerative diseases, nerve fiber injuries and disorders related to nerve fiber losses.
3. The method of claim 2, wherein said neurodegenerative disease is selected from the group consisting of motomeuronal diseases (MND), ALS, Alzheimer's disease, Parkinson's disease, progressive bulbar palsy, progressive muscular atrophy, HIV-related dementia and spinal muscular atrophy(ies), Down's Syndrome, Huntington's Disease, Creutzfeldt-Jacob Disease, Gerstmann-Straeussler Syndrome, kuru, Scrapie, transmissible mink encephalopathy, other unknown prion diseases, multiple system atrophy, Riley-Day familial dysautonomia wherein said nerve fiber injuries are selected from the group consisting of spinal cord injury(ies), brain injuries related to raised intracranial pressure, trauma, secondary damage due to increased intracranial pressure, infection, infarction, exposure to toxic agents, malignancy and paraneoplastic syndromes and wherein said disorders related to nerve fiber losses are selected from the group consisting of paresis of nervus facialis, nervus medianus, nervus ulnaris, nervus axillaris, nervus thoracicus longus, nervus radialis and for of other peripheral nerves.
4. The method of claim 1, wherein said disease or condition associated with seizures is epilepsy.
5. The method of claim 1, wherein said disease or conditions associated with angiogenic disorders are selected from the group consisting of ischemic disorders, infarction, disorders leading to the progression of vascular plaque formation in cardio-vascular, cerebrovascular and/or nephro-vascular disorders or disorders wherein the permeability of the blood-brain barrier has to be regulated.
6. The method of claim 1, wherein said disorders of the cardio-vascular system comprises disorders of the blood-brain barrier, brain oedema, secondary brain damages due to increased intracranial pressure, infection, infarction, ischemia, hypoxia, hypoglycemia, exposure to toxic agents, malignancy, paraneoplastic syndromes.
7. The method of a modulator of a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative for the preparation of a pharmaceutical composition for remyelinization and for modifying and/or altering the differentiation of neuronal stem cells and/or their progenitors.
8. (canceled)
9. The method of claim 1, wherein said modulator is an antibody or a fragment or a derivative thereof, is an aptamer, is a specific receptor molecule capable of interacting with a polypeptide having or comprising the amino acid sequence of SEQ ID NO: 18, 20, 23 or 25 or with a functional fragment or derivative thereof or is a specific nucleic acid molecule interacting with a polynucleotide encoding said polypeptide.
10. The method of a polypeptide having or comprising the amino acid sequence of SEQ ID NOs 18, 20, 23 or 25 or of a functional fragment or derivative thereof or of a polynucleotide encoding such polypeptide or fragment as derivative for the preparation of a pharmaceutical composition for preventing or treating diseases or conditions associated with excessive collateral sprouting of nerve fibres tumor growth or formation of tumor metastases, for preventing, alleviating or treating diseases or conditions associated with the activity of autoreactive immune cells or with overreactive inflammatory cells, for the treatment of inflammation processes and/or allergies, for wound healing or for the suppression/alleviation of scar formation, of a functional fragment or derivative thereof or of a polynucleotide encoding said polypeptide or fragment or derivative as a marker of stem cells, and for alleviating, preventing and/or treating homoestatic and/or bleeding disorders and/or vascular damage.
11.-15. (canceled)
16. The method of claim 10, wherein said polypeptide or fragment or derivative is soluble.
17. The method of an antibody or a fragment or a derivative thereof, is an aptamer, is a specific receptor molecule capable of interacting with a polypeptide having or comprising the amino acid sequence of SEQ ID NO: 18, 20, 23 or 25 or with a functional fragment or derivative thereof, or is a specific nucleic acid molecule interacting with a polynucleotide encoding said polypeptide for the preparation of a diagnostic composition for detecting neurological, neurodegenerative disorders or dispositions thereto.
US12/939,823 2000-12-22 2010-11-04 Use of rgm and its modulators Abandoned US20110112280A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US12/939,823 US20110112280A1 (en) 2000-12-22 2010-11-04 Use of rgm and its modulators
US13/493,005 US8680239B2 (en) 2000-12-22 2012-06-11 Use of RGM and its modulators

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
EP00128356.3 2000-12-22
EP00128356 2000-12-22
PCT/EP2001/015289 WO2002051438A2 (en) 2000-12-22 2001-12-21 Use of repulsive guidance molecule (rgm) and its modulators
US10/451,586 US7981420B2 (en) 2000-12-22 2001-12-21 Therapeutic use of antibodies directed against repulsive guidance molecule (RGM)
US12/939,823 US20110112280A1 (en) 2000-12-22 2010-11-04 Use of rgm and its modulators

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
US10451586 Continuation 2001-12-21
PCT/EP2001/015289 Continuation WO2002051438A2 (en) 2000-12-22 2001-12-21 Use of repulsive guidance molecule (rgm) and its modulators
US10/451,586 Continuation US7981420B2 (en) 2000-12-22 2001-12-21 Therapeutic use of antibodies directed against repulsive guidance molecule (RGM)

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/493,005 Continuation US8680239B2 (en) 2000-12-22 2012-06-11 Use of RGM and its modulators

Publications (1)

Publication Number Publication Date
US20110112280A1 true US20110112280A1 (en) 2011-05-12

Family

ID=8170794

Family Applications (3)

Application Number Title Priority Date Filing Date
US10/451,586 Expired - Fee Related US7981420B2 (en) 2000-12-22 2001-12-21 Therapeutic use of antibodies directed against repulsive guidance molecule (RGM)
US12/939,823 Abandoned US20110112280A1 (en) 2000-12-22 2010-11-04 Use of rgm and its modulators
US13/493,005 Expired - Lifetime US8680239B2 (en) 2000-12-22 2012-06-11 Use of RGM and its modulators

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US10/451,586 Expired - Fee Related US7981420B2 (en) 2000-12-22 2001-12-21 Therapeutic use of antibodies directed against repulsive guidance molecule (RGM)

Family Applications After (1)

Application Number Title Priority Date Filing Date
US13/493,005 Expired - Lifetime US8680239B2 (en) 2000-12-22 2012-06-11 Use of RGM and its modulators

Country Status (6)

Country Link
US (3) US7981420B2 (en)
EP (1) EP1355666B1 (en)
JP (2) JP4986370B2 (en)
AU (1) AU2002231736A1 (en)
ES (1) ES2390425T3 (en)
WO (1) WO2002051438A2 (en)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100028340A1 (en) * 2008-02-29 2010-02-04 Abbott Gmbh & Co. Kg Antibodies against the rgm a protein and uses thereof
US20100322948A1 (en) * 2007-09-06 2010-12-23 Abbott Gmbh & Co. Kg Bone morphogenetic protein (BMP)-binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and use of same
US20110135664A1 (en) * 2009-12-08 2011-06-09 Abbott Gmbh & Co. Kg Monoclonal antibodies against the rgm a protein for use in the treatment of retinal nerve fiber layer degeneration
US8680239B2 (en) 2000-12-22 2014-03-25 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Use of RGM and its modulators
US8906864B2 (en) 2005-09-30 2014-12-09 AbbVie Deutschland GmbH & Co. KG Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use
US9102722B2 (en) 2012-01-27 2015-08-11 AbbVie Deutschland GmbH & Co. KG Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration
US10055540B2 (en) 2015-12-16 2018-08-21 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US20190275118A1 (en) * 2013-09-17 2019-09-12 University Health Network Agents directed against a cis rgma/neogenin interaction or lipid rafts and use of the same in methods of treatment
US11264117B2 (en) 2017-10-10 2022-03-01 Gritstone Bio, Inc. Neoantigen identification using hotspots
US11885815B2 (en) 2017-11-22 2024-01-30 Gritstone Bio, Inc. Reducing junction epitope presentation for neoantigens

