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WO1995022344A1 - Therapeutic use of myelin-associated glycoprotein (mag) - Google Patents

Therapeutic use of myelin-associated glycoprotein (mag) Download PDF

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Publication number
WO1995022344A1
WO1995022344A1 PCT/CA1995/000089 CA9500089W WO9522344A1 WO 1995022344 A1 WO1995022344 A1 WO 1995022344A1 CA 9500089 W CA9500089 W CA 9500089W WO 9522344 A1 WO9522344 A1 WO 9522344A1
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Prior art keywords
mag
growth
neurons
myelin
inhibition
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PCT/CA1995/000089
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French (fr)
Inventor
Lisa Joan Mckerracher
Samuel David
Peter Erich Braun
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Mcgill University
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Priority to AU17035/95A priority Critical patent/AU1703595A/en
Publication of WO1995022344A1 publication Critical patent/WO1995022344A1/en
Priority to SE9703032A priority patent/SE9703032D0/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the use of a myelin associated glycoprotein (MAG) for the regulation of growth of neurons in peripheral nervous system (PNS) or central nervous system (CNS).
  • MAG myelin associated glycoprotein
  • CNS central nervous system
  • a major barrier to axonal regeneration in the CNS of mammals is the presence of growth inhibitory molecules. It is not known whether there is one or several inhibitory pro ⁇ teins present in myelin membranes, and the primary sequence has not yet been identified for any myelin associated protein inhibitor. The axon growth inhibi ⁇ tory property of CNS myelin has been well established, but the inhibitory proteins remain unknown. It is well documented that myelin in the CNS of adult mam- mals inhibits the growth of neurons in vi tro (P. Caroni, M.E. Schwab, (1988) Neuron, 1:85-96; M.E. Schwab, P.
  • Inhibitory activity can be extracted from SDS gels with proteins of approximately 250 and 35 kDa. Further, many in vivo experiments suggest that myelin- derived inhibitors block regeneration in vivo (Schnell and Schwab, (1990) Nature, 243:268; Schnell et al., (1994) Nature, 367:170).
  • tenascin molecules secreted by oligodendro- cytes, that have growth inhibitory properties, have been identified as 160 and 180 kDa homologues of tenascin (Peshiva et al., (1989) JOB, 109:1765-1788; Fuss et al., (1993) JCB, 120:1237-1249; Lochter et al., (1991) JCB, 113:1159-1171).
  • the tenascin-like proteins are secreted and, therefore, are not expected to be present in myelin.
  • myelin-derived inhibitory protein It would be highly desirable to be provided with such a myelin-derived inhibitory protein to be able to find reagents to overcome growth inhibition which would allow regenerative regrowth of damaged axons in the mammalian CNS.
  • myelin asso ⁇ ciated glycoprotein (MAG) as one of the major molecu- lar components involved in contact-mediated growth inhibition on myelin.
  • the growth inhibitory property of MAG was determined by: 1) demonstrating that MAG chromatographed with partially purified myelin-derived inhibitory activity; 2) testing the ability of recom- binant MAG to inhibit neurite growth.
  • MAG myelin associated glycopro ⁇ tein
  • a method to suppress the inhibition of neuron growth comprising the steps of delivering, to the nerve growth environ- ment, a myelin-associated glycoprotein antagonist in an amount effective to reverse said inhibition.
  • an assay method useful to identify MAG antagonist agents that suppress inhibition of neuron growth comprising the steps of: a) culturing neurons on a growth permissive substrate that incorporates a growth-inhibiting amount of MAG; and b) exposing the cultured neurons of step a) to a candidate MAG antagonist agent in an amount and for a period sufficient prospectively to permit growth of the neurons; thereby identifying as MAG antagonists the can ⁇ didates of step b) which elicit neurite outgrowth from the cultured neurons of step a) .
  • a method for inhibiting neuron growth comprising the steps of introducing into the growth environment of the neurons a growth inhibiting amount of MAG or a MAG agonist.
  • Antist refers to a pharmaceutical agent having myelin associated glycoprotein biological activity of inhibiting the neurite outgrowth of neu ⁇ rons cultured on a permissive substrate or inhibiting the regeneration of damaged neurons. It would be desirable to inhibit neuron growth in cases of epilepsy, neuroblastoma, and neuromas, a disease state in a mammal which includes neurite outgrowth or other neural growth of an abnormal sort which causes pain at the end of an amputated limb.
  • Agonists which may be used in accordance with the present invention include without limitation a MAG fragment, an analog of MAG or of the MAG fragment, a derivative of either MAG, the MAG fragment or said analog, an anti-idiotypic MAG antibody or a binding fragment thereof, MAG ectodo- main, and a pharmaceutical agent.
  • Antagonist refers to a pharmaceutical agent which in accordance with the present invention which inhibits at least one biological activity normally associated with MAG, that is blocking or suppressing the inhibition of neuron growth.
  • Antagonists which may be used in accordance with the present invention include without limitation a MAG antibody or a binding fragment of said antibody, a MAG fragment, a deriva ⁇ tive of MAG or of a MAG fragment, an analog of MAG or of a MAG fragment or of said derivative, and a pharma ⁇ ceutical agent, and is further characterized by the property of suppressing MAG-mediated inhibition of neurite outgrowth.
  • the agonist or antagonist of MAG in accordance with the present invention is not limited to MAG or its derivatives, but also includes the therapeutic application of all agents, referred herein as pharma ⁇ ceutical agents, which alter the biological activity of the neuronal receptor for MAG such that growth of neurons or their axons is suppressed.
  • the receptor can be identified with known technologies by those skilled in the art (Mason, (1994) Curr. Biol . , 4:1158- 1161) and its association with MAG or fragments thereof can be determined.
  • the neuronal receptor for MAG may or may not be the same as cell surface mole ⁇ cules that recognize and bind MAG in an adhesion assay (Kelm et al., (1994) Curr. Biol .
  • an effective amount or “growth-inhibit ⁇ ing amount” refers to the amount of pharmaceutical agent required to produce a desired agonist or antago ⁇ nist effect of the MAG biological activity.
  • the pre ⁇ cise effective amount will vary with the nature of pharmaceutical agent used and may be determined by one of ordinary skill in the art with only routine experi ⁇ mentation.
  • MAG biological activity refers to cellular events triggered by MAG, being of either biochemical or biophysical nature.
  • the following list is provided, without limitation, which discloses some of the known activities associ ⁇ ated with MAG: contact-mediated growth inhibition on myelin, inhibition of neuron growth, inhibition of neurite outgrowth, adhesion to neuronal cells, and promotion of neurite outgrowth from newborn dorsal root ganglion neurons.
  • Fig. 1 illustrates the neurite outgrowth from Dil-labeled NG108-15 cells on myelin and MAG substrates X 160;
  • Fig. 2 illustrates a quantitative analysis of neurite outgrowth from NG108-15 cells on different substrates
  • Fig. 3 is the analysis of growth inhibition after separation of myelin proteins by DEAE anion exchange chromatography.
  • the strategy pursued was the purification of myelin inhibitors by non-denaturing extraction and column chromatography. This approach has allowed the identi ⁇ fication of a known protein of unknown function as a major myelin-derived growth inhibitor.
  • the myelin-associated glycoprotein was identified as a major inhibitory molecules present in myelin.
  • the primary sequence of the protein has previously been reported (Arquint et al., (1987) PNAS, 84:600-604; Salzer et al. , (1987) JOB, 104:957-965).
  • This protein is present in both peripheral nervous system (PNS) and CNS myelin, an observation consistent with findings that PNS myelin also has inhibitory properties (Fig. 2).
  • Myelin-derived inhibitory activity was assessed by bioassays performed in tissue culture.
  • Cells grow neurites on "permissive" substrates such as a polylys- ine or laminin substrate (Fig. 1A) .