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003089608A2 (en) * 2002-04-18 2003-10-30 The General Hospital Corporation Drg11-responsive (dragon) gene family
AU2003280420A1 (en) * 2002-06-26 2004-01-19 Yale University Modulators and modulation of the interacton between rgm and neogenin
EP1380644A1 (en) * 2002-07-08 2004-01-14 Kylix B.V. The use of specified TCF target genes to identify drugs for the treatment of cancer, in particular colorectal cancer, in which TCF/beta-catenin/WNT signalling plays a central role
WO2004092405A2 (en) 2003-04-15 2004-10-28 Xenon Pharmaceuticals Inc. Juvenile hemochromatosis gene (hfe2a), expression products and uses thereof
US20070253946A1 (en) * 2004-03-11 2007-11-01 Bioclues, Inc. Axon Regeneration Promoter
US20060063208A1 (en) 2004-08-02 2006-03-23 Woolf Clifford J DRG11-responsive (DRAGON) gene and uses thereof
EP1858541B1 (en) 2005-02-16 2012-07-25 The General Hospital Corporation Use of bmp antagonists to regulate hepcidin-mediated iron metabolism and treat iron deficiency
JPWO2008038599A1 (en) * 2006-09-25 2010-01-28 国立大学法人 千葉大学 Axon regeneration promoter
US8895002B2 (en) 2007-04-09 2014-11-25 The General Hospital Corporation Hemojuvelin fusion proteins and uses thereof
KR102284780B1 (en) 2009-12-09 2021-08-02 미쓰비시 타나베 파마 코퍼레이션 T cell activation inhibitor, pharmaceutical composition containing same, and screening method for t cell activation inhibiting substance
KR101297037B1 (en) * 2010-03-26 2013-08-14 숙명여자대학교산학협력단 Peptides for Promotion of Angiogenesis and the use thereof
BR112017004883A2 (en) 2014-09-10 2017-12-05 Abbvie Deutschland rgma fragment-based diagnostic assay
PL3290441T3 (en) 2015-04-28 2020-05-18 Mitsubishi Tanabe Pharma Corporation Rgma binding protein and use thereof
CA3169224A1 (en) 2015-09-23 2017-03-30 University Health Network Methods, compounds and compositions for modulating blood brain barrier integrity and re-myelination
JP7449859B2 (en) * 2018-07-10 2024-03-14 田辺三菱製薬株式会社 Method for preventing or treating pain associated with peripheral neuropathy or a disease in which peripheral neuropathy or astrocyte disorder is observed

Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030087394A1 (en) * 2001-08-31 2003-05-08 Arun Sharma Insulin related transcription factor and uses thereof
US20030212001A1 (en) * 2002-03-27 2003-11-13 Peri Krishna G. Methods and compounds for prevention and treatment of elevated intraocular pressure and related conditions
US20040038292A1 (en) * 2001-06-18 2004-02-26 Burslem Martyn Frank Wound healing biomarkers
US20040071711A1 (en) * 2000-11-06 2004-04-15 Roy Bicknell Imaging, diagnosis and treatment of disease
US20050197284A9 (en) * 2002-06-11 2005-09-08 Murat Digicaylioglu Neuroprotective synergy of erythropoietin and insulin-like growth factors
US20070122491A1 (en) * 2005-10-14 2007-05-31 Alltech, Inc. Methods and compositions for altering cell function
US20070155687A1 (en) * 2005-10-14 2007-07-05 Alltech, Inc. Methods and compositions for altering cell function
US20080004255A1 (en) * 2005-10-14 2008-01-03 Alltech, Inc. Methods and compositions for altering cell function
US20080008692A1 (en) * 2005-10-14 2008-01-10 Alltech, Inc. Methods and compositions for altering cell function
US20080105705A1 (en) * 2006-10-18 2008-05-08 Schmidt Norman G Device to allow for cleaning access in semi-solid metering machines
US20080135582A1 (en) * 2006-10-02 2008-06-12 Schmidt Norman G Device for controlled metering of semi solid food products
US20080160034A1 (en) * 2004-08-25 2008-07-03 Genentech, Inc. Novel Gene Disruptions, Compositions and Methods Relating Thereto
US20080213791A1 (en) * 2005-11-08 2008-09-04 Euclid Diagnostics Llc Materials and methods for assaying for methylation of CpG islands associated with genes in the evaluation of cancer
US20080279859A1 (en) * 2006-11-21 2008-11-13 Abbott Laboratories NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST THE NOGO-66 RECEPTOR (NgR) AND USES THEREOF
US20090028852A1 (en) * 2004-11-16 2009-01-29 Trustees Of Boston University Roles For Dual Endothelin-1/Angiotensin II Receptor (DEAR) In Hypertension and Angiogenesis
US20090220588A1 (en) * 2008-02-21 2009-09-03 Massachusetts Institute Of Technology Simultaneous Delivery of Receptors and/or Co-Receptors for Growth Factor Stability and Activity
US20090220589A1 (en) * 2008-03-01 2009-09-03 Abraxis Bioscience, Llc Treatment, diagnostic, and method for discovering antagonist using sparc specific mirnas
US20090269356A1 (en) * 2006-03-08 2009-10-29 David Epstein Complement Binding Aptamers and Anti-C5 Agents Useful in the Treatment of Ocular Disorders
US20090311253A1 (en) * 2008-06-03 2009-12-17 Abbott Laboratories Dual Variable Domain Immunoglobulins and Uses Thereof
US20100183588A1 (en) * 2004-11-22 2010-07-22 Jean Plouet Mutated Netrin-4, Fragments Thereof And Their Use As Medicines

Family Cites Families (238)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4394448A (en) 1978-02-24 1983-07-19 Szoka Jr Francis C Method of inserting DNA into living cells
US5179017A (en) 1980-02-25 1993-01-12 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4634665A (en) 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
DE3378250D1 (en) 1982-04-22 1988-11-24 Ici Plc Continuous release formulations
US4510245A (en) 1982-11-18 1985-04-09 Chiron Corporation Adenovirus promoter system
GB8308235D0 (en) 1983-03-25 1983-05-05 Celltech Ltd Polypeptides
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5807715A (en) 1984-08-27 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin
US5128326A (en) 1984-12-06 1992-07-07 Biomatrix, Inc. Drug delivery systems based on hyaluronans derivatives thereof and their salts and methods of producing same
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
DE229046T1 (en) 1985-03-30 1987-12-17 Marc Genf/Geneve Ch Ballivet METHOD FOR OBTAINING DNA, RNS, PEPTIDES, POLYPEPTIDES OR PROTEINS BY DNA RECOMBINANT METHOD.
US6492107B1 (en) 1986-11-20 2002-12-10 Stuart Kauffman Process for obtaining DNA, RNA, peptides, polypeptides, or protein, by recombinant DNA technique
US4980286A (en) 1985-07-05 1990-12-25 Whitehead Institute For Biomedical Research In vivo introduction and expression of foreign genetic material in epithelial cells
US5618920A (en) 1985-11-01 1997-04-08 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US4968615A (en) 1985-12-18 1990-11-06 Ciba-Geigy Corporation Deoxyribonucleic acid segment from a virus
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5635600A (en) 1986-07-07 1997-06-03 Trustees Of Dartmouth College Bifunctional and heteroantibodies specific for the high affinity Fc receptor for immunoglobulin G on human mononuclear phagocytes
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
DE3750503T2 (en) 1986-10-22 1995-02-09 Abbott Lab Chemiluminescent acridinium and phenantridinium salts.
EP0279582A3 (en) 1987-02-17 1989-10-18 Pharming B.V. Dna sequences to target proteins to the mammary gland for efficient secretion
DE3883899T3 (en) 1987-03-18 1999-04-22 Sb2 Inc CHANGED ANTIBODIES.
US5258498A (en) 1987-05-21 1993-11-02 Creative Biomolecules, Inc. Polypeptide linkers for production of biosynthetic proteins
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
US4880078A (en) 1987-06-29 1989-11-14 Honda Giken Kogyo Kabushiki Kaisha Exhaust muffler
US5006309A (en) 1988-04-22 1991-04-09 Abbott Laboratories Immunoassay device with liquid transfer between wells by washing
US5089424A (en) 1988-06-14 1992-02-18 Abbott Laboratories Method and apparatus for heterogeneous chemiluminescence assay
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5241070A (en) 1988-09-26 1993-08-31 Ciba Corning Diagnostics Corp. Nucleophilic polysubstituted aryl acridinium esters and uses thereof
EP0368684B2 (en) 1988-11-11 2004-09-29 Medical Research Council Cloning immunoglobulin variable domain sequences.
US5063081A (en) 1988-11-14 1991-11-05 I-Stat Corporation Method of manufacturing a plurality of uniform microfabricated sensing devices having an immobilized ligand receptor
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5028535A (en) 1989-01-10 1991-07-02 Biosite Diagnostics, Inc. Threshold ligand-receptor assay
US5939272A (en) 1989-01-10 1999-08-17 Biosite Diagnostics Incorporated Non-competitive threshold ligand-receptor assays
GB8901334D0 (en) 1989-01-21 1989-03-15 Oakland Design Products Ltd Improvements relating to broadhead arrows
US5703055A (en) 1989-03-21 1997-12-30 Wisconsin Alumni Research Foundation Generation of antibodies through lipid mediated DNA delivery
CA2016841C (en) 1989-05-16 1999-09-21 William D. Huse A method for producing polymers having a preselected activity
WO1990014443A1 (en) 1989-05-16 1990-11-29 Huse William D Co-expression of heteromeric receptors
CA2016842A1 (en) 1989-05-16 1990-11-16 Richard A. Lerner Method for tapping the immunological repertoire
US5139400A (en) 1989-10-11 1992-08-18 Ide Russell D Progressive cavity drive train
AU642932B2 (en) 1989-11-06 1993-11-04 Alkermes Controlled Therapeutics, Inc. Protein microspheres and methods of using them
FR2656431B1 (en) 1989-12-22 1994-06-10 Essilor Int METHOD AND SOLUTION FOR DECONTAMINATING A FLEXIBLE LENS, ESPECIALLY OF THE HYDROPHILIC TYPE.
US5780225A (en) 1990-01-12 1998-07-14 Stratagene Method for generating libaries of antibody genes comprising amplification of diverse antibody DNAs and methods for using these libraries for the production of diverse antigen combining molecules
EP1690935A3 (en) 1990-01-12 2008-07-30 Abgenix, Inc. Generation of xenogeneic antibodies
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5922615A (en) 1990-03-12 1999-07-13 Biosite Diagnostics Incorporated Assay devices comprising a porous capture membrane in fluid-withdrawing contact with a nonabsorbent capillary network
US5427908A (en) 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
WO1992002551A1 (en) 1990-08-02 1992-02-20 B.R. Centre Limited Methods for the production of proteins with a desired function
CA2048302A1 (en) 1990-08-15 1992-02-16 Victoria P. Meucci Solubilization reagent for biological test samples
US5135875A (en) 1990-08-15 1992-08-04 Abbott Laboratories Protein precipitation reagent
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
ES2246502T3 (en) 1990-08-29 2006-02-16 Genpharm International, Inc. TRANSGENIC NON-HUMAN ANIMALS ABLE TO PRODUCE HETEROLOGICAL ANTIBODIES.
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5877397A (en) 1990-08-29 1999-03-02 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
WO1992005282A1 (en) 1990-09-14 1992-04-02 Biosite Diagnostics, Inc. Antibodies to complexes of ligand receptors and ligands and their utility in ligand-receptor assays
US5698426A (en) 1990-09-28 1997-12-16 Ixsys, Incorporated Surface expression libraries of heteromeric receptors
ATE164395T1 (en) 1990-12-03 1998-04-15 Genentech Inc METHOD FOR ENRICHMENT OF PROTEIN VARIANTS WITH MODIFIED BINDING PROPERTIES
GB9101134D0 (en) 1991-01-18 1991-02-27 R B S Improvements in and relating to accommodation for animals
ATE177841T1 (en) 1991-04-10 1999-04-15 Biosite Diagnostics Inc CROSSTALK OR CROSSTALK INHIBITORS AND THEIR USE
CA2108147C (en) 1991-04-10 2009-01-06 Angray Kang Heterodimeric receptor libraries using phagemids
JP3290988B2 (en) 1991-04-11 2002-06-10 バイオサイト・ダイアグノスティックス・インコーポレイテッド Novel conjugates and assays for simultaneous detection of multiple ligands
DE69232706T2 (en) 1991-05-01 2002-11-28 Jackson H M Found Military Med METHOD FOR TREATING INFECTIOUS RESPIRATORY DISEASES
EP0519596B1 (en) 1991-05-17 2005-02-23 Merck & Co. Inc. A method for reducing the immunogenicity of antibody variable domains
CA2069530A1 (en) 1991-06-03 1992-12-04 Cass J. Grandone Reagent pack for immunoassays
ES2136092T3 (en) 1991-09-23 1999-11-16 Medical Res Council PROCEDURES FOR THE PRODUCTION OF HUMANIZED ANTIBODIES.
DK1024191T3 (en) 1991-12-02 2008-12-08 Medical Res Council Preparation of autoantibodies displayed on phage surfaces from antibody segment libraries
EP0571613B1 (en) 1991-12-13 2003-09-17 Xoma Corporation Methods and materials for preparation of modified antibody variable domains and therapeutic uses thereof
GB9201755D0 (en) 1992-01-28 1992-03-11 British Bio Technology Compounds
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
US5912015A (en) 1992-03-12 1999-06-15 Alkermes Controlled Therapeutics, Inc. Modulated release from biocompatible polymers
US5733743A (en) 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5352803A (en) 1992-03-30 1994-10-04 Abbott Laboratories 5(6)-methyl substituted fluorescein derivatives
DE69328622T2 (en) 1992-03-30 2001-02-08 Abbott Lab REAGENTS AND METHOD FOR DETECTING AND QUANTIFYING THYROXIN IN LIQUID SAMPLES
US6019944A (en) 1992-05-21 2000-02-01 Biosite Diagnostics, Inc. Diagnostic devices and apparatus for the controlled movement of reagents without membranes
US6143576A (en) 1992-05-21 2000-11-07 Biosite Diagnostics, Inc. Non-porous diagnostic devices for the controlled movement of reagents
EP0603366B1 (en) 1992-07-10 1998-12-23 Horst Warneke Assembly line for producing steel coffers for ceiling and/wall constructions from sheet metal plates
US5934272A (en) 1993-01-29 1999-08-10 Aradigm Corporation Device and method of creating aerosolized mist of respiratory drug
US5824799A (en) 1993-09-24 1998-10-20 Biosite Diagnostics Incorporated Hybrid phthalocyanine derivatives and their uses
US5565352A (en) 1993-11-24 1996-10-15 Arch Development Corporation Deubiquitinating enzyme: compositions and methods
US5827690A (en) 1993-12-20 1998-10-27 Genzyme Transgenics Corporatiion Transgenic production of antibodies in milk
WO1995024220A1 (en) 1994-03-07 1995-09-14 Medarex, Inc. Bispecific molecules having clinical utilities
US5525490A (en) 1994-03-29 1996-06-11 Onyx Pharmaceuticals, Inc. Reverse two-hybrid method
US5541109A (en) 1994-04-19 1996-07-30 Rhone-Poulenc Rorer Pharmaceuticals Inc. Expression cloning of c-src SH3-domain binding proteins
US5516637A (en) 1994-06-10 1996-05-14 Dade International Inc. Method involving display of protein binding pairs on the surface of bacterial pili and bacteriophage