  • Purified bovine CNS myelin used as a tissue culture substrate permit ⁇ ted cell attachment, but inhibited cAMP-induced neu ⁇ rite outgrowth from NG108-15 cells (Fig. IB).
  • Cells grown on 4 ⁇ g of myelin were rounded and did not grow neurites (Fig. IB).
  • When cells were grown on recombi- nant MAG they did not spread or differentiate neurites Fig. 1C; Attia S. et al. , (1993) J. Neurochem.
  • Bovine corpus callosum or sciatic nerve was homogenized with a DounceTM homogenizer, and myelin was purified as described by W.T. Norton et al. ((1973) J. Neurochem. , 21:749-757) .
  • Myelin with 4 ⁇ g or 8 ⁇ g protein was added to polylysine-coated 96 well plates, dried and washed with PBS before plating the cells.
  • NG108 cells readily accessible to those skilled in the art, a cell line that extends neurites in response to cAMP (D.G.
  • cAMP primed NG108 cells were labeled with the lipophilic dye Di-I(1, 1 '-dioctadecyl-3, 3, 3 * , 3 ' -tetra- methylindocarbocyanine perchlorate) by incubating the cultures with 1% solution of the dye in culture medium for 2 hr. The cells were then rinsed and maintained in culture for 3 hr. prior to using them for the neu ⁇ rite outgrowth assay. Dil-labeled NG108 cells were added to the wells at a density of 1000 cells/well, and cultured for 24 hr.
  • DMEM Dulbecco's minimal essen- tial medium
  • the cultures were then fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer for 30 min., washed and stored in buffer, the percentage of NG108 cells extending neu- rites longer than 1 cell body diameter was estimated with a Zeiss AxiomatTM inverted microscope equipped with fluorescence optics.
  • myelin-derived inhibitory activity was solubilized in octylglucoside and chroma- tographed on a DEAE anion exchange column.
  • Bovine brain myelin was extracted for 2 hr. at 20°C with 1% octylglucoside (1 ml per mg of protein) in 0.2 M phosphate buffer (pH 6.8) containing 0.1 M Na2S ⁇ 4, 1 mM EDTA, 1 mM dithiothreitol and a cocktail of protease inhibitors.
  • the extract was clarified by centrifugation at 400,000 g.min. and applied to a col ⁇ umn of DEAE-SepharoseTM (Pharmacia; 1.3 cm X 10 cm).
  • Fig. 3 A profile of growth inhibition present in the column fractions is shown in Fig. 3. Percent inhibition was calculated as (C - E/C x 100) where C was the average number of cells with neurites on polylysine substrates, and E was the average number of cells with neurites on the test substrate.
  • Western blots of the inhibitory frac ⁇ tions with anti-MAG antibody (GenS3; Nobile-Orazio et al., (1984) Neurology, 34:1336-1342) showed that the inhibitory peaks that eluted in low salt corresponded to the elution profile for MAG (Fig. 3).
  • FIG. 3 is a Western blots of column fractions probed with anti-MAG antibody.
  • the inhibitory activity eluted at the highest salt concentrations (Fig. 3, fractions 23-26) was not enriched in MAG, and likely represents a different inhibitory protein activity, possibly the one reported by Schwab (M.E. Schwab, (1993) Annu . Rev. Neurosci . , 16:565).
  • MAG may be an inhibi ⁇ tor of axon growth.
  • Direct evidence for this role was obtained by showing that recombinant MAG was a potent inhibitor of neurite outgrowth from NG108 cells (Fig 1C; Fig. 2). Heating recombinant MAG at 80°C for 1 hr. abolished this activity (Fig. ID; Fig. 2).
  • Comparison of cells grown on polylysine, CNS myelin, peripheral nerve myelin, recombinant MAG (MAG), dena- tured MAG (denat. MAG), and bovine serum albumin (BSA) demonstrate the potent growth inhibition of PNS and CNS, myelin and MAG.
  • Results are the mean of 3 experiments done in triplicate; 2 experiments for denatured MAG (Fig. 2). In these experiments, no cells extended neurites when plated on MAG compared to 73%+ 4 on denatured MAG, or to 69% ⁇ 4 on BSA, used as a control. Because MAG is a component of myelin pro ⁇ pokerd by both Schwann cells and CNS glial cells, neu ⁇ rite growth inhibition present in peripheral nerve and CNS myelin were compared. Purified bovine CNS and peripheral nerve myelin plated at 4 ⁇ g protein/well inhibited neurite outgrowth equally (Fig. 2).
  • MAG is reported here as having potent neurite growth-inhibitory activity, its sequence homology with adhesion molecules of the immunoglobulin family has led others to investigate a possible role for MAG in cell adhesion (M. Arquint et al. , (1987) Proc . Natl . Acad. Sci . U. S. A. , 84:600; M. Poltorak et al., (1987) J. Cell Biol . , 105:1893; J.L. Salzer et al., (1987) J. Cell Biol . , 104:957; Kelm, S. et al. , (1994) Current Biology, 4:965-972; Schachner et al. , (1994) Curr. Opinion Neurobiol . , 4:726-734; Doherty et ⁇ al., (1994) Curr. Opinion Neurobiol . , 4:49-55).
  • the results of the present invention were cor- related as follows with the growth inhibitory proper ⁇ ties of MAG with experimental evidence that the MAG extracellular domain may mediate adhesion to axons.
  • the finding that recombinant MAG lacking the L2/HNK-1 carbohydrate epitope is a potent neurite out- growth inhibitor suggests that there may be functional differences among carbohydrate variants of MAG.
  • observations that MAG-containing liposomes bind to neurons specifically suggests a receptor- mediated interaction between neurons and MAG.
  • such experiments do not reveal how MAG binding may affect growth cone dynamics.
  • adhesive prop ⁇ erties of MAG have been examined when it was expressed in heterologous, living cells. In these experiments, cell adhesion molecules or extracellular matrix compo- nents that are known to override the growth inhibition on myelin may be present.
  • MAG growth inhibitory properties
  • myelin debris in the CNS is not removed very quickly after injury (G. Stoll et al., (1989) J. Neurosci . , 9:2327- 2335).
  • peripheral nerve myelin is asso ⁇ ciated with molecules such as laminin that can over ⁇ ride the inhibitory effects of myelin.
  • CNS neurons will regenerate in a peripheral nerve environment, they show little capacity to elon ⁇ gate in the CNS.
  • Growth inhibition by MAG which is a prominent glycoprotein component of the total myelin protein (R.H. Quarles, D.R. Coleman, J.L.
  • MAG is a major growth inhibitory protein in myelin
  • agents and therapies that suppress MAG-medi ⁇ ated inhibition of nerve growth.
  • the invention provides an assay method adapted to identify MAG antagonist, that is agents that block or suppress the growth-inhibiting action of MAG.
  • the assay is a tissue culture assay that measures neurite out ⁇ growth as a convenient end-point, and accordingly uses nerve cells that extend neurites when grown on a per- missive substrate.
  • Nerve cells suitable in this regard include neuroblastoma cells of the NG108 line ⁇ age, such as NG108-15, as well as other neuronal cell lines such as PC12 cells (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA, ATCC accession No.
  • CRL 1721 human neuroblastoma cells, and primary cultures of CNS or PNS neurons taken from embryonic, postnatal or adult animals.
  • the nerve cells for instance about 10 ⁇ cells-microwell or equivalent, are cultured on a growth permissive sub- strate, such as polylysine or laminin, that is over- layed with a growth-inhibiting amount of MAG.
  • the MAG incorporated in the culture is suitably myelin- extracted MAG, although forms of MAG -other than endogenous forms can be used provided they exhibit the MAG property of inhibiting neuron growth when added to a substrate that is otherwise growth permissive.