WO1996002561A1 (en) 1994-07-20 1996-02-01 The General Hospital Corporation Interaction trap systems for detecting protein interactions
US6111166A (en) 1994-09-19 2000-08-29 Medarex, Incorporated Transgenic mice expressing human Fcα and β receptors
DE69532857T2 (en) 1994-10-27 2005-04-28 Genentech Inc., San Francisco Neurotrophic AL-1 factor, a ligand related to the EPH tyrosine kinase receptor
GB9425232D0 (en) 1994-12-14 1995-02-08 Secr Defence Method of authenticating watermarked paper
US6130364A (en) 1995-03-29 2000-10-10 Abgenix, Inc. Production of antibodies using Cre-mediated site-specific recombination
US6091001A (en) 1995-03-29 2000-07-18 Abgenix, Inc. Production of antibodies using Cre-mediated site-specific recombination
US6019968A (en) 1995-04-14 2000-02-01 Inhale Therapeutic Systems, Inc. Dispersible antibody compositions and methods for their preparation and use
AU705616B2 (en) 1995-04-21 1999-05-27 Cell Genesys, Inc. Generation of large genomic DNA deletions
US6410690B1 (en) 1995-06-07 2002-06-25 Medarex, Inc. Therapeutic compounds comprised of anti-Fc receptor antibodies
JP2000507912A (en) 1995-08-31 2000-06-27 アルカームズ コントロールド セラピューティックス,インコーポレイテッド Sustained release composition of active agent
US5747262A (en) 1995-10-16 1998-05-05 The Regents Of The University Of California Neurological drug screens
US20020136725A1 (en) 1996-01-17 2002-09-26 Smithkline Beecham Corporation Antithrombotic agents
JP2978435B2 (en) 1996-01-24 1999-11-15 チッソ株式会社 Method for producing acryloxypropyl silane
DE122004000003I2 (en) 1996-02-09 2007-03-29 Abbott Biotech Ltd Human antibodies which bind to human TNFalpha
DK0885002T3 (en) 1996-03-04 2011-08-22 Penn State Res Found Materials and methods for enhancing cellular internalization
US5714352A (en) 1996-03-20 1998-02-03 Xenotech Incorporated Directed switch-mediated DNA recombination
US5994619A (en) 1996-04-01 1999-11-30 University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts, As Represented By Its Amherst Campus Production of chimeric bovine or porcine animals using cultured inner cell mass cells
US5874064A (en) 1996-05-24 1999-02-23 Massachusetts Institute Of Technology Aerodynamically light particles for pulmonary drug delivery
US5855913A (en) 1997-01-16 1999-01-05 Massachusetts Instite Of Technology Particles incorporating surfactants for pulmonary drug delivery
US5985309A (en) 1996-05-24 1999-11-16 Massachusetts Institute Of Technology Preparation of particles for inhalation
US6699658B1 (en) 1996-05-31 2004-03-02 Board Of Trustees Of The University Of Illinois Yeast cell surface display of proteins and uses thereof
US5922845A (en) 1996-07-11 1999-07-13 Medarex, Inc. Therapeutic multispecific compounds comprised of anti-Fcα receptor antibodies
US5916771A (en) 1996-10-11 1999-06-29 Abgenix, Inc. Production of a multimeric protein by cell fusion method
FR2754461B1 (en) 1996-10-16 1999-02-12 Husson Olivier MANUALLY REMOVABLE ATTACHMENT FOR IN-LINE SKATE WITH A HOLDING LEG THAT CONTROLS A BRAKING SYSTEM
US6113855A (en) 1996-11-15 2000-09-05 Biosite Diagnostics, Inc. Devices comprising multiple capillarity inducing surfaces
WO1998025947A1 (en) 1996-12-11 1998-06-18 Bristol-Myers Squibb Company Prokaryotic two-hybrid system
US6017517A (en) 1996-12-18 2000-01-25 The Dial Corporation Method for treating human nails
US7368531B2 (en) * 1997-03-07 2008-05-06 Human Genome Sciences, Inc. Human secreted proteins
US5947124A (en) 1997-03-11 1999-09-07 Biosite Diagnostics Incorporated Diagnostic for determining the time of a heart attack
US6440455B1 (en) 1997-09-02 2002-08-27 Children's Medical Center Corporation Methods for modulating the axonal outgrowth of central nervous system neurons
US5989463A (en) 1997-09-24 1999-11-23 Alkermes Controlled Therapeutics, Inc. Methods for fabricating polymer-based controlled release devices
SE512663C2 (en) 1997-10-23 2000-04-17 Biogram Ab Active substance encapsulation process in a biodegradable polymer
WO1999028349A2 (en) 1997-12-02 1999-06-10 Medarex, Inc. CELLS EXPRESSING ANTI-Fc RECEPTOR BINDING COMPONENTS
US6464686B1 (en) 1998-01-21 2002-10-15 Abbott Laboratories Polyurethane feeding tube and associated adaptors
DK1071700T3 (en) 1998-04-20 2010-06-07 Glycart Biotechnology Ag Glycosylation modification of antibodies to enhance antibody-dependent cellular cytotoxicity
TWI242563B (en) 1998-04-30 2005-11-01 Tanox Inc Monoclonal agonist antibodies which specifically bind to or interact with human G-CSF receptor
DK1135498T3 (en) 1998-11-18 2008-05-26 Genentech Inc Antibody variants with higher binding affinity than parental antibodies
PL210435B1 (en) 1998-12-23 2012-01-31 Amgen Fremont Inc Human monoclonal antibodies to ctla-4
US6231768B1 (en) 1999-01-19 2001-05-15 Nalco Chemical Company Rheology modification of settled solids in mineral processing
US6914128B1 (en) 1999-03-25 2005-07-05 Abbott Gmbh & Co. Kg Human antibodies that bind human IL-12 and methods for producing
ES2568898T3 (en) 1999-04-09 2016-05-05 Kyowa Hakko Kirin Co., Ltd. Procedure to control the activity of an immunofunctional molecule
WO2000073801A2 (en) 1999-05-28 2000-12-07 Ludwig Institute For Cancer Research Breast, gastric and prostate cancer associated antigens and uses therefor
EP1396543A3 (en) 1999-07-08 2004-03-31 Research Association for Biotechnology Primers for synthesizing full length cDNA clones and their use
CN102766210B (en) 1999-08-24 2015-07-08 E.R.施贵宝&圣斯有限责任公司 Human CTLA-4 antibodies and their uses
AU2001237958A1 (en) * 2000-01-31 2001-08-07 Human Genome Sciences, Inc. 22 human secreted proteins
WO2001064749A2 (en) 2000-02-28 2001-09-07 Idec Pharmaceuticals Corporation Method for preparing anti-mif antibodies
US6800455B2 (en) * 2000-03-31 2004-10-05 Scios Inc. Secreted factors
CA2403425C (en) 2000-04-11 2013-08-27 Genentech, Inc. Multivalent antibodies and uses therefor
AU2001274888A1 (en) 2000-05-19 2001-12-03 Human Genome Sciences, Inc. Nucleic acids, proteins, and antibodies
US7449308B2 (en) 2000-06-28 2008-11-11 Glycofi, Inc. Combinatorial DNA library for producing modified N-glycans in lower eukaryotes
EP2119793A1 (en) 2000-06-28 2009-11-18 Glycofi, Inc. Methods for producing modified glycoproteins
US6925389B2 (en) 2000-07-18 2005-08-02 Correlogic Systems, Inc., Process for discriminating between biological states based on hidden patterns from biological data
EP1355666B1 (en) 2000-12-22 2012-06-13 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Use of repulsive guidance molecule (RGM) and its modulators
KR100923514B1 (en) 2000-12-28 2009-10-27 알투스 파마슈티컬스 인코포레이티드 Crystals of whole antibodies and fragments thereof and methods for making and using them
US20060084794A1 (en) 2001-04-12 2006-04-20 Human Genome Sciences, Inc. Albumin fusion proteins
US6890763B2 (en) 2001-04-30 2005-05-10 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1350 daltons
JP2005512512A (en) * 2001-07-05 2005-05-12 インサイト・ゲノミックス・インコーポレイテッド Secreted protein
WO2003016466A2 (en) 2001-08-17 2003-02-27 Eli Lilly And Company ANTI-Aβ ANTIBODIES
US20040142325A1 (en) 2001-09-14 2004-07-22 Liat Mintz Methods and systems for annotating biomolecular sequences
DE60232672D1 (en) 2001-10-01 2009-07-30 Dyax Corp MULTILACKED EUKARYONTIC DISPLAY VECTORS AND THEIR USES
IL161412A0 (en) 2001-10-25 2004-09-27 Genentech Inc Glycoprotein compositions
CA2467597A1 (en) 2001-12-03 2003-06-12 Abgenix, Inc. Anti-cd45rb antibodies for use in treating autoimmune disease and transplant rejection
US8435529B2 (en) 2002-06-14 2013-05-07 Immunomedics, Inc. Combining radioimmunotherapy and antibody-drug conjugates for improved cancer therapy
US7419821B2 (en) 2002-03-05 2008-09-02 I-Stat Corporation Apparatus and methods for analyte measurement and immunoassay
US7193069B2 (en) 2002-03-22 2007-03-20 Research Association For Biotechnology Full-length cDNA
WO2003089608A2 (en) 2002-04-18 2003-10-30 The General Hospital Corporation Drg11-responsive (dragon) gene family
WO2003095978A2 (en) 2002-05-09 2003-11-20 Surromed, Inc. Methods for time-alignment of liquid chromatography-mass spectrometry data
IL149820A0 (en) 2002-05-23 2002-11-10 Curetech Ltd Humanized immunomodulatory monoclonal antibodies for the treatment of neoplastic disease or immunodeficiency
AU2003280420A1 (en) 2002-06-26 2004-01-19 Yale University Modulators and modulation of the interacton between rgm and neogenin
US20040018577A1 (en) 2002-07-29 2004-01-29 Emerson Campbell John Lewis Multiple hybrid immunoassay
TWI323265B (en) 2002-08-06 2010-04-11 Glaxo Group Ltd Antibodies
US7541440B2 (en) 2002-09-30 2009-06-02 Immunomedics, Inc. Chimeric, human and humanized anti-granulocyte antibodies and methods of use
WO2004050032A2 (en) 2002-12-02 2004-06-17 Abgenix, Inc. Antibodies against drugs of abuse
MXPA05005925A (en) 2002-12-02 2006-02-08 Abgenix Inc Antibodies directed to phospholipase a2 and uses thereof.
EP1440981A3 (en) 2003-01-21 2005-11-23 Research Association for Biotechnology Full-length human cdna
US20060104968A1 (en) 2003-03-05 2006-05-18 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases
GB0306309D0 (en) 2003-03-19 2003-04-23 Glaxo Group Ltd Method of treatment
WO2004092405A2 (en) 2003-04-15 2004-10-28 Xenon Pharmaceuticals Inc. Juvenile hemochromatosis gene (hfe2a), expression products and uses thereof
EP2829283B1 (en) 2003-04-30 2017-06-07 Universität Zürich Methods for treating cancer using an immunotoxin
US20080274112A1 (en) 2003-08-07 2008-11-06 Lee Daniel H S Nogo Receptor Antagonists
WO2005016111A2 (en) 2003-08-08 2005-02-24 Abgenix, Inc. Antibodies directed to parathyroid hormone (pth) and uses thereof
US20060003391A1 (en) 2003-08-11 2006-01-05 Ring Brian Z Reagents and methods for use in cancer diagnosis, classification and therapy
AU2004280333A1 (en) 2003-08-22 2005-04-21 Medimmune, Llc Humanization of antibodies
US7723099B2 (en) 2003-09-10 2010-05-25 Abbott Point Of Care Inc. Immunoassay device with immuno-reference electrode
US7682833B2 (en) 2003-09-10 2010-03-23 Abbott Point Of Care Inc. Immunoassay device with improved sample closure
EP1697417A2 (en) 2003-11-07 2006-09-06 Curagen Corporation Antibodies against secretory leukocyte protease inhibitor
EP1711530B1 (en) 2003-12-22 2009-08-26 Glaxo Group Limited Nogo-a neutralising immunoglobulins for treatment of neurological diseases
US20110171126A1 (en) 2010-01-11 2011-07-14 Center For Molecular Medicine And Immunology Enhanced Cytotoxicity of Anti-CD74 and Anti-HLA-DR Antibodies with Interferon-Gamma
US20070253946A1 (en) 2004-03-11 2007-11-01 Bioclues, Inc. Axon Regeneration Promoter
AU2005233387B2 (en) 2004-04-15 2011-05-26 Glycofi, Inc. Production of galactosylated glycoproteins in lower eukaryotes
US20060063208A1 (en) 2004-08-02 2006-03-23 Woolf Clifford J DRG11-responsive (DRAGON) gene and uses thereof
US20090123413A1 (en) 2004-08-23 2009-05-14 Britta Hardy Use of bat monoclonal antibody for immunotherapy
AR051800A1 (en) 2004-12-15 2007-02-07 Wyeth Corp BETA ANTIBODIES USED TO IMPROVE COGNITION
EP1677113A1 (en) 2004-12-29 2006-07-05 Max-Delbrück-Centrum für Molekulare Medizin (MDC) Method for the identification of protein-protein interactions in disease related protein networks
EP1858541B1 (en) 2005-02-16 2012-07-25 The General Hospital Corporation Use of bmp antagonists to regulate hepcidin-mediated iron metabolism and treat iron deficiency
US7504225B2 (en) 2005-05-12 2009-03-17 Applied Genomics, Inc. Reagents and methods for use in cancer diagnosis, classification and therapy
PT1904104E (en) 2005-07-08 2013-11-21 Biogen Idec Inc Sp35 antibodies and uses thereof
US7612181B2 (en) 2005-08-19 2009-11-03 Abbott Laboratories Dual variable domain immunoglobulin and uses thereof
JP5364870B2 (en) 2005-08-19 2013-12-11 アッヴィ・インコーポレイテッド Dual variable domain immunoglobulins and uses thereof
JP2009510002A (en) 2005-09-30 2009-03-12 アボット ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディトゲゼルシャフト Binding domains of proteins of the repulsion-inducing molecule (RGM) protein family, and functional fragments thereof, and uses thereof
AU2006300951A1 (en) 2005-10-11 2007-04-19 Domantis Limited Antibody polypeptide library screening and selected antibody polypeptides
AR056857A1 (en) 2005-12-30 2007-10-24 U3 Pharma Ag DIRECTED ANTIBODIES TO HER-3 (RECEIVER OF THE HUMAN EPIDERMAL GROWTH FACTOR-3) AND ITS USES
US7420040B2 (en) 2006-02-24 2008-09-02 Arius Research Inc. Cytotoxicity mediation of cells evidencing surface expression of TROP-2
EP1865071A1 (en) 2006-06-09 2007-12-12 Rheinische Friedrich-Wilhelms-Universität Bonn Method for early diagnosis of proliferative diabetic retinopathy
US7883855B2 (en) 2006-07-21 2011-02-08 Abbott Laboratories Immunosuppressant drug extraction reagent for immunoassays
WO2008133722A2 (en) 2006-11-10 2008-11-06 Ucb Pharma S.A. Anti human sclerostin antibodies
US20100036091A1 (en) 2006-11-10 2010-02-11 Amgen Inc. Antibody-based diagnostics and therapeutics
KR101343916B1 (en) 2006-12-21 2013-12-20 삼성전자주식회사 Marker for diagnosing lymph node micrometastasis of lung cancer, kit comprising primer for the marker, microarray comprising the marker or antibody against the marker, and method for diagnosing lymph node micrometastasis of lung cancer
EP2114443A4 (en) 2006-12-29 2011-08-10 Abbott Lab Dual-specific il-1a/ il-1b antibodies
US8128926B2 (en) 2007-01-09 2012-03-06 Biogen Idec Ma Inc. Sp35 antibodies and uses thereof
EP1947114A1 (en) 2007-01-19 2008-07-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Mutated netrin 4, fragments thereof and uses thereof as drugs
BRPI0809594A2 (en) 2007-04-03 2019-08-27 Micromet Ag polypeptide, nucleic acid sequence, vector, host, process for producing a polypeptide, pharmaceutical composition, use of a polypeptide, method for preventing, treating or ameliorating a disease in an individual in need thereof, kit, method for the identification of a polypeptide (s)
US8163279B2 (en) 2007-04-13 2012-04-24 Stemline Therapeutics, Inc. IL3Rα antibody conjugates and uses thereof
WO2009006543A1 (en) 2007-07-02 2009-01-08 Euclid Diagnostics Llc Methods for evaluating the methylation status of a polynucleotide
US8070827B2 (en) 2007-07-03 2011-12-06 Histogenics Corporation Method for use of a double-structured tissue implant for treatment of tissue defects
US20090054984A1 (en) 2007-08-20 2009-02-26 Histogenics Corporation Method For Use Of A Double-Structured Tissue Implant For Treatment Of Tissue Defects
EP2182887B1 (en) 2007-08-20 2016-12-14 Histogenics Corporation A method for improvement of differentiation of mesenchymal stem cells using a double-structured tissue implant
EP2033971A1 (en) 2007-09-06 2009-03-11 Abbott GmbH & Co. KG Bone Morphogenetic Protein (BMP) binding domains of proteins of the Repulsive Guidance Molecule (RGM) protein family and functional fragments thereof and their application
US7981415B2 (en) 2007-09-07 2011-07-19 Cisthera, Inc. Humanized PAI-1 antibodies
CA2701189C (en) 2007-10-11 2017-05-16 Biogen Idec Ma Inc. Methods for treating pressure induced optic neuropathy, preventing neuronal degeneration and promoting neuronal cell survival via administration of lingo-1 antagonists and trkb agonists
WO2009064854A2 (en) 2007-11-13 2009-05-22 Cogenesys, Inc Humanized antibodies against tl1a
GB2456390A (en) 2008-01-15 2009-07-22 Glaxo Group Ltd Bipolar disorder treatments
AU2009213738B2 (en) 2008-02-11 2015-01-22 Curetech Ltd. Monoclonal antibodies for tumor treatment
US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
DE102008014880A1 (en) 2008-03-12 2009-09-17 Eberhard-Karls-Universität Tübingen Use of a polypeptide with the activity of repulsive guidance molecule A as an antiinflammatory agent
US8314213B2 (en) 2008-04-18 2012-11-20 Xencor, Inc. Human equivalent monoclonal antibodies engineered from nonhuman variable regions
WO2009140383A2 (en) 2008-05-13 2009-11-19 Archemix Corp. Aptamers that bind to p-selectin and their use as coagulation, thrombotic, inflammatory, and metastatic disease therapeutics
US9109026B2 (en) 2008-06-03 2015-08-18 Abbvie, Inc. Dual variable domain immunoglobulins and uses thereof
CA2729949A1 (en) 2008-07-08 2010-01-14 Abbott Laboratories Prostaglandin e2 dual variable domain immunoglobulins and uses thereof
WO2010006184A2 (en) 2008-07-11 2010-01-14 Emory University Small-molecule inhibitors of hif and angiogenesis
WO2010006189A2 (en) 2008-07-11 2010-01-14 Emory University Small-molecule inhibitors of hif and angiogenesis
US20100184033A1 (en) 2008-07-16 2010-07-22 West Michael D Methods to accelerate the isolation of novel cell strains from pluripotent stem cells and cells obtained thereby
WO2010007144A2 (en) 2008-07-18 2010-01-21 Centre National De La Recherche Scientifique New mutated netrin 4 proteins, fragments thereof and their uses as drugs
EP2326356B1 (en) 2008-08-07 2017-10-11 Exogenesis Corporation Medical device for bone implant and method for producing such a device
RU2011117210A (en) 2008-09-30 2012-11-10 Эбботт Лэборетриз (Us) IMPROVED RNA DISPLAY METHODS
JP2012504416A (en) 2008-09-30 2012-02-23 アボット・ラボラトリーズ Improved antibody library
US8268314B2 (en) 2008-10-08 2012-09-18 Hoffmann-La Roche Inc. Bispecific anti-VEGF/anti-ANG-2 antibodies
US20110293526A1 (en) 2008-11-20 2011-12-01 University Of Southern California Compositions and methods to modulate hair growth
ES2602459T3 (en) 2008-12-26 2017-02-21 Kyowa Hakko Kirin Co., Ltd. Anti-CD4 antibody
US20100254979A1 (en) 2009-03-06 2010-10-07 Cisthera, Incorporated Humanized PAI-1 Antibodies and Uses Thereof
US20110020221A1 (en) 2009-04-09 2011-01-27 The Johns Hopkins University Cancer stem cell expression patterns and compounds to target cancer stem cells
UY32603A (en) 2009-05-01 2010-12-31 Abbott Lab IMMUNOGLOBULIN WITH DUAL VARIABLE DOMAIN AND USES OF THE SAME
TWI483736B (en) 2009-10-23 2015-05-11 Millennium Pharm Inc Anti-gcc antibody molecules and related compositions and methods
UA109888C2 (en) 2009-12-07 2015-10-26 ANTIBODY OR ANTIBODILITY ANTIBODY OR ITS BINDING TO THE β-CLOTE, FGF RECEPTORS AND THEIR COMPLEXES
CA2780069C (en) 2009-12-08 2018-07-17 Abbott Gmbh & Co. Kg Monoclonal antibodies against the rgm a protein for use in the treatment of retinal nerve fiber layer degeneration
CA2789629A1 (en) 2010-02-10 2011-08-18 Immunogen, Inc. Cd20 antibodies and uses thereof
AU2011235328A1 (en) 2010-04-01 2012-09-27 Immunomedics, Inc. Antibody-based depletion of antigen-presenting cells and dendritic cells