  • Suitable MAG alternatives include, for example, the MAG ectodomain reported by Salzer et al. ((1989) Develop. Neurosci . , 11:377-390) and by Attia et al. ((1993) J. Neurochem. , 61:718-726), and other growth- inhibiting MAG fragments.
  • candidate MAG antagonists i.e., compounds that block the growth-inhibiting effect of MAG
  • candidate MAG antagonists are added to the MAG-containing tissue culture preferably in amounts sufficient to neutralize the MAG growth-inhibiting activity, that is between 1.5 and 15 ⁇ g of MAG antagonists per well containing a density of 1000 NG108-15 cells/well cultured for 24 hr. in Dulbecco's minimal essential medium. After culturing for a period sufficient for neurite outgrowth, e.g. 3- 7 days, the culture is evaluated for neurite outgrowth, and MAG antagonists are thereby revealed as those candidates which elicit neurite outgrowth.
  • candidates selected as MAG antagonists are those which elicit neurite outgrowth to a statisti ⁇ cally significant extent, e.g., in at least 50%, more desirably at least 60%, e.g. 70%, per 1,000 cultured neurons.
  • assay tests include without limitation the following: 1) The growth cone collapse assay that is used to assess growth inhibi ⁇ tory activity of collapsin (Raper, J.A., and Kapfhammer, J.P., (1990) Neuron, 2:21-29; Luo et al. , (1993) Cell , 75:217-227) and of various other inhibi- tory molecules (Igarashi, M. et al. , (1993) Science, 259:77-79) whereby the test substance is added to the culture medium and a loss of elaborate growth cone morphology is scored. 2) The use of patterned sub ⁇ strates to assess substrate preference (Walter, J.
  • Useful MAG antagonists include antibodies to MAG and the binding fragments of those antibodies. Antibodies which are either monoclonal or polyclonal can be produced which recognize MAG and its various epitopes using now routine procedures. For the rais ⁇ ing of antibody, various host animals can be immunized by injection with MAG or fragment thereof, including but not limited to rabbits, mice, rats, etc.
  • adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinmitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette- Guerin) .
  • a monoclonal antibody to an epitope of MAG can be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • the monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric human- mouse (or other species) monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g. Teng et al. , (1983) Proc . Natl . Acad. Sci . U. S. A. , 80:7308-7312; Kozbor et al., (1983) Immunology Today, 4:72-79; Olsson et al., (1982) Meth . Enzymol . , 92:3-16).
  • Chimeric antibody molecules may be prepared containing a mouse antigen- binding domain with human contact regions (Morrision et al., (1984) Proc . Natl . Acad. Sci . U. S. A. , 81:6851; Takeda et al., (1985) Nature, 314:452).
  • a molecular clone of an antibody to a MAG epi ⁇ tope can be prepared by known techniques. Recombinant DNA methodology may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof (see e.g., Maniatis et al., (1982) In Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • MAG antibody molecules may be purified by known techniques, such as immunoabsorption or immu- noaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof, etc.
  • MAG antibody fragments which contain the idio- type of the molecule can be generated by known tech ⁇ niques.
  • fragments include but are not limited to: the F (ab' )2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab, fragments which can be generated by reducing the disulfide bridges of the F(ab'>2 fragment, and the two Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • Monoclonal antibodies known to react with human MAG may be tested for their usefulness to serve as MAG antagonists (Nobile-Orazio et al. , (1984) Neurology, 34:1336-1342; Doberson et al., (1985) Neurochem. Res . , 10:499-513).
  • MAG antagonist candidates for evaluation in the assay are fragments, analogs and derivatives of MAG. Such candidates may interfere with MAG-mediated growth inhibition as competitive but non-functional mimics of endogenous MAG. From the reported amino acid sequence of MAG and from the cloned DNA coding for it, it will be appreciated that MAG fragments can be produced either by peptide syn ⁇ thesis or by recombinant DNA expression of either a truncated MAG, such as the 67kD ectodomain described by Attia S. et al. ((1993) J. Neurochem.
  • MAG or MAG fragments can be gener- ated also by recombinant DNA techniques or by peptide synthesis, and will incorporate one or more, e.g. 1-5, L- or D-amino acid substitutions.
  • Derivatives of MAG, MAG fragments and MAG analogs can be generated by chemical reaction of the parent substance to incorpo ⁇ rate the desired derivatizing group, such as N-termi- nal, C-terminal and intra-residue modifying groups that have the effect of masking or stabilizing the substance or target amino acids within it.
  • candidate MAG antagonists include those that are derived from a determination of the functionally active region(s) of MAG contained within, but not exclusively limited to, the known immunoglobulin-like domains.
  • any oth ⁇ ers to be prepared against epitopes in the ectodomain when found to be function-blocking in in vi tro assays, can be used to map the active regions of the polypep- tide as has been reported (Fahrig et al., (1993) Europ. , J. Neurosci . , 5:1118-1126; Tropak et al. , (1994) J. Neurochem. , 62:854-862).
  • it can be determined which of the five immunoglobulin-like domains of MAG is/are recognized by neuronal receptors and/or are involved in inhibition of neurite out- growth.
  • synthetic peptides can be prepared to be assayed as candidate antagonists of the MAG effect.
  • Derivatives of these can be prepared, including those with selected amino acid substitutions to provide desirable properties to enhance their effectiveness as antagonists of the MAG candidate functional regions of MAG can also be determined by the preparation of altered forms of the MAG ectodomain using recombinant DNA technologies to produce deletion or insertion mutants that can be expressed in various cell types as chimaeric proteins that contain the Fc portion of immunoglobulin G (Kelm et al., (1994) Curr. Biol . , 4:965-972).
  • candidate mutant forms of MAG can be expressed on cell surfaces by transfection of various cultured cell types.
  • the MAG antagonist is formulated as a pharmaceutical composition which contains the MAG antagonist in an amount effective to suppress MAG-mediated inhibition of nerve growth, in combination with a suitable phar ⁇ maceutical carrier.
  • Such compositions are useful, in accordance with another aspect of the invention, to suppress MAG-inhibited nerve growth in patients diag ⁇ nosed with a variety of neurological disorders, condi- tions and ailments of the PNS and the CNS where treat ⁇ ment to increase neurite extension, growth, or regen ⁇ eration is desired, e.g., in patients with nervous system damage.
  • MAG antagonists can be treated with such MAG antagonists.
  • disorders include but are not limited to Strokes, Alzheimer's disease, Down's syndrome, Creutzfeldt-Jacob disease, kuru, Gerstman-Straussler syndrome, scrapie, transmissible mink encephalopathy, Huntington's disease, Riley-Day familial dysautonomia, multiple system atrophy, amyotropic lateral sclerosis or Lou Gehrig's disease, progressive supranuclear palsy, Parkinson's disease and the like.
  • the MAG antagonists may be used to promote the regeneration of CNS pathways, fiber systems and tracts.
  • MAG antagonists are used to promote the regen ⁇ eration of nerve fibers over long distances following spinal cord damage.
  • MAG and related compounds that retain the MAG property of inhibiting neuron growth are used therapeutically to treat conditions in which suppression of undesired neuronal growth is desired. These include for example the treatment of tumors of nerve tissue and of conditions resulting from uncontrolled nerve sprouting such as is associated with epilepsy and in the spinal cord after nerve injury.
  • patients with neuro- blastoma, and particularly with neuropathies associ ⁇ ated with circulating MAG antibody can be treated with MAG or MAG agonist.
  • Useful for nerve growth suppression are pharma ⁇ ceutical compositions that contain, in an amount effective to suppress nerve growth, either MAG or a MAG agonist in combination with an acceptable carrier.
  • MAG can be obtained either by extraction from myelin as described above or, more practically, by recombi ⁇ nant DNA expression of MAG-encoding DNA in the manner reported by Attia S. et al. ((1993) J. Neurochem. , 61:718-726).