Patent Citations (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7498034B2 (en) * 2000-11-06 2009-03-03 Cancer Research Technology Limited Imaging, diagnosis and treatment of disease
US20040071711A1 (en) * 2000-11-06 2004-04-15 Roy Bicknell Imaging, diagnosis and treatment of disease
US7582440B2 (en) * 2000-11-06 2009-09-01 Cancer Research Technology Limited Imaging, diagnosis and treatment of disease
US20090191572A1 (en) * 2000-11-06 2009-07-30 Cancer Research Technology Limited Imaging, diagnosis and treatment of disease
US20080219924A1 (en) * 2000-11-06 2008-09-11 Cancer Research Technology Limited Imaging, diagnosis and treatment of disease
US20080274045A9 (en) * 2000-11-06 2008-11-06 Cancer Research Technology Limited, A United Kingdom Corporation Imaging, diagnosis and treatment of disease
US20080145359A1 (en) * 2000-11-06 2008-06-19 Cancer Research Technology Limited Imaging, diagnosis and treatment of disease
US20070025913A1 (en) * 2000-11-06 2007-02-01 Cancer Research Technology Limited, A United Kingdom Corporation Imaging, diagnosis and treatment of disease
US20040038292A1 (en) * 2001-06-18 2004-02-26 Burslem Martyn Frank Wound healing biomarkers
US7524492B2 (en) * 2001-08-31 2009-04-28 Joslin Diabetes Center, Inc. Insulin related transcription factor and uses thereof
US20030087394A1 (en) * 2001-08-31 2003-05-08 Arun Sharma Insulin related transcription factor and uses thereof
US20080081337A1 (en) * 2001-08-31 2008-04-03 Joslin Diabetes Center, Inc. Insulin related transcription factor and uses thereof
US6864239B2 (en) * 2002-03-27 2005-03-08 Theratechnologies Inc. Methods and compounds for prevention and treatment of elevated intraocular pressure and related conditions
US20030212001A1 (en) * 2002-03-27 2003-11-13 Peri Krishna G. Methods and compounds for prevention and treatment of elevated intraocular pressure and related conditions
US20060292613A1 (en) * 2002-03-27 2006-12-28 Theratechnologies Inc. Methods and compounds for prevention and treatment of elevated intraocular pressure and related conditions
US7094761B2 (en) * 2002-03-27 2006-08-22 Theratechnologies Inc. Methods and compounds for prevention and treatment of elevated intraocular pressure and related conditions
US20050059604A1 (en) * 2002-03-27 2005-03-17 Theratechnologies Inc. Methods and compounds for prevention and treatment of elevated intraocular pressure and related conditions
US20090093409A1 (en) * 2002-06-11 2009-04-09 Burnham Institute For Medical Research Neuroprotective synergy of erythropoietin and insulin-like growth factors
US7439063B2 (en) * 2002-06-11 2008-10-21 Burnham Institute For Medical Research Neuroprotective synergy of erythropoietin and insulin-like growth factors
US20050197284A9 (en) * 2002-06-11 2005-09-08 Murat Digicaylioglu Neuroprotective synergy of erythropoietin and insulin-like growth factors
US20080160034A1 (en) * 2004-08-25 2008-07-03 Genentech, Inc. Novel Gene Disruptions, Compositions and Methods Relating Thereto
US20090028852A1 (en) * 2004-11-16 2009-01-29 Trustees Of Boston University Roles For Dual Endothelin-1/Angiotensin II Receptor (DEAR) In Hypertension and Angiogenesis
US20100183588A1 (en) * 2004-11-22 2010-07-22 Jean Plouet Mutated Netrin-4, Fragments Thereof And Their Use As Medicines
US20080008692A1 (en) * 2005-10-14 2008-01-10 Alltech, Inc. Methods and compositions for altering cell function
US20080004255A1 (en) * 2005-10-14 2008-01-03 Alltech, Inc. Methods and compositions for altering cell function
US20070155687A1 (en) * 2005-10-14 2007-07-05 Alltech, Inc. Methods and compositions for altering cell function
US20070122491A1 (en) * 2005-10-14 2007-05-31 Alltech, Inc. Methods and compositions for altering cell function
US20080213791A1 (en) * 2005-11-08 2008-09-04 Euclid Diagnostics Llc Materials and methods for assaying for methylation of CpG islands associated with genes in the evaluation of cancer
US20090269356A1 (en) * 2006-03-08 2009-10-29 David Epstein Complement Binding Aptamers and Anti-C5 Agents Useful in the Treatment of Ocular Disorders
US20080135582A1 (en) * 2006-10-02 2008-06-12 Schmidt Norman G Device for controlled metering of semi solid food products
US20080105705A1 (en) * 2006-10-18 2008-05-08 Schmidt Norman G Device to allow for cleaning access in semi-solid metering machines
US20080279859A1 (en) * 2006-11-21 2008-11-13 Abbott Laboratories NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST THE NOGO-66 RECEPTOR (NgR) AND USES THEREOF
US20090220588A1 (en) * 2008-02-21 2009-09-03 Massachusetts Institute Of Technology Simultaneous Delivery of Receptors and/or Co-Receptors for Growth Factor Stability and Activity
US20090220589A1 (en) * 2008-03-01 2009-09-03 Abraxis Bioscience, Llc Treatment, diagnostic, and method for discovering antagonist using sparc specific mirnas
US20090311253A1 (en) * 2008-06-03 2009-12-17 Abbott Laboratories Dual Variable Domain Immunoglobulins and Uses Thereof

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8680239B2 (en) 2000-12-22 2014-03-25 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Use of RGM and its modulators
US8906864B2 (en) 2005-09-30 2014-12-09 AbbVie Deutschland GmbH & Co. KG Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use
US20100322948A1 (en) * 2007-09-06 2010-12-23 Abbott Gmbh & Co. Kg Bone morphogenetic protein (BMP)-binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and use of same
US20100028340A1 (en) * 2008-02-29 2010-02-04 Abbott Gmbh & Co. Kg Antibodies against the rgm a protein and uses thereof
US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
US9605069B2 (en) 2008-02-29 2017-03-28 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM a protein and uses thereof
US20110135664A1 (en) * 2009-12-08 2011-06-09 Abbott Gmbh & Co. Kg Monoclonal antibodies against the rgm a protein for use in the treatment of retinal nerve fiber layer degeneration
US9175075B2 (en) 2009-12-08 2015-11-03 AbbVie Deutschland GmbH & Co. KG Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein
US9365643B2 (en) 2012-01-27 2016-06-14 AbbVie Deutschland GmbH & Co. KG Antibodies that bind to repulsive guidance molecule A (RGMA)
US9102722B2 (en) 2012-01-27 2015-08-11 AbbVie Deutschland GmbH & Co. KG Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration
US10106602B2 (en) 2012-01-27 2018-10-23 AbbVie Deutschland GmbH & Co. KG Isolated monoclonal anti-repulsive guidance molecule A antibodies and uses thereof
US20190275118A1 (en) * 2013-09-17 2019-09-12 University Health Network Agents directed against a cis rgma/neogenin interaction or lipid rafts and use of the same in methods of treatment
US11497796B2 (en) * 2013-09-17 2022-11-15 University Health Network Agents directed against a cis RGMa/neogenin interaction or lipid rafts and use of the same in methods of treatment
US10055540B2 (en) 2015-12-16 2018-08-21 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US10847252B2 (en) 2015-12-16 2020-11-24 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US10847253B2 (en) 2015-12-16 2020-11-24 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US11183286B2 (en) 2015-12-16 2021-11-23 Gritstone Bio, Inc. Neoantigen identification, manufacture, and use
US11264117B2 (en) 2017-10-10 2022-03-01 Gritstone Bio, Inc. Neoantigen identification using hotspots
US11885815B2 (en) 2017-11-22 2024-01-30 Gritstone Bio, Inc. Reducing junction epitope presentation for neoantigens