  • Useful MAG agonists are those compounds which, when added to the permissive substrate described above, suppress the growth of neuronal cells.
  • MAG agonists are those compounds which cause a statistically significant reduction in the number of neuronal cells that extend neurites, relative to control cells not exposed to the agonist.
  • Candidate MAG agonists include fragments of MAG that incorporate the ectodomain, including the ectodomain per se and other N- and/or C-terminally truncated fragments of MAG or the ectodomain, as well as analogs thereof in which amino acids, e.g. from 1 to 10 residues, are substituted, particularly conser ⁇ vatively, and derivatives of MAG or MAG fragments in which the N- and/or C-terminal residues are derivat- ized by chemical stabilizing groups.
  • Such MAG ago ⁇ nists can also include anti-idiotypes of MAG antibod ⁇ ies and their binding fragments.
  • can- didate MAG agonists include specific regions of the MAG ectodomain, and analogs or derivatives of these. These can be identified by using the same technologies described above for identification of MAG regions that serve as inhibitors of neurite outgrowth.
  • the MAG-related derivatives, analogs, and frag ⁇ ments of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene of protein level. For example, MAG-encoding DNA can be modified by any of numerous strategies known in the art (Maniatis et al.
  • the MAG-encoding gene can be mutated in vi tro or in vivo, for instance in the man- ner applied for production of the ectodomain, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonucle- ase sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mut- agenesis known in the art can be used, including but not limited to, in vi tro site-directed mutagenesi ⁇ (Hutchinson, et al., (1978) J. Biol . Che . , 253:6551), use of TABTM linkers (Pharmacia), etc.
  • MAG MAG agonist
  • MAG antagonist various known delivery systems can be used, such as encapsulation in liposomes or semiperme- able membranes, expression by suitably transformed or transfected glial cells, oligodendroglial cells, fibroblasts, etc. according to the procedure knownto those skilled in the art (Lindvall et al. , (1994) Curr. Opinion Neurobiol . , 4:752-757).
  • Linkage to ligands such as antibodies can be used to target delivery to myelin and to other therapeutically rele- vant sites in vivo .
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and
  • cells secreting MAG antago ⁇ nist activity for example, and not by way of limita ⁇ tion
  • hybridoma cells, excapsulated in a suitable bio ⁇ logical membrane may be implanted in a patient so as to provide a continuous source of MAG inhibitor.

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Abstract

The present invention relates to the use of a myelin associated glycoprotein (MAG) for the regulation of growth of neurons in peripheral nervous system (PNS) or central nervous system (CNS). The present invention relates to MAG agonists to inhibit neuron growth and to MAG antagonists to suppress the inhibition of neuron growth. The present invention also relates to an assay method useful to identify MAG antagonist agents that suppress inhibition of neuron growth, comprising the steps of: (a) culturing neurons on a growth permissive substrate that incorporates a growth-inhibiting amount of MAG; and (b) exposing the cultured neurons of step (a) to a candidate MAG antagonist agent in an amount and for a period sufficient prospectively to permit growth of said neurons; thereby identifying as MAG antagonists said candidates of step (b) which elicit neurite outgrowth from said cultured neurons of step (a).

Description

THERAPEUTIC USE OP YELIN-ASSOCIATED GLYCOPROTEIN (MAG) BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to the use of a myelin associated glycoprotein (MAG) for the regulation of growth of neurons in peripheral nervous system (PNS) or central nervous system (CNS).
(b) Description of Prior Art
Neurons injured in the central nervous system (CNS) of mammals do not regenerate. A major barrier to axonal regeneration in the CNS of mammals is the presence of growth inhibitory molecules. It is not known whether there is one or several inhibitory pro¬ teins present in myelin membranes, and the primary sequence has not yet been identified for any myelin associated protein inhibitor. The axon growth inhibi¬ tory property of CNS myelin has been well established, but the inhibitory proteins remain unknown. It is well documented that myelin in the CNS of adult mam- mals inhibits the growth of neurons in vi tro (P. Caroni, M.E. Schwab, (1988) Neuron, 1:85-96; M.E. Schwab, P. Caroni, (1988) J. Neurosci . , 8:2382-2393; M.E. Schwab, Annu . Rev. Neurosci . , (1993) 16:565; C. Bandtlow et al., (1990) J. Neurosci., 10:3838-3848) . Inhibitory activity can be extracted from SDS gels with proteins of approximately 250 and 35 kDa. Further, many in vivo experiments suggest that myelin- derived inhibitors block regeneration in vivo (Schnell and Schwab, (1990) Nature, 243:268; Schnell et al., (1994) Nature, 367:170).
In addition, molecules secreted by oligodendro- cytes, that have growth inhibitory properties, have been identified as 160 and 180 kDa homologues of tenascin (Peshiva et al., (1989) JOB, 109:1765-1788; Fuss et al., (1993) JCB, 120:1237-1249; Lochter et al., (1991) JCB, 113:1159-1171). The tenascin-like proteins are secreted and, therefore, are not expected to be present in myelin.
Of the present candidates, two proteins are antigenically related, and two others are secreted and do not likely co-purify with myelin (P. Pesheva et al., J. Cell Biol. 109, 1765 (1989); B. Fuss et al . , J. Cell
Biol. 5, 1237 (1993)).
To date none of the inhibitory proteins in myelin have been identified at the molecular level.
It would be highly desirable to be provided with such a myelin-derived inhibitory protein to be able to find reagents to overcome growth inhibition which would allow regenerative regrowth of damaged axons in the mammalian CNS.
SUMMARY OF THE INVENTION
There is now provided evidence for a novel myelin-derived inhibitory protein which can block regeneration of damaged neurons in the mammalian CNS and PNS.
In accordance with the present invention, another protein has now been identified, myelin asso¬ ciated glycoprotein (MAG) as one of the major molecu- lar components involved in contact-mediated growth inhibition on myelin. The growth inhibitory property of MAG was determined by: 1) demonstrating that MAG chromatographed with partially purified myelin-derived inhibitory activity; 2) testing the ability of recom- binant MAG to inhibit neurite growth.
In accordance with the present invention, there is provided the use of a myelin associated glycopro¬ tein (MAG) for the regulation of growth of neurons in PNS or CNS. In accordance with another aspect of the present invention, there is provided a method to suppress the inhibition of neuron growth, comprising the steps of delivering, to the nerve growth environ- ment, a myelin-associated glycoprotein antagonist in an amount effective to reverse said inhibition.
In accordance with another aspect of the present invention, there is provided an assay method useful to identify MAG antagonist agents that suppress inhibition of neuron growth, comprising the steps of: a) culturing neurons on a growth permissive substrate that incorporates a growth-inhibiting amount of MAG; and b) exposing the cultured neurons of step a) to a candidate MAG antagonist agent in an amount and for a period sufficient prospectively to permit growth of the neurons; thereby identifying as MAG antagonists the can¬ didates of step b) which elicit neurite outgrowth from the cultured neurons of step a) .
In a further aspect of the present invention, there is provided a method for inhibiting neuron growth, comprising the steps of introducing into the growth environment of the neurons a growth inhibiting amount of MAG or a MAG agonist.
For the purpose of the present invention the following terms are defined below.
"Agonist" refers to a pharmaceutical agent having myelin associated glycoprotein biological activity of inhibiting the neurite outgrowth of neu¬ rons cultured on a permissive substrate or inhibiting the regeneration of damaged neurons. It would be desirable to inhibit neuron growth in cases of epilepsy, neuroblastoma, and neuromas, a disease state in a mammal which includes neurite outgrowth or other neural growth of an abnormal sort which causes pain at the end of an amputated limb. Agonists which may be used in accordance with the present invention include without limitation a MAG fragment, an analog of MAG or of the MAG fragment, a derivative of either MAG, the MAG fragment or said analog, an anti-idiotypic MAG antibody or a binding fragment thereof, MAG ectodo- main, and a pharmaceutical agent.
"Antagonist" refers to a pharmaceutical agent which in accordance with the present invention which inhibits at least one biological activity normally associated with MAG, that is blocking or suppressing the inhibition of neuron growth. Antagonists which may be used in accordance with the present invention include without limitation a MAG antibody or a binding fragment of said antibody, a MAG fragment, a deriva¬ tive of MAG or of a MAG fragment, an analog of MAG or of a MAG fragment or of said derivative, and a pharma¬ ceutical agent, and is further characterized by the property of suppressing MAG-mediated inhibition of neurite outgrowth.
The agonist or antagonist of MAG in accordance with the present invention is not limited to MAG or its derivatives, but also includes the therapeutic application of all agents, referred herein as pharma¬ ceutical agents, which alter the biological activity of the neuronal receptor for MAG such that growth of neurons or their axons is suppressed. The receptor can be identified with known technologies by those skilled in the art (Mason, (1994) Curr. Biol . , 4:1158- 1161) and its association with MAG or fragments thereof can be determined. The neuronal receptor for MAG may or may not be the same as cell surface mole¬ cules that recognize and bind MAG in an adhesion assay (Kelm et al., (1994) Curr. Biol . , 4:965-972). Once the active MAG-recognition domain of the receptor(s) is/are known, appropriate peptides or their analogs can be designed and prepared to serve as agonist or antagonist of the MAG-receptor interaction. The term "effective amount" or "growth-inhibit¬ ing amount" refers to the amount of pharmaceutical agent required to produce a desired agonist or antago¬ nist effect of the MAG biological activity. The pre¬ cise effective amount will vary with the nature of pharmaceutical agent used and may be determined by one of ordinary skill in the art with only routine experi¬ mentation.
As used herein, the terms "MAG biological activity" refers to cellular events triggered by MAG, being of either biochemical or biophysical nature. The following list is provided, without limitation, which discloses some of the known activities associ¬ ated with MAG: contact-mediated growth inhibition on myelin, inhibition of neuron growth, inhibition of neurite outgrowth, adhesion to neuronal cells, and promotion of neurite outgrowth from newborn dorsal root ganglion neurons.
In general, the abbreviations used herein for designating the amino acids are based on the conven- tional one-letter abbreviations as indicated below:
Alanine A
Arginine R
Asparagine N
Aspartic Acid D
Cysteine C
Glutamine Q
Glutamic Acid E
Glycine G
Histidine H
Isoleucine I Leucine L
Lysine K
Methionine M
Phenylalanine F
Proline P
Serine S
Threonine T
Tryptophane W
Tyrosine Y
Valine V
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the neurite outgrowth from Dil-labeled NG108-15 cells on myelin and MAG substrates X 160;
Fig. 2 illustrates a quantitative analysis of neurite outgrowth from NG108-15 cells on different substrates; and
Fig. 3 is the analysis of growth inhibition after separation of myelin proteins by DEAE anion exchange chromatography.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, to search for the myelin-associated inhibitory molecules the strategy pursued was the purification of myelin inhibitors by non-denaturing extraction and column chromatography. This approach has allowed the identi¬ fication of a known protein of unknown function as a major myelin-derived growth inhibitor.
In accordance with the present invention, the myelin-associated glycoprotein (MAG) was identified as a major inhibitory molecules present in myelin. The primary sequence of the protein has previously been reported (Arquint et al., (1987) PNAS, 84:600-604; Salzer et al. , (1987) JOB, 104:957-965). This protein is present in both peripheral nervous system (PNS) and CNS myelin, an observation consistent with findings that PNS myelin also has inhibitory properties (Fig. 2).
Myelin-derived inhibitory activity was assessed by bioassays performed in tissue culture. Cells grow neurites on "permissive" substrates such as a polylys- ine or laminin substrate (Fig. 1A) . Purified bovine CNS myelin used as a tissue culture substrate permit¬ ted cell attachment, but inhibited cAMP-induced neu¬ rite outgrowth from NG108-15 cells (Fig. IB). Cells grown on 4 μg of myelin were rounded and did not grow neurites (Fig. IB). When cells were grown on recombi- nant MAG they did not spread or differentiate neurites (Fig. 1C; Attia S. et al. , (1993) J. Neurochem. , 61:718-726), but the treatment of MAG with heat at 80°C for 1 hour abolished its inhibitory properties (Fig. ID). Bovine corpus callosum or sciatic nerve was homogenized with a Dounce™ homogenizer, and myelin was purified as described by W.T. Norton et al. ((1973) J. Neurochem. , 21:749-757) . Myelin with 4μg or 8μg protein was added to polylysine-coated 96 well plates, dried and washed with PBS before plating the cells. NG108 cells readily accessible to those skilled in the art, a cell line that extends neurites in response to cAMP (D.G. Puro et al., (1976) Proc. Natl . Acad. Sci . , 73:3544-3548), were plated at 1000 cells/well in triplicate. After 24 hours the cultures were fixed with 4% paraformaldehyde, 0.1% glutaralde- hyde, stained with 0.5% cresyl violet, and the per¬ centage of cells with neurites of 1 cell body diameter or longer was determined. In some experiments neurite outgrowth was assayed with Di-I labeled NG108 cells as follows. cAMP primed NG108 cells were labeled with the lipophilic dye Di-I(1, 1 '-dioctadecyl-3, 3, 3 * , 3 ' -tetra- methylindocarbocyanine perchlorate) by incubating the cultures with 1% solution of the dye in culture medium for 2 hr. The cells were then rinsed and maintained in culture for 3 hr. prior to using them for the neu¬ rite outgrowth assay. Dil-labeled NG108 cells were added to the wells at a density of 1000 cells/well, and cultured for 24 hr. in Dulbecco's minimal essen- tial medium (DMEM) containing 5% fetal bovine serum, ImM dibutyryl cAMP. The cultures were then fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer for 30 min., washed and stored in buffer, the percentage of NG108 cells extending neu- rites longer than 1 cell body diameter was estimated with a Zeiss Axiomat™ inverted microscope equipped with fluorescence optics.
On a polylysine substrate, 70% ± 2. (n=26) of the plated cells extended neurites compared with 2% ± 0.6 (n=17) on 8μg myelin and 4% ± 1 (n=15) on 4μg mye¬ lin.
To characterize the growth inhibitory molecules present in CNS myelin, myelin-derived inhibitory activity was solubilized in octylglucoside and chroma- tographed on a DEAE anion exchange column.
Bovine brain myelin was extracted for 2 hr. at 20°C with 1% octylglucoside (1 ml per mg of protein) in 0.2 M phosphate buffer (pH 6.8) containing 0.1 M Na2Sθ4, 1 mM EDTA, 1 mM dithiothreitol and a cocktail of protease inhibitors. The extract was clarified by centrifugation at 400,000 g.min. and applied to a col¬ umn of DEAE-Sepharose™ (Pharmacia; 1.3 cm X 10 cm). After several washes, elution was effected with a NaCl gradient (0.2 M to 2.0 M) containing 1% octylglu- coside, 50 mM Tris HC1 (pH 7) and 1 mM dithiothreitol. Fractions (2.0 ml) were collected and protein concen¬ trations were estimated by the Bradford protein assay (Bio-Rad. Toronto, Ont). Aliquots of 70 μl were tested in culture. Proteins were prepared for SDS- PAGE by precipitation with trichloracetic acid, removal of detergent with acetone, and dispersion in boiling sample buffer.
Several peaks of inhibitory activity were eluted with a salt gradient. A profile of growth inhibition present in the column fractions is shown in Fig. 3. Percent inhibition was calculated as (C - E/C x 100) where C was the average number of cells with neurites on polylysine substrates, and E was the average number of cells with neurites on the test substrate. Western blots of the inhibitory frac¬ tions with anti-MAG antibody (GenS3; Nobile-Orazio et al., (1984) Neurology, 34:1336-1342) showed that the inhibitory peaks that eluted in low salt corresponded to the elution profile for MAG (Fig. 3). Fig. 3 is a Western blots of column fractions probed with anti-MAG antibody. The inhibitory activity eluted at the highest salt concentrations (Fig. 3, fractions 23-26) was not enriched in MAG, and likely represents a different inhibitory protein activity, possibly the one reported by Schwab (M.E. Schwab, (1993) Annu . Rev. Neurosci . , 16:565).
These data suggested that MAG may be an inhibi¬ tor of axon growth. Direct evidence for this role was obtained by showing that recombinant MAG was a potent inhibitor of neurite outgrowth from NG108 cells (Fig 1C; Fig. 2). Heating recombinant MAG at 80°C for 1 hr. abolished this activity (Fig. ID; Fig. 2). Comparison of cells grown on polylysine, CNS myelin, peripheral nerve myelin, recombinant MAG (MAG), dena- tured MAG (denat. MAG), and bovine serum albumin (BSA) demonstrate the potent growth inhibition of PNS and CNS, myelin and MAG. Results are the mean of 3 experiments done in triplicate; 2 experiments for denatured MAG (Fig. 2). In these experiments, no cells extended neurites when plated on MAG compared to 73%+ 4 on denatured MAG, or to 69%± 4 on BSA, used as a control. Because MAG is a component of myelin pro¬ duced by both Schwann cells and CNS glial cells, neu¬ rite growth inhibition present in peripheral nerve and CNS myelin were compared. Purified bovine CNS and peripheral nerve myelin plated at 4μg protein/well inhibited neurite outgrowth equally (Fig. 2). The observed growth inhibition by MAG, and its presence in peripheral nerve myelin, accords with recent observa- tions that unlesioned sciatic nerve has growth inhibi¬ tory properties (K.S. Bedi et al. , (1992) Europ. J. Neurosci , 4:193).
Although MAG is reported here as having potent neurite growth-inhibitory activity, its sequence homology with adhesion molecules of the immunoglobulin family has led others to investigate a possible role for MAG in cell adhesion (M. Arquint et al. , (1987) Proc . Natl . Acad. Sci . U. S. A. , 84:600; M. Poltorak et al., (1987) J. Cell Biol . , 105:1893; J.L. Salzer et al., (1987) J. Cell Biol . , 104:957; Kelm, S. et al. , (1994) Current Biology, 4:965-972; Schachner et al. , (1994) Curr. Opinion Neurobiol . , 4:726-734; Doherty et ■al., (1994) Curr. Opinion Neurobiol . , 4:49-55).
The results of the present invention were cor- related as follows with the growth inhibitory proper¬ ties of MAG with experimental evidence that the MAG extracellular domain may mediate adhesion to axons. First, the finding that recombinant MAG lacking the L2/HNK-1 carbohydrate epitope is a potent neurite out- growth inhibitor suggests that there may be functional differences among carbohydrate variants of MAG. Further, observations that MAG-containing liposomes bind to neurons specifically suggests a receptor- mediated interaction between neurons and MAG. However, such experiments do not reveal how MAG binding may affect growth cone dynamics. Finally, adhesive prop¬ erties of MAG have been examined when it was expressed in heterologous, living cells. In these experiments, cell adhesion molecules or extracellular matrix compo- nents that are known to override the growth inhibition on myelin may be present.
The growth inhibitory properties of MAG may be of critical importance after nerve injury in the CNS. In contrast to injured peripheral nerves, myelin debris in the CNS is not removed very quickly after injury (G. Stoll et al., (1989) J. Neurosci . , 9:2327- 2335). In addition, peripheral nerve myelin is asso¬ ciated with molecules such as laminin that can over¬ ride the inhibitory effects of myelin. Although it is known that CNS neurons will regenerate in a peripheral nerve environment, they show little capacity to elon¬ gate in the CNS. Growth inhibition by MAG, which is a prominent glycoprotein component of the total myelin protein (R.H. Quarles, D.R. Coleman, J.L. Salzer and B.D. Trapp., (1992) In: Myelin : Biology and Chemistry, R.E. Martenson, ed. CRC Press), may thus contribute significantly to the failure of injured axons to regrow within the CNS.
Based on the present evidence that MAG is a major growth inhibitory protein in myelin, one can now identify agents and therapies that suppress MAG-medi¬ ated inhibition of nerve growth. Further, one can exploit the growth inhibiting properties of MAG, or MAG agonists, to suppress undesired nerve growth. Without the critical finding that MAG has growth inhibitory properties, these strategies would not be developed.
More particularly, the invention provides an assay method adapted to identify MAG antagonist, that is agents that block or suppress the growth-inhibiting action of MAG. In its most convenient form, the assay is a tissue culture assay that measures neurite out¬ growth as a convenient end-point, and accordingly uses nerve cells that extend neurites when grown on a per- missive substrate. Nerve cells suitable in this regard include neuroblastoma cells of the NG108 line¬ age, such as NG108-15, as well as other neuronal cell lines such as PC12 cells (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA, ATCC accession No. CRL 1721), human neuroblastoma cells, and primary cultures of CNS or PNS neurons taken from embryonic, postnatal or adult animals. The nerve cells, for instance about 10^ cells-microwell or equivalent, are cultured on a growth permissive sub- strate, such as polylysine or laminin, that is over- layed with a growth-inhibiting amount of MAG. The MAG incorporated in the culture is suitably myelin- extracted MAG, although forms of MAG -other than endogenous forms can be used provided they exhibit the MAG property of inhibiting neuron growth when added to a substrate that is otherwise growth permissive. Suitable MAG alternatives include, for example, the MAG ectodomain reported by Salzer et al. ((1989) Develop. Neurosci . , 11:377-390) and by Attia et al. ((1993) J. Neurochem. , 61:718-726), and other growth- inhibiting MAG fragments.
In this assay, candidate MAG antagonists, i.e., compounds that block the growth-inhibiting effect of MAG, are added to the MAG-containing tissue culture preferably in amounts sufficient to neutralize the MAG growth-inhibiting activity, that is between 1.5 and 15 μg of MAG antagonists per well containing a density of 1000 NG108-15 cells/well cultured for 24 hr. in Dulbecco's minimal essential medium. After culturing for a period sufficient for neurite outgrowth, e.g. 3- 7 days, the culture is evaluated for neurite outgrowth, and MAG antagonists are thereby revealed as those candidates which elicit neurite outgrowth. Desirably, candidates selected as MAG antagonists are those which elicit neurite outgrowth to a statisti¬ cally significant extent, e.g., in at least 50%, more desirably at least 60%, e.g. 70%, per 1,000 cultured neurons.
Other assay tests that could be used include without limitation the following: 1) The growth cone collapse assay that is used to assess growth inhibi¬ tory activity of collapsin (Raper, J.A., and Kapfhammer, J.P., (1990) Neuron, 2:21-29; Luo et al. , (1993) Cell , 75:217-227) and of various other inhibi- tory molecules (Igarashi, M. et al. , (1993) Science, 259:77-79) whereby the test substance is added to the culture medium and a loss of elaborate growth cone morphology is scored. 2) The use of patterned sub¬ strates to assess substrate preference (Walter, J. et al., (1987) Development, 101:909-913; Stahl et al., (1990) Neuron, 5:735-743) or avoidance of test sub¬ strates (Ethell, D.W. et al., (1993) Dev. Brain Res . , 72:1-8). 3) The expression of recombinant proteins on a heterologous cell surface, and the transfected cells are used in co-culture experiments. The ability of the neurons to extend neurites on the transfected cells is assessed (Mukhopadhyay et al., (1994) Neuron, 13:757-767). 4) The use of sections of tissue, such as sections of CNS white matter, to assess molecules that may modulate growth inhibition (Carbonetto et al., (1987) J. Neuroscience, 7:610-620; Savlo, T. and Schwab, M.E., (1989) J. Neurosci . , 9:1126-1133). 5) Neurite retraction assays whereby test substrates are applied to differentiated neural cells for their abil- ity to induce or inhibit the retraction of previously extended neurites (Jalnink et al., (1994) J. Cell Biol . , 126:801-810; Suidan, H.S. et al. , (1992) Neuron, 8:363-375; Smalheiser, N. (1993) J.
Neurochem. , 61:340-343). 6) The repulsion of cell- cell interactions by cell aggregation assays (Kelm, S. et al., (1994) Current Biology, 4:965-972; Brady- Kainay, S. et al. , (1993) J. Cell Biol . , 4:961-972). 7) The use of nitrocellulose to prepare substrates for growth assays to assess the ability of neural cells to extend neurites on the test substrate (Laganeur, C. and Lemmon, V., (1987) PNAS, 84:7753-7757; Dou, C-L and Levine, J.M., (1994) J. Neuroscience, 14:7616- 7628) .
Useful MAG antagonists include antibodies to MAG and the binding fragments of those antibodies. Antibodies which are either monoclonal or polyclonal can be produced which recognize MAG and its various epitopes using now routine procedures. For the rais¬ ing of antibody, various host animals can be immunized by injection with MAG or fragment thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinmitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette- Guerin) . A monoclonal antibody to an epitope of MAG can be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not lim- ited to the hybridoma technique originally described by Kδhler and Milstein ((1975) Nature, 256:495-497), and the more recent human B cell hybridoma technique (Kozbor et al., (1983) Immunology Today, 4:72) and EBV-hybridoma technique (Cole et al. , (1985) In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In a particular embodiment, the procedure described by Nobile-Orazio et al. ((1984) Neurology, 34:1336-1342) may be used to obtain antibodies which recognize recombinant MAG (Attia S. et al., (1993) J. Neurochem. , 61:718-726).
The monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric human- mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g. Teng et al. , (1983) Proc . Natl . Acad. Sci . U. S. A. , 80:7308-7312; Kozbor et al., (1983) Immunology Today, 4:72-79; Olsson et al., (1982) Meth . Enzymol . , 92:3-16). Chimeric antibody molecules may be prepared containing a mouse antigen- binding domain with human contact regions (Morrision et al., (1984) Proc . Natl . Acad. Sci . U. S. A. , 81:6851; Takeda et al., (1985) Nature, 314:452).
A molecular clone of an antibody to a MAG epi¬ tope can be prepared by known techniques. Recombinant DNA methodology may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof (see e.g., Maniatis et al., (1982) In Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). For use, MAG antibody molecules may be purified by known techniques, such as immunoabsorption or immu- noaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof, etc.
MAG antibody fragments which contain the idio- type of the molecule can be generated by known tech¬ niques. For example, such fragments include but are not limited to: the F (ab' )2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab, fragments which can be generated by reducing the disulfide bridges of the F(ab'>2 fragment, and the two Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
Monoclonal antibodies known to react with human MAG may be tested for their usefulness to serve as MAG antagonists (Nobile-Orazio et al. , (1984) Neurology, 34:1336-1342; Doberson et al., (1985) Neurochem. Res . , 10:499-513).
Also suitable as MAG antagonist candidates for evaluation in the assay are fragments, analogs and derivatives of MAG. Such candidates may interfere with MAG-mediated growth inhibition as competitive but non-functional mimics of endogenous MAG. From the reported amino acid sequence of MAG and from the cloned DNA coding for it, it will be appreciated that MAG fragments can be produced either by peptide syn¬ thesis or by recombinant DNA expression of either a truncated MAG, such as the 67kD ectodomain described by Attia S. et al. ((1993) J. Neurochem. , 61:718-726) or of 72kD intact MAG could be prepared using standard recombinant procedures, that can then be digested enzymically in either a random or a site-selective manner. Analogs of MAG or MAG fragments can be gener- ated also by recombinant DNA techniques or by peptide synthesis, and will incorporate one or more, e.g. 1-5, L- or D-amino acid substitutions. Derivatives of MAG, MAG fragments and MAG analogs can be generated by chemical reaction of the parent substance to incorpo¬ rate the desired derivatizing group, such as N-termi- nal, C-terminal and intra-residue modifying groups that have the effect of masking or stabilizing the substance or target amino acids within it. In specific embodiments of the invention, candidate MAG antagonists include those that are derived from a determination of the functionally active region(s) of MAG contained within, but not exclusively limited to, the known immunoglobulin-like domains. The antibodies mentioned above and any oth¬ ers to be prepared against epitopes in the ectodomain, when found to be function-blocking in in vi tro assays, can be used to map the active regions of the polypep- tide as has been reported (Fahrig et al., (1993) Europ. , J. Neurosci . , 5:1118-1126; Tropak et al. , (1994) J. Neurochem. , 62:854-862). Thus, it can be determined which of the five immunoglobulin-like domains of MAG is/are recognized by neuronal receptors and/or are involved in inhibition of neurite out- growth. When those are known, synthetic peptides can be prepared to be assayed as candidate antagonists of the MAG effect. Derivatives of these can be prepared, including those with selected amino acid substitutions to provide desirable properties to enhance their effectiveness as antagonists of the MAG candidate functional regions of MAG can also be determined by the preparation of altered forms of the MAG ectodomain using recombinant DNA technologies to produce deletion or insertion mutants that can be expressed in various cell types as chimaeric proteins that contain the Fc portion of immunoglobulin G (Kelm et al., (1994) Curr. Biol . , 4:965-972). Alternatively, candidate mutant forms of MAG can be expressed on cell surfaces by transfection of various cultured cell types. All of the above forms of MAG, and forms that may be gener¬ ated by technologies not limited to the above, can be tested for the presence of functional regions that inhibit or suppress neurite outgrowth, and can be used to design and prepare peptides to serve as antago- nists.
In accordance with an aspect of the invention, the MAG antagonist is formulated as a pharmaceutical composition which contains the MAG antagonist in an amount effective to suppress MAG-mediated inhibition of nerve growth, in combination with a suitable phar¬ maceutical carrier. Such compositions are useful, in accordance with another aspect of the invention, to suppress MAG-inhibited nerve growth in patients diag¬ nosed with a variety of neurological disorders, condi- tions and ailments of the PNS and the CNS where treat¬ ment to increase neurite extension, growth, or regen¬ eration is desired, e.g., in patients with nervous system damage. Patients suffering from traumatic dis¬ orders (including but not limited to spinal cord inju- ries, spinal cord lesions, surgical nerve lesions or other CNS pathway lesions) damage secondary to infarc¬ tion, infection, exposure to toxic agents, malignancy, paraneoplastic syndromes, or patients with various types of degenerative disorders of the central nervous system (Cutler, (1987) In: Scientific American
Medicines, vol. 2, Scientific American Inc., N.Y., pp. 11-1-11-13) can be treated with such MAG antagonists. Examples of such disorders include but are not limited to Strokes, Alzheimer's disease, Down's syndrome, Creutzfeldt-Jacob disease, kuru, Gerstman-Straussler syndrome, scrapie, transmissible mink encephalopathy, Huntington's disease, Riley-Day familial dysautonomia, multiple system atrophy, amyotropic lateral sclerosis or Lou Gehrig's disease, progressive supranuclear palsy, Parkinson's disease and the like. The MAG antagonists may be used to promote the regeneration of CNS pathways, fiber systems and tracts. Administra¬ tion of antibodies directed to an epitope of MAG, or the binding portion thereof, or cells secreting such antibodies can also be used to inhibit MAG function in patients. In a particular embodiment of the inven¬ tion, the MAG antagonist is used to promote the regen¬ eration of nerve fibers over long distances following spinal cord damage. In accordance with another aspect of the inven¬ tion, MAG and related compounds that retain the MAG property of inhibiting neuron growth (herein referred to as MAG agonists) are used therapeutically to treat conditions in which suppression of undesired neuronal growth is desired. These include for example the treatment of tumors of nerve tissue and of conditions resulting from uncontrolled nerve sprouting such as is associated with epilepsy and in the spinal cord after nerve injury. In one embodiment, patients with neuro- blastoma, and particularly with neuropathies associ¬ ated with circulating MAG antibody, can be treated with MAG or MAG agonist.
Useful for nerve growth suppression are pharma¬ ceutical compositions that contain, in an amount effective to suppress nerve growth, either MAG or a MAG agonist in combination with an acceptable carrier. MAG can be obtained either by extraction from myelin as described above or, more practically, by recombi¬ nant DNA expression of MAG-encoding DNA in the manner reported by Attia S. et al. ((1993) J. Neurochem. , 61:718-726). Useful MAG agonists are those compounds which, when added to the permissive substrate described above, suppress the growth of neuronal cells. Particularly useful MAG agonists are those compounds which cause a statistically significant reduction in the number of neuronal cells that extend neurites, relative to control cells not exposed to the agonist. Candidate MAG agonists include fragments of MAG that incorporate the ectodomain, including the ectodomain per se and other N- and/or C-terminally truncated fragments of MAG or the ectodomain, as well as analogs thereof in which amino acids, e.g. from 1 to 10 residues, are substituted, particularly conser¬ vatively, and derivatives of MAG or MAG fragments in which the N- and/or C-terminal residues are derivat- ized by chemical stabilizing groups. Such MAG ago¬ nists can also include anti-idiotypes of MAG antibod¬ ies and their binding fragments.
In specific embodiments of the invention, can- didate MAG agonists include specific regions of the MAG ectodomain, and analogs or derivatives of these. These can be identified by using the same technologies described above for identification of MAG regions that serve as inhibitors of neurite outgrowth. The MAG-related derivatives, analogs, and frag¬ ments of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene of protein level. For example, MAG-encoding DNA can be modified by any of numerous strategies known in the art (Maniatis et al. , (1982) In Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), such as by cleavage at appropri¬ ate sites with restriction endonuclease(s) , subjected to enzymatic modifications if desired, isolated, and ligated in vitro.
Additionally, the MAG-encoding gene can be mutated in vi tro or in vivo, for instance in the man- ner applied for production of the ectodomain, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonucle- ase sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mut- agenesis known in the art can be used, including but not limited to, in vi tro site-directed mutagenesiε (Hutchinson, et al., (1978) J. Biol . Che . , 253:6551), use of TAB™ linkers (Pharmacia), etc. For delivery of either MAG, MAG agonist or MAG antagonist, various known delivery systems can be used, such as encapsulation in liposomes or semiperme- able membranes, expression by suitably transformed or transfected glial cells, oligodendroglial cells, fibroblasts, etc. according to the procedure knownto those skilled in the art (Lindvall et al. , (1994) Curr. Opinion Neurobiol . , 4:752-757). Linkage to ligands such as antibodies can be used to target delivery to myelin and to other therapeutically rele- vant sites in vivo . Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and
• intranasal routes, and infusion into ventricles or a site of operation (e.g. for spinal cord lesions) or tumor removal. Likewise, cells secreting MAG antago¬ nist activity, for example, and not by way of limita¬ tion, hybridoma cells, excapsulated in a suitable bio¬ logical membrane may be implanted in a patient so as to provide a continuous source of MAG inhibitor. While the invention has been described in con¬ nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any vari- ations, uses or adaptations of the invention follow¬ ing, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WE CLAIM :
1. The use of a myelin associated glycoprotein (MAG), biologically active variants or fragments thereof, for the regulation of growth of neurons in PNS or CNS, wherein MAG has an apparent molecular weight of 100 kDa in its glycosylated form, wherein the polypep- tide is 67 kDa or 72 kDa, depending on the isoforms, which comprises about 0.1 to 1.0% of total myelin pro¬ tein.
2. The use of a myelin associated glycoprotein (MAG), biologically active variants or fragments thereof, for raising antibodies or ligands thereof which overcome growth inhibition, wherein MAG has an apparent molecular weight of 100 kDa in its glycosy¬ lated form, wherein the polypeptide is 67 kDa or 72 kDa, depending on the isoforms.
3. A method effective to suppress the inhibition of neuron growth, comprising the step of delivering a MAG antagonist to the nerve growth environment in an amount effective to reverse said inhibition.
4. A method according to claim 3, wherein said MAG antagonist is selected from a MAG antibody or a binding fragment of said antibody, a MAG fragment, a derivative of MAG or of a MAG fragment, an analog of MAG or of a MAG fragment or of said derivative, and a pharmaceuti¬ cal agent, and is further characterized by the property of suppressing MAG-mediated inhibition of neurite out¬ growth.
5. A method according to claim 4, wherein said MAG antagonist is a MAG antibody or a binding fragment thereof.
6. A method according to claim 3, 4 or 5, wherein said MAG antagonist is delivered to the growth environ¬ ment of a CNS neuron requiring growth or regeneration as a result of spinal cord injury, spinal cord lesions, surgical nerve lesions, damage secondary to infarction, infection, exposure to toxic agents and malignancy.
7. A method according to claim 3, 4 or 5, wherein said MAG antagonist is delivered to a patient having a medical condition selected from Strokes, Alzheimer's disease, Down's syndrome, Creutzfeldt-Jacob disease, kuru, Gerstman-Straussler syndrome, scrapie, transmissible mink encephalopathy, Huntington ' s disease, Riley-Day familial dysautonomia, multiple system atrophy, amyotropic lateral sclerosis or Lou Gehrig's disease, progressive supranuclear palsy, Parkinson's disease.
8. An assay method useful to identify MAG antago¬ nist agents that suppress inhibition of neuron growth, comprising the steps of: a) culturing neurons on a growth permissive sub¬ strate that incorporates a growth-inhibiting amount of MAG; and b) exposing the cultured neurons of step a) to a candidate MAG antagonist agent in an amount and for a period sufficient prospectively to permit growth of said neurons; thereby identifying as MAG antagonists said candidates of step b) which elicit neurite outgrowth from said cultured neurons of step a).
9. A method for inhibiting neuron growth, compris¬ ing the step of introducing into the neuron growth environment a growth-inhibiting amount of a neuron growth inhibitor selected from MAG and a MAG agonist.
10. A method according to claim 9, wherein said inhibitor is MAG.
11. A method according to claim 10, wherein said inhibitor is a MAG agonist having MAG-biological activ¬ ity of inhibiting neurite outgrowth from neurons cul¬ tured on a permissive substrate, and is selected from a MAG fragment, an analog of MAG or of the MAG fragment, a derivative of either MAG, the MAG fragment or said analog, an anti-idiotypic MAG antibody or a binding fragment thereof, and a pharmaceutical agent.
12. A method according to claim 10, wherein said MAG agonist is the MAG ectodomain.
13. A method according to claim 9, 10, 11 or 12, wherein said inhibitor is delivered to a patient afflicted with a medical condition selected from epi¬ lepsy, neuroblastoma and neuromas.
PCT/CA1995/000089 1994-02-21 1995-02-17 Therapeutic use of myelin-associated glycoprotein (mag) WO1995022344A1 (en)

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