Also Published As

Publication number Publication date
US20130072667A1 (en) 2013-03-21
US20040102376A1 (en) 2004-05-27
US8680239B2 (en) 2014-03-25
JP2010065045A (en) 2010-03-25
AU2002231736A1 (en) 2002-07-08
ES2390425T3 (en) 2012-11-12
EP1355666B1 (en) 2012-06-13
WO2002051438A3 (en) 2003-08-14
US7981420B2 (en) 2011-07-19
WO2002051438A2 (en) 2002-07-04
JP2004525875A (en) 2004-08-26
EP1355666A2 (en) 2003-10-29
JP4986370B2 (en) 2012-07-25

Similar Documents

Publication Publication Date Title
US8680239B2 (en) Use of RGM and its modulators
US20130101605A1 (en) Modulators and modulation of the interaction between rgm and neogenin
JPH09505725A (en) Semaphorin gene group
US20070117166A1 (en) Use of ULIP proteins in the diagnosis and therapy of cancers and paraneoplastic neurological syndromes
JPH09507744A (en) A novel fission phosphoprotein: mitocin
US20110137013A1 (en) Novel collagen-like protein clac, precursor thereof and genes encoding the same
US20100233180A1 (en) Antibodies that bind to an epitope on the huntington&#39;s disease protein
CA2245956A1 (en) Monoclonal antibodies specific to endothelial cell cadherins and uses thereof
US7790673B2 (en) Methods and compositions relating to cystatin C
JP4399260B2 (en) Nerve regeneration peptide and its use in the treatment of brain injury
JP2001506847A (en) Junctional adhesion molecule (JAM), tight junction transmembrane protein
US20130336988A1 (en) Methods for treating early stage or mild neurological disorders
US7820375B2 (en) Use of a protein of the CRMP family for treating diseases related to the immune system
US6559288B1 (en) Ninjurin
JP4482235B2 (en) Antibodies against mammalian Langerhans cell antigens and their use
EP1101820A1 (en) Pentraxin I and Pentraxin receptor, inhibitors of said proteins and pharmaceutical compositions containing said compounds
US20020115069A1 (en) Eh domain containing genes and proteins
JP4503287B2 (en) Methods for inhibiting the growth of astrocytes and astrocyte tumor cells, methods for increasing neuronal survival, and uses thereof
US20100129288A1 (en) Gliomedin, Fragments Thereof and Methods of Using Same
US20230123187A1 (en) Methods and compositions for treatment of atherosclerosis
US20090258023A1 (en) Tgf-beta superfamily binding proteins and modulation of bone formation and loss
ES2369354T3 (en) USE OF A CRMPS FAMILY PROTEIN FOR THE TREATMENT OF DISEASES LINKED TO THE IMMUNE SYSTEM.

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION