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US20020072493A1 - Activated T cells, nervous system-specific antigens and their uses - Google Patents

Activated T cells, nervous system-specific antigens and their uses Download PDF

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US20020072493A1
US20020072493A1 US09/893,348 US89334801A US2002072493A1 US 20020072493 A1 US20020072493 A1 US 20020072493A1 US 89334801 A US89334801 A US 89334801A US 2002072493 A1 US2002072493 A1 US 2002072493A1
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Michal Eisenbach-Schwartz
Ehud Hauben
Irun Cohen
Pierre Beserman
Alon Mosonego
Gila Moalem
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Yeda Research and Development Co Ltd
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Yeda Research and Development Co Ltd
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Priority claimed from IL12455098A external-priority patent/IL124550A0/xx
Priority claimed from PCT/US1998/014715 external-priority patent/WO1999034827A1/fr
Priority claimed from US09/218,277 external-priority patent/US20030108528A1/en
Application filed by Yeda Research and Development Co Ltd filed Critical Yeda Research and Development Co Ltd
Priority to US09/893,348 priority Critical patent/US20020072493A1/en
Assigned to YEDA RESEARCH AND DEVELOPMENT CO. LTD. reassignment YEDA RESEARCH AND DEVELOPMENT CO. LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: COHEN, IRUN R., EISENBACH-SCHWARTZ, MICHAL, HAUBEN, EHUD, BESERMAN, PIERRE, MOALEM, GILA, MONSONEGO, ALON
Publication of US20020072493A1 publication Critical patent/US20020072493A1/en
Priority to AU2002314509A priority patent/AU2002314509A1/en
Priority to PCT/IL2002/000518 priority patent/WO2003002602A2/fr
Priority to US10/810,653 priority patent/US7560102B2/en
Priority to US11/563,630 priority patent/US20080279869A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/0007—Nervous system antigens; Prions
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/414—Nervous system antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • 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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/47—Brain; Nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to compositions and methods for the promotion of nerve regeneration or prevention or inhibition of neuronal degeneration to ameliorate the effects of injury or disease of the nervous system (NS).
  • activated T cells, an NS-specific antigen or peptide derived therefrom or a nucleotide sequence encoding an NS-specific antigen can be used to promote nerve regeneration or to confer neuroprotection and prevent or inhibit neuronal degeneration caused by injury or disease of nerves within the central nervous system or peripheral nervous system of a human subject.
  • the compositions of the present invention may be administered alone or may be optionally administered in any desired combination.
  • APC antigen-presenting cell
  • BSA bovine serum albumin
  • CAP compound action potential
  • CFA complete Freund's adjuvant
  • CNS central nervous system
  • 4-Di-10-Asp 4-(4-didecylamino)styryl)-N-methylpyridinium iodide
  • EAE experimental autoimmune encephalomyelitis
  • FCS fetal calf serum
  • IFA incomplete Freund's adjuvant
  • MAG myelin-associated glycoprotein
  • MBP myelin basic protein
  • MOG myelin oligodendrocyte glycoprotein
  • NS nervous system
  • OVA ovalbumin
  • PBS phosphate-buffered saline
  • PLP proteolipid it protein
  • PNS peripheral nervous system
  • RGC retinal ganglion cells
  • TCR T-cell receptor.
  • the nervous system comprises the central (CNS) and the peripheral (PNS) nervous system.
  • the CNS is composed of the brain, the spinal cord and the visual system; the PNS consists of all of the other neural elements, namely the nerves and ganglia outside of the brain and spinal cord.
  • Damage to the nervous system may result from a traumatic injury, such as penetrating trauma or blunt trauma, or a disease or disorder including, but not limited to Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis (ALS), diabetic neuropathy, senile dementia, stroke and ischemia.
  • a traumatic injury such as penetrating trauma or blunt trauma
  • a disease or disorder including, but not limited to Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis (ALS), diabetic neuropathy, senile dementia, stroke and ischemia.
  • the restricted communication between the CNS and blood-borne macrophages affects the capacity of axotomized axons to regrow; transplants of activated macrophages can promote CNS regrowth (Lazarov-Spiegler et al, 1996; Rapalino et al, 1998).
  • T cells have been shown to enter the CNS parenchyma, irrespective of their antigen specificity, but only T cells capable of reacting with a CNS antigen seem to persist there (Hickey et al, 1991; Werkele, 1993; Kramer et al, 1995).
  • T cells reactive to antigens of CNS white matter such as myelin basic protein (MBP)
  • MBP myelin basic protein
  • EAE paralytic disease experimental autoimmune encephalomyelitis
  • Anti-MPB T cells may also be involved in the human disease multiple sclerosis (Ota et al, 1990; Martin, 1997).
  • anti-MBP T cell clones are present in the immune systems of healthy subjects (Burns et al, 1983; Pette et al, 1990; Martin et al, 1990; Schluesener et al, 1985).
  • Activated T cells which normally patrol the intact CNS, transiently accumulate at sites of CNS white matter lesions (Hirschberg et al, 1998).
  • a catastrophic consequence of CNS injury is that the primary damage is often compounded by the gradual secondary loss of adjacent neurons that apparently were undamaged, or only marginally damaged, by the initial injury (Faden et al, 1992; Faden, 1993; McIntosh, 1993).
  • the primary lesion causes changes in extracellular ion concentrations, elevation of amounts of free radicals, release of neurotransmitters, depletion of growth factors, and local inflammation These changes trigger a cascade of destructive events in the adjacent neurons that initially escaped the primary injury (Lynch et al, 1994; Bazan et al, 1995; Wu et al, 1994).
  • This secondary damage is mediated by activation of voltage-dependent or agonist-gated channels, ion leaks, activation of calcium-dependent enzymes such as proteases, lipases and nucleases, mitochondrial dysfunction and energy depletion, culminating in neuronal cell death (Yoshina et al, 1991; Hovda et al, 1991; Zivin et al, 1991; Yoles et al, 1992).
  • the widespread loss of neurons beyond the loss caused directly by the primary injury has been called “secondary degeneration.”
  • CNS injury Another tragic consequence of CNS injury is that neurons in the mammalian CNS do not undergo spontaneous regeneration following an injury. Thus, a CNS injury causes permanent impairment of motor and sensory functions.
  • spinal shock Spinal cord lesions, regardless of the severity of the injury, initially result in a complete functional paralysis known as spinal shock. Some spontaneous recovery from spinal shock may be observed, starting a few days after the injury and tapering off within three to four weeks. The less severe the insult, the better the functional outcome. The extent of recovery is a function of the amount of undamaged tissue minus the loss due to secondary degeneration. Recovery from injury would be improved by neuroprotective treatment that could reduce secondary degeneration.
  • NS-specific activated T cells could be used to protect nervous system tissue from secondary degeneration which may follow damage caused by injury or disease of the CNS or PNS.
  • the mechanism of action of such NS-specific T cells has yet to be discovered, but the massive accumulation of exogenously administered T cells at the site of CNS injury suggests that the presence of T cells at the site of injury plays a prominent role in neuroprotection. It appears, however, that the accumulation, though a necessary condition, is not sufficient for the purpose, as T cells specific to the non-self antigen ovalbumin also accumulate at the site, but have no neuroprotective effect (Hirschberg et al, 1998).
  • NS-specific activated T cells can be carried out also with a natural or synthetic NS-specific antigen such as MAG, S-100, ⁇ -amyloid, Thy-1, P0, P2, a neurotransmitter receptor, and preferably human MBP, human proteolipid protein (PLP), and human oligodendrocyte glycoprotein (MOG), or with a peptide derived from an NS-specific antigen such as a peptide comprising amino acids 51-70 of MBP or amino acids 35-55 of MOG.
  • a natural or synthetic NS-specific antigen such as MAG, S-100, ⁇ -amyloid, Thy-1, P0, P2, a neurotransmitter receptor, and preferably human MBP, human proteolipid protein (PLP), and human oligodendrocyte glycoprotein (MOG), or with a peptide derived from an NS-specific antigen such as a peptide comprising amino acids 51-70 of MBP or amino acids 35-55 of MOG.
  • the present invention is directed to methods and compositions for promotion of nerve regeneration or for neuroprotection and prevention or inhibition of neuronal degeneration to ameliorate the effects of injury to, or disease of, the nervous system (NS).
  • NS nervous system
  • the present invention is based in part on the inventors' unexpected discovery that activated T cells that recognize an antigen of the NS of the patient promote nerve regeneration or confer neuroprotection.
  • “neuroprotection” refers to the prevention or inhibition of degenerative effects of injury or disease in the NS. Since it was thought until recently that immune cells do not participate in nervous system repair, it was quite surprising to discover that NS-specific activated T cells and also the NS-specific antigens themselves and peptides derived therefrom can be used to promote nerve regeneration or to protect nervous system tissue from secondary degeneration which may follow damage caused by injury or disease of the CNS or PNS.
  • the invention relates to a method for promoting nerve regeneration or for conferring neuroprotection and preventing or inhibiting neuronal degeneration in the central nervous system or peripheral nervous system for ameliorating the effects of injury or disease, comprising administering to an individual in need thereof at least one ingredient selected from the group consisting of:
  • the invention relates to a pharmaceutical composition for promoting nerve regeneration or for neuroprotection and prevention or inhibition of neuronal degeneration in the CNS or PNS for ameliorating the effects of injury or disease in the NS, comprising a therapeutically effective amount of at least one ingredient selected from the group consisting of (a) to (e) above or any combination of (a)-(e).
  • NS-specific antigen refers to an antigen of the NS that specifically activates T cells such that following activation the activated T cells accumulate at a site of injury or disease in the NS of the patient.
  • NS-specific antigens include, but are not limited to, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), S-100, ⁇ -amyloid, Thy-1, P0, P2, neurotransmitter receptors, the protein Nogo (Nogo-A, Nogo-B and Nogo-C) and the Nogo receptor (NgR).
  • MBP myelin basic protein
  • MOG myelin oligodendrocyte glycoprotein
  • PBP proteolipid protein
  • MAG myelin-associated glycoprotein
  • S-100 ⁇ -amyloid
  • Thy-1 Thy-1, P0, P2
  • P2 myelin-associated glycoprotein
  • FIG. 1 is a bar graph showing the presence of T cells in uninjured optic nerve or in injured optic nerve one week after injury.
  • Adult Lewis rats were injected with activated T cells of the anti-MBP (T MBP ), anti-OVA (T OVA ), anti-p277 (a peptide of the human hsp60) (Tp 277 ) lines, or with PBS, immediately after unilateral crush injury of the optic nerve.
  • T MBP anti-MBP
  • T OVA anti-OVA
  • Tp277 a peptide of the human hsp60
  • Tp 277 anti-p277 lines
  • the histogram shows the mean number of T cells per mm 2 ⁇ s.e.m., counted in two to three sections of each nerve. Each group contained three to four rats. The number of T cells was considerably higher in injured nerves of rats injected with anti-MBP, anti-OVA or anti-p277 T cells; statistical analysis (one-way ANOVA) showed significant differences between T cell numbers in injured optic nerves of rats injected with anti-MBP, anti-OVA, or anti-p277 T cells and the T cell numbers in injured optic nerves of rats injected with PBS (P ⁇ 0.001); and between injured optic nerves and uninjured optic nerves of rats injected with anti-MBP, anti-OVA, or anti-p277 T cells (P ⁇ 0.001).
  • FIG. 2 is a bar graph illustrating that T cells specific to MBP, but not to OVA or p277, protect neurons from secondary degeneration.
  • rats were injected with anti-MBP, anti-OVA or anti-p277 T cells, or with PBS.
  • the neurotracer dye 4-Di-10-Asp was applied to optic nerves distal to the site of the injury, immediately after injury (for assessment of primary damage) or two weeks later (for assessment of secondary degeneration). Five days after dye application, the retinas were excised and flat-mounted. Labeled retinal ganglion cells (RGCs) from three to five randomly selected fields in each retina (all located at approximately the same distance from the optic disk) were counted by fluorescence microscopy.
  • RRCs retinal ganglion cells
  • RGC survival in each group of injured nerves was expressed as the percentage of the total number of neurons spared after the primary injury (42% of neurons remained undamaged after the primary injury).
  • the neuroprotective effect of anti-MBP T cells compared with that of PBS was significant (P ⁇ 0.001, one-way ANOVA).
  • Anti-OVA T cells or anti-p277 T cells did not differ significantly from PBS in their effects on the protection of neurons that had escaped primary injury (P>0.05, one-way ANOVA).
  • the results are a summary of five experiments. Each group contained five to ten rats.
  • FIGS. 3 present photomicrographs of retrogradely labeled retinas of injured optic nerves of rats.
  • rats were injected with PBS (FIG. 3A) or with activated anti-p277 T cells (FIG. 3B) or activated anti-MBP T cells (FIG. 3C).
  • the neurotracer dye 4-Di-10-Asp was applied to the optic nerves, distal to the site of injury. After 5 days, the retinas were excised and flat-mounted. Labeled (surviving) RGCs, located at approximately the same distance from the optic disk in each retina, were photographed.
  • FIGS. 4 are graphs showing that clinical severity of EAE is not influenced by an optic nerve crush injury.
  • Lewis rats either uninjured (dash line) or immediately after optic nerve crush injury (solid line), were injected with activated anti-MBP T cells. EAE was evaluated according to a neurological paralysis scale. [Data points represent ⁇ s.e.m.] These results represent a summary of three experiments. Each group contained five to nine rats.
  • FIG. 4B shows that the number of RGCs in the uninjured optic nerve is not influenced by injection of anti-MBP T cells. Two weeks after the injection of anti-MBP T cells or PBS, 4-Di-10-Asp was applied to the optic nerves.
  • FIG. 5 is a bar graph showing that T cells specific to p51-70 of MBP protect neurons from secondary degeneration.
  • rats were injected with anti-MBP T cells, anti-p51-70 T cells, or PBS.
  • 4-Di-10-Asp was applied to optic nerves distal to the site of the injury, immediately after injury (for assessment of primary damage) or two weeks later (for assessment of secondary degeneration).
  • Five days after dye application the retinas were excised and flat-mounted. Labeled RGCs from three to five randomly selected fields in each retina (all located at approximately the same distance from the optic disk) were counted by fluorescence microscopy.
  • RGC survival in each group of injured nerves was expressed as the percentage of the total number of neurons spared after primary injury. Compared with that of PBS treatment, the neuroprotective effects of anti-MBP and of anti-p51-70 T cells were significant (P ⁇ 0.001, one-way ANOVA).
  • FIGS. 6 are graphs showing that anti-MBP T cells increase the compound action potential (CAP) amplitudes of injured optic nerves.
  • CAP compound action potential
  • FIGS. 7 are graphs showing recovery of voluntary motor activity as a function of time after contusion, with and without injection of autoimmune anti-MBP T cells.
  • FIG. 7A Twelve rats were deeply anesthetized and laminectomized, and then subjected to a contusion insult produced by a 10 gram weight dropped from a height of 50 mm. Six of the rats, selected at random, were then inoculated i.p. with 10 7 anti-MBP T cells and the other six were inoculated with PBS. At the indicated time points, locomotor behavior in an open field was scored by observers blinded to the treatment received by the rats. Results are expressed as the mean values for each group. The vertical bars indicate SEM.
  • FIG. 7B A similar experiment using five PBS-treated animals and six animals treated with anti-MBP T cells were all subjected to a more severe contusion. At the indicated time points, locomotor behavior in an open field was scored. The results are expressed as the mean values for each group. The vertical bars indicate SEM. Rats in the treated group are represented by open circles and rats in the control group are represented by black circles. Horizontal bars show the median values. The inset shows the median plateau values of the two groups.
  • FIGS. 8 show retrograde labeling of cell bodies at the red nucleus in rats treated with autoimmune anti-MBP T cells ( 8 A) and in control injured ( 8 B) rats.
  • 8 A autoimmune anti-MBP T cells
  • 8 B control injured rats
  • Three months after contusion and treatment with anti-MBP T cells some rats from both the treated and the control groups were re-anesthetized and a dye was applied below the site of the contusion. After five to seven days, the rats were again deeply anesthetized and their brains were excised, processed, and cryosectioned. Sections taken through the red nucleus were inspected and analyzed qualitatively and quantitatively under fluorescent and confocal microscopes.
  • FIG. 9 is a series of photographs showing diffusion-weighted imaging of contused spinal cord treated with anti-MBP T cells.
  • Spinal cords of MBP-T cell-treated and PBS-treated animals (with locomotion scores of 10 and 8, respectively) were excised under deep anesthesia, immediately fixed in 4% paraformaldehyde solution, and placed into 5 mm NMR tubes.
  • Diffusion anisotropy was measured in a Bruker DMX 400 widebore spectrometer using a microscopy probe with a 5-mm Helmholtz coil and actively shielded magnetic field gradients.
  • a multislice pulsed gradient spin echo experiment was performed with 9 axial slices, with the central slice positioned at the center of the spinal injury.
  • Images were acquired with TE of 31 ms, TR of 2000 ms, a diffusion time of 15 ms, a diffusion gradient duration of 3 ms, field of view 0.6 mm, matrix size 128 ⁇ 128, slice thickness 0.5 mm, and slice separation of 1.18 mm.
  • Four diffusion gradient values of 0, 28, 49, and 71 g/cm were applied along the read direction (transverse diffusion) or along the slice direction (longitudinal diffusion). Diffusion anisotropy is manifested by increased signal intensity in the images with the highest transverse diffusion gradient relative to the longitudinal diffusion gradient.
  • the excised spinal cords of a PBS-treated rat and in the rat treated with MBP-T cells were subjected to diffusion-weighted MRI analysis.
  • FIG. 10 is a graph illustrating inhibition of secondary degeneration after optic nerve crush injury in adult rats. See text, Example 3, for experimental details. Rats were injected intradermally through the footpads with a 21-mer peptide based on MOG amino acid residues 35-55 (MOG p35-55) ((50 ⁇ /animal, chemically synthesized at the Weizmann Institute of Science, Rehovot, Israel) or PBS ten days prior to optic nerve crush injury or MOG p35-55 in the absence of crush injury. MOG p35-55 was administered with IFA. Surviving optic nerve fibers were monitored by retrograde labeling of RGCs. The number of RGCs in rats injected with PBS or MOG p35-55 was expressed as a percentage of the total number of neurons in rats injected with MOG p35-55 in the absence of crush injury.
  • MOG p35-55 MOG amino acid residues 35-55
  • FIG. 11 is a graph illustrating inhibition in adult rats of secondary degeneration after optic nerve crush injury by MBP. See text, Example 4, for experimental details.
  • MBP Sigma, Israel
  • MBP (1 mg in 0.5 ml saline) was administered orally to adult rats by gavage using a blunt needle.
  • MBP was administered 5 times, i.e., every third day beginning two weeks prior to optic nerve crush injury.
  • Surviving optic nerve fibers were monitored by retrograde labeling of RGCs. The number of RGCs in treated rats was expressed as a percentage of the total number of neurons in untreated rats following the injury.
  • FIGS. 12 (A-F) show expression of B7 co-stimulatory molecules in intact and injured rat optic nerve.
  • Optic nerves were excised from adult Lewis rats before ( 12 A, 12 B) and three days after injury ( 12 C, 12 D, 12 E) and analyzed immunohistochemically for expression of the B7 co-stimulatory molecule.
  • the site of injury was delineated by GFAP staining.
  • calibrated cross-action forceps the right optic nerve was subjected to a mild crush injury 1-2 mm from the eye. The uninjured cointralateral nerve was left undisturbed. Immunohistochemical analysis of optic nerve antigens was performed as follows.
  • the sections were washed again and incubated with rhodamine isothiocyanate-conjugated goat anti-mouse IgG (with minimal cross-reaction to rat, human, bovine and horse serum protein) go (Jackson ImmunoResearch, West Grove, Pa.), for one hour at room is temperature. All washing solutions contained PBS and 0.05% Tween-20. All diluting solutions contained PBS containing 3% fetal calf serum and 2% bovine serum albumin. The sections were treated with glycerol containing 1,4-diazobicyclo-(2,2,2)-octane and were then viewed with a Zeiss microscope.
  • B7.2 positive cells after injury, from a rounded ( 12 A, 12 B) to a star-like shape ( 12 C, 12 D).
  • the B7.2 positive cells were present at a higher density closer to the injury site ( 12 E).
  • Expression of B7.1 was detectable only from day seven and only at the injured site ( 12 F).
  • FIGS. 13 A-C show immunohistochemical analysis of T cells, macrophages or microglia, and B7.2 co-stimulatory molecules in the injured optic nerves of rats fed MBP.
  • Lewis rats aged 6-8 weeks were fed 1 mg of bovine MBP (Sigma, Israel) (2 mg MBP/ml PBS) or 0.5 ml PBS only every other day by gastric intubation using a stainless steel feeding needle (Thomas Scientific, Swedesboro, N.J.) (Chen et al, 1994).
  • T cells were excised and prepared for immunohistochemical analysis of T cells using mouse monoclonal antibodies to T cell receptor 11, diluted 1:25, macrophages or microglia using anti-ED1 antibodies (Serotek, Oxford, U.K) diluted 1:250, astrocytes using anti-GFAP antibodies and B7.2 co-stimulatory molecules as described for FIG. 12.
  • T cells or in ED-1 positive cells between injured optic nerves of PBS-fed ( 13 A) and MBP-fed ( 13 B) rats.
  • the number of B7.2 positive cells at the site of injury of MBP-fed rats ( 13 C) should be noted, as compared with injured controls (see FIG. 12E above).
  • FIG. 14 is a graph showing the slowing of neuronal degeneration in rats with orally induced tolerance to MBP.
  • Lewis rats were fed 1 mg MBP daily, or every other day, or 4 times a day at two-hour intervals for five consecutive days.
  • Control animals were given PBS or the non-self antigen OVA Sigma, Israel).
  • FIG. 15 shows the nucleotide sequence of rat MBP gene, SEQ ID NO:1, Genbank accession number M25889 (Schaich et al, 1986).
  • FIG. 16 shows the nucleotide sequence of human MBP gene, SEQ ID NO:2, Genbank accession number M13577 (Kamholz et al, 1986).
  • FIGS. 17 (A-F) show the nucleotide sequences of human PLP gene exons 1-7, SEQ ID NOs:3-8, respectively, Genbank accession numbers M15026-M15032, respectively (Diehl et al, 1986).
  • FIG. 18 shows the nucleotide sequence of human MOG gene, SEQ ID NO:9, Genbank accession number Z48051 (Roth et al, submitted (Jan. 17, 1995) Roth, CNRS UPR 8291, CIGH, CHU Purpan,ière, France, 31300; Gonzalez et al, 1996).
  • FIG. 19 shows the nucleotide sequence of rat PLP gene and variant, SEQ ID NO:10, Genbank accession number M16471 (Nave et al, 1987).
  • FIG. 20 shows the nucleotide sequence of rat MAG gene, SEQ ID NO:11, Genbank accession number M14871 (Arquint et al, 1987).
  • FIG. 21 shows the amino acid sequence of human MBP, SEQ ID NO:12, Genbank accession number 307160 (Kamholz et al, 1986).
  • FIG. 22 shows the amino acid sequence of human PLP, SEQ ID NO:13, Genbank accession number 387028.
  • FIG. 23 shows the amino acid sequence of human MOG, SEQ ID NO:14, Genbank accession number 793839 (Roth et al, 1995; Roth Submitted (JAN. 17, 1995) Roth CNRS UPR 8291, CIGH, CHU Purpan,ière, France, 31300; Gonzalez et al, 1996).
  • FIGS. 24 show that post-traumatic immunization with Nogo peptide p472 emulsified in CFA promotes functional recovery from spinal cord contusion in comparison to PBS+CFA-treated rats.
  • Spinal cords of male SPD rats were laminectomized at the level of T9 and a 10-g rod was dropped onto the laminectomized cord from a height of 50 mm (FIG. 24A) or of 25 mm (FIG. 24B). See text, Example 5, for experimental details.
  • FIG. 25 show that post-traumatic immunization with Nogo peptide p472 emulsified in CFA promotes functional recovery from spinal cord contusion in comparison to PBS-treated or PBS+CFA-treated rats.
  • Spinal cords of female SPD rats were laminectomized at the level of T9 and a 10-g rod was dropped onto the laminectomized cord from a height of 50 mm. See text, Example 5, for experimental details.
  • the present invention relates to compositions and methods for promoting nerve regeneration or for conferring neuroprotection and preventing or inhibiting neuronal degeneration in the CNS or PNS for ameliorating the effects of injury or disease, comprising administering to an individual in need thereof at least one ingredient selected from the group consisting of:
  • NS-specific activated T cells and T-cell banks NS-specific antigens, analogs thereof, peptides derived therefrom and analogs and derivatives thereof of said peptides; nucleotide sequences encoding NS-specific antigens, analogs thereof, peptides derived therefrom and analogs thereof; therapeutic uses; and formulations and modes of administration.
  • NS-specific activated T cells can be used in an amount which is effective to confer neuroprotection for ameliorating or inhibiting the effects of injury or disease of the CNS or PNS that result in NS degeneration or for promoting regeneration in the NS, in particular the CNS, as described in the section on therapeutic uses hereinafter.
  • administering may optionally be in combination with an NS-specific antigen or an analog thereof or a peptide derived therefrom or an analog or derivative of said peptide.
  • oral administration of NS-specific antigen or an analog thereof or a peptide derived therefrom or an analog or derivative thereof can be combined with active immunization to build up a critical T-cell response immediately after injury.
  • Activation of T cells is initiated by interaction of a TCR complex with a processed antigenic peptide bound to a MHC molecule on the surface of an antigen-presenting cell (APC).
  • APC antigen-presenting cell
  • the term “activated T cells” includes both (i) T cells that have been activated by exposure to a cognate antigen or peptide derived therefrom or derivative thereof; and (ii) progeny of such activated T cells.
  • a “cognate antigen” is an antigen that is specifically recognized by the TCR of a T cell that has been previously exposed to the antigen.
  • the T cell which has been previously exposed to the antigen may be activated by a mitogen, such as phytohemagglutinin (PHA) or concanavalin A (Con A).
  • PHA phytohemagglutinin
  • Con A concanavalin A
  • NS-specific activated T cell refers to an activated T cell having specificity for an antigen of the NS, said NS-specific antigen being an antigen of the NS that specifically activates T cells such that these activated T cells will accumulate at a site of injury or disease in the NS of the patient.
  • the NS-specific antigen used to confer the specificity to the T cells may be a self NS-antigen of the patient or a non-self NS-antigen of another individual or even of another species, or an analog of said NS-antigen, or a peptide derived from said NS-antigen or from said analog thereof, or an analog or derivative of said peptide, all as described in the section on NS-specific antigens, analogs thereof, peptides derived therefrom and analogs and derivatives thereof of said peptides hereinafter, as long as the activated T cell recognizes an antigen in the NS of the patient.
  • the T cells which are used in accordance with the present invention for the treatment of neural damage or degeneration caused by such disease are preferably not activated against the same autoimmune antigen involved in the disease. While the prior art has described methods of treating autoimmune diseases by administering activated T cells to create a tolerance to the autoimmune antigen, the T cells of the present invention are not administered in such a way as to create tolerance, but are administered in such a way as to create accumulation of the T cells at the site of injury or disease so as to facilitate neural regeneration or to inhibit neural degeneration.
  • the prior art also discloses uses of immunotherapy against tumors, including brain tumors, by administering T cells specific to an NS antigen in the tumor so that such T cells may induce an immune system attack against the tumors.
  • the present invention is not intended to comprehend such prior art techniques.
  • the present invention is intended to comprehend the inhibition of neural degeneration or the enhancement of neural regeneration in patients with brain tumors by means other than the prior art immunotherapy of brain tumors.
  • NS-specific activated T cells which are activated to an NS-antigen of the patient other than an antigen which is involved in the tumor, would be expected to be useful for the purpose of the present invention and would not have been suggested by known immunotherapy techniques.
  • the NS-specific activated T cells are preferably autologous, most preferably of the CD4 and/or CD8 phenotypes, but they may also be semi-allogeneic T cells or allogeneic T cells from related donors, e.g., siblings, parents, children, or from donors with the same HLA type (HLA-matched) or a very similar HLA type (HLA-partially matched), or even from unrelated donors.
  • related donors e.g., siblings, parents, children, or from donors with the same HLA type (HLA-matched) or a very similar HLA type (HLA-partially matched), or even from unrelated donors.
  • the present invention also comprehends the use of semi-allogeneic T cells for neuroprotection.
  • the T cells may be prepared as short- or long-term lines and stored by conventional cryopreservation methods for thawing and administration, either immediately or after culturing for 1-3 days, to a subject suffering from injury to the CNS and in need of T-cell neuroprotection.
  • T cells can recognize a specific antigen epitope presented by foreign APC, provided that the APC expresses the MHC molecule, class I or class II, to which the specific responding T-cell population is restricted, along with the antigen epitope recognized by the T cells.
  • a semi-allogeneic population of T cells that can recognize at least one allelic product of the subject's MHC molecules, preferably a class II HLA-DR or HLA-DQ or other HLA molecule, and that is specific for a NS-associated antigen epitope, will be able to recognize the NS antigen in the subject's area of NS damage and produce the needed neuro-protective effect.
  • the semi-allogeneic T cells will be able to migrate and accumulate at the CNS site in need of neuroprotection and will be activated to produce the desired effect.
  • semi-allogeneic T cells will be rejected by the subject's immune system, but that rejection requires about two weeks to develop. Hence, the semi-allogeneic T cells will have the two-week window of opportunity needed to exert neuroprotection. After two weeks, the semi-allogeneic T cells will be rejected from the body of the subject, but that rejection is advantageous to the subject because it will rid the subject of the foreign T cells and prevent any untoward consequences of the activated T cells.
  • the semi-allogeneic T cells thus provide an important safety factor and are a preferred embodiment.
  • HLA class II molecules are shared by most individuals in a population. For example, about 50% of the Jewish population express the HLA-DR5 gene. Thus, a bank of specific T cells reactive to NS-antigen epitopes that are restricted to HLA-DR5 would be useful in 50% of that population. The entire population can be covered essentially by a small number of additional T cell lines restricted to a few other prevalent HLA molecules, such as DR1, DR4, DR2, etc. Thus, a functional bank of uniform T cell lines can be prepared and stored for immediate use in almost any individual in a given population.
  • Such a bank of T cells would overcome any technical problems in obtaining a sufficient number of specific T cells from the subject in need of neuroprotection during the open window of treatment opportunity.
  • the semi-allogeneic T cells will be safely rejected after accomplishing their role of neuroprotection.
  • This aspect of the invention does not contradict, and is in addition to the use of autologous T cells as described herein.
  • the NS-specific activated T cells are preferably non-attenuated, although attenuated NS-specific activated T cells may be used.
  • T cells may be attenuated using methods well-known in the art including, but not limited to, by gamma-irradiation, e.g.,, 1.5-10.0 Rads (Ben-Nun et al, 1981; Ben-Nun and Cohen, 1982); and/or by pressure treatment, for example as described in U.S. Pat. No. 4,996,194 (Cohen et al); and/or by chemical cross-linking with an agent such as formaldehyde, glutaraldehyde and the like, for example as described in U.S. Pat. No.
  • NS-specific activated T cells are isolated as described below. T cells can be isolated and purified according to methods known in the art (Mor and Cohen, 1995). For an illustrative example, see Example 1, Materials and Methods.
  • Circulating T cells of a subject which recognize an NS-antigen are isolated and expanded using known procedures (Burns et al, 1983; Pette et al, 1990; Martin et al, 1990; Schluesener et al, 1985; Suruhan-Dires Keneli et al, 1993, which are incorporated herein by reference in their entirety).
  • T cells are isolated and the NS-specific activated T cells are then expanded.
  • the isolated T cells may be activated by exposure of the cells to one or more of a variety of natural or synthetic NS-specific antigens or epitopes as described in section on NS-specific antigens, analogs thereof, peptides derived therefrom and analogs and derivatives thereof of said peptides hereinafter.
  • the T cells may be activated by culturing in medium to which at least one suitable growth promoting factor has been added, such as cytokines, e.g., TNF- ⁇ , IL-2 and/or IL-4.
  • the NS-specific activated T cells endogenously produce a substance that ameliorates the effects of injury or disease in the NS.
  • the NS-specific activated T cells endogenously produce a substance that stimulates other cells, including, but not limited to, transforming growth factor- ⁇ (TGF- ⁇ ), nerve growth factor (NGF), neurotrophic factor 3(NT 3), neurotrophic factor 4/5 (NT-4/5), brain derived neurotrophic factor (BDNF); IFN- ⁇ and IL-6, wherein the other cells, directly or indirectly, ameliorate the effects of injury or disease.
  • TGF- ⁇ transforming growth factor- ⁇
  • NEF nerve growth factor
  • NNF neurotrophic factor 3(NT 3)
  • NT-4/5 neurotrophic factor 4/5
  • BDNF brain derived neurotrophic factor
  • IFN- ⁇ IFN- ⁇ and IL-6
  • T cells Following their proliferation in vitro, the T cells are administered to a mammalian, preferably a human, subject. T cell expansion is preferably performed using peptides corresponding to sequences in a non-pathogenic, NS-specific, self-protein.
  • a subject can initially be immunized with an NS-specific antigen using a non-pathogenic peptide of the self-protein.
  • a T-cell preparation can be prepared from the blood of such immunized subjects, preferably from T cells selected for their specificity towards the NS-specific antigen. The selected T cells can then be stimulated to produce a T cell line specific to the self-antigen (Ben-Nun and Cohen, 1982).
  • NS-specific antigen activated T cells obtained as described above, can be used immediately or may be preserved for later use, e.g., by cryopreservation as described below.
  • NS-specific activated T cells may also be obtained using previously cryopreserved T cells, i.e., after thawing the cells, the T cells may be incubated with NS-specific antigen, optimally together with thymocytes, to obtain a preparation of NS-specific activated T cells.
  • the T cells can be preserved, e.g., by cryopreservation, either before or after culture.
  • Cryopreservation agents which can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959; Ashwood-Smith, 1961), polyvinylpyrrolidone (Rinfret, 1960), glycerol, polyethylene glycol (Sloviter and Ravdin, 1962), albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al, 1962), D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al, 1960), amino acids (Phan The Tran and Bender, 1960), methanol, acetamide, glycerol monoacetate (Lovelock, 1954), inorganic salts (Phan The Tran and Bender, 1960 and 1961) and DMSO combined with hydroxyethyl starch and human serum albumin (Zaroulis
  • a controlled cooling rate is critical. Different cryoprotective agents (Rapatz et al, 1968) and different cell types have different optimal cooling rates. See, e.g., Rowe and Rinfret, 1962; Rowe, 1966; Lewis et al, 1967; Mazur, 1970) for effects of cooling velocity on survival of cells and on their transplantation potential. The heat of fusion phase where water turns to ice should be minimal.
  • the cooling procedure can be carried out by use of, e.g., a programmable freezing device or a methanol bath procedure.
  • Programmable freezing apparatuses allow determination of optimal cooling rates and facilitate standard reproducible cooling.
  • Programmable controlled-rate freezers such as Cryomed or Planar permit tuning of the freezing regimen to the desired cooling rate curve.
  • samples can be cryogenically stored in mechanical freezers, such as freezers that maintain a temperature of about ⁇ 80° C. or about ⁇ 20° C.
  • samples can be cryogenically stored in liquid nitrogen ( ⁇ 196° C.) or its vapor.
  • Frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37-47° C.) and chilled immediately upon thawing. It may be desirable to treat the cells in order to prevent cellular clumping upon thawing. To prevent clumping, various procedures can be used, including but not limited to the addition before or after freezing of DNAse (Spitzer et al, 1980), low molecular weight dextran and citrate, citrate, hydroxyethyl starch (Stiff et al, 1983), or acid citrate dextrose (Zaroulis and Senseman, 1980), etc.
  • cryoprotective agent if toxic in humans, should be removed prior to therapeutic use of the thawed T cells.
  • One way in which to remove the cryoprotective agent is by dilution to an insignificant concentration.
  • T cells Once frozen T cells have been thawed and recovered, they are used to promote neuroprotection as described herein with respect to non-frozen T cells. Once thawed, the T cells may be used immediately, assuming that they were activated prior to freezing. Preferably, however, the thawed cells are cultured before injection to the patient in order to eliminate non-viable cells. Furthermore, in the course of this culturing over a period of about one to three days, an appropriate activating agent can be added so as to activate the cells, if the frozen cells were resting T cells, or to help the cells achieve a higher rate of activation if they were activated prior to freezing. Usually, time is available to allow such a culturing step prior to administration as the T cells may be administered as long as a week after injury, and possibly longer, and still maintain their neuro-regenerative and neuroprotective effect.
  • patients can be treated by administering autologous or semi-allogeneic T lymphocytes sensitized to at least one appropriate NS-antigen.
  • therapy should be administered as soon as possible after the primary injury to maximize the chances of success, preferably within about one week.
  • a bank with autologous, semi-allogeneic or allogeneic T cells can be established for future use.
  • the invention provides cell banks that can be established to store NS-sensitized T cells for neuroprotective treatment of individuals at a later time, as needed.
  • autologous T cells may be obtained from an individual and the cell bank will contain personal vaults of autologous T lymphocytes prepared for future use for neuroprotective therapy against secondary degeneration in case of NS injury.
  • T lymphocytes are isolated from the blood, sensitized to a NS-antigen, and the cells are then frozen and suitably stored under the person's name, identity number, and blood group, in a cell bank until needed.
  • autologous stem cells of the CNS can be processed and stored for potential use by an individual patient in the event of traumatic disorders of the NS such as ischemia or mechanical injury, as well as for treating neurodegenerative conditions such as Alzheimer's disease or Parkinson's disease.
  • allogeneic or semi-allogeneic T cells may be stored such that a bank of T cells of each of the most common MHC-class II types are present.
  • the semi-allogeneic or allogeneic T cells are stored frozen for use by any individual who shares one MHC type II molecule with the source of the T cells.
  • the cells are preferably stored in an activated state after exposure to an NS-antigen or peptide derived therefrom. However, the cells may also be stored in a resting state and activated once they are thawed and prepared for use.
  • the cell lines of the bank are preferably cryopreserved. The cell lines are prepared in any way which is well known in the art. Once the cells are thawed, they are preferably cultured prior to injection in order to eliminate non-viable cells. During this culturing, the cells can be activated or reactivated using the same NS-antigen or peptide as used in the original activation.
  • activation may be achieved by culturing in the presence of a mitogen, such as phytohemagglutinin (PHA) or concanavalin A (preferably the former). This will place the cells into an even higher state of activation.
  • a mitogen such as phytohemagglutinin (PHA) or concanavalin A (preferably the former).
  • PHA phytohemagglutinin
  • concanavalin A preferably the former
  • NS-specific antigen refers to an antigen of the NS that specifically activates T cells such that following activation the activated T cells accumulate at a site of injury or disease in the NS of the patient.
  • the NS-specific antigen used according to the present invention may be an antigen obtained from NS tissue, preferably from tissue at a site of CNS injury or disease. It may be a crude NS-tissue preparation, e.g., derived from NS tissue obtained from mammalian NS that may include cells, both living or dead cells, membrane fractions of such cells or tissue, etc., and may be obtained by an NS biopsy or necropsy from a mammal, preferably human, tissue including, but not limited to, from a site of CNS injury; from cadavers; and from cell lines grown in culture.
  • the NS-specific antigen is an isolated or purified antigen.
  • the NS-specific antigen may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of antigens.
  • the functional properties may be evaluated using any suitable assay.
  • an NS-specific antigen may be a protein obtained by genetic engineering, chemically synthesized, etc.
  • natural or synthetic NS-specific antigens include, without being limited to, myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), myelin-associated glycoprotein (MAG), S-100, ⁇ -amyloid, Thy-1, P0, P2, neurotransmitter receptors, Nogo and Nogo receptor (NgR).
  • MBP myelin basic protein
  • PBP proteolipid protein
  • MOG myelin oligodendrocyte glycoprotein
  • MAG myelin-associated glycoprotein
  • S-100 S-100
  • ⁇ -amyloid Thy-1
  • P0, P2 neurotransmitter receptors
  • NgR Nogo and Nogo receptor
  • NS-specific antigens include but are not limited to, human MBP, depicted in FIG. 21 (SEQ ID NO:12); human PLP, depicted in FIG. 22 (SEQ ID NO:13); human MOG, depicted in FIG.
  • analogs of NS-specific antigens including, but not being limited to, those molecules comprising regions that are substantially homologous to the full-length NS-specific antigen, or fragments thereof.
  • these analogs will have at least 60% or 70% or 80% or 90% or 95% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art or whose encoding nucleic acid is capable of hybridizing to a coding nucleotide sequence of the full-length NS-specific antigen, under high stringency, moderate stringency, or low stringency conditions.
  • Computer programs for determining homology may include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al, 1990; Thompson, et al, 1994; Higgins, et al, 1996).
  • NS-specific antigen analogs of the invention can be produced by various methods known in the art.
  • the manipulations which result in their production can occur at the gene or protein level.
  • a cloned gene sequence can be modified by any of numerous strategies known in the art (Maniatis, 1990).
  • the sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the coding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, et al, 1978), etc.
  • Manipulations may also be made at the protein level. Included within the scope of the invention are NS-specific antigen derivatives which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • the invention relates to peptides derived from NS-specific antigens or from analogs thereof and to analogs or derivatives of said peptides, which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length NS-specific antigen.
  • Such functional activities include, but are not limited to, antigenicity (ability to bind, or compete with an NS-antigen for binding, to an anti-NS-specific antibody), immunogenicity (ability to generate antibody which binds to an NS-specific protein), and ability to interact with T cells, resulting in activation comparable to that obtained using the corresponding full-length NS-specific antigen.
  • the crucial test is that the antigen which is used for activating the T cells causes the T cells to be capable of recognizing an antigen in the NS of the mammal (patient) being treated.
  • the NS-antigen derived peptide may be either: (1) an immunogenic peptide, i.e., a peptide that can elicit a human T-cell response detected by a T-cell proliferation assay or by cytokine, e.g., IFN- ⁇ , IL-2, IL-4 or IL-10, production, or (2) a “cryptic epitope” (also designated herein as “immunosilent” or “non-immunodominant” epitope), i.e., a peptide that by itself can induce a T-cell immune response that is not induced by the whole antigen protein (see Moalem et al, 1999).
  • an immunogenic peptide i.e., a peptide that can elicit a human T-cell response detected by a T-cell proliferation assay or by cytokine, e.g., IFN- ⁇ , IL-2, IL-4 or IL-10, production
  • a peptide derived from a NS-specific antigen preferably has a sequence comprised within the NS-specific antigen sequence and has at least 10, 13, 15, 18, 20 or 50 contiguous amino acids of the NS-specific antigen sequence.
  • the peptide derived from an NS-specific antigen is a “cryptic epitope” of the antigen.
  • a cryptic epitope activates specific T cells after an animal is immunized with the particular peptide, but not with the whole antigen.
  • Cryptic epitopes for use in the present invention include, but are not limited to, peptides of the MBP sequence: peptides p11-30, p51-70, p87-99, p91-110, p131-150, and p151-170.
  • Such cryptic epitopes are particularly preferred as T cells activated thereby will accumulate at the injury site, but are particularly weak in autoimmunity. Thus, they would be expected to have fewer side effects.
  • the peptide derived from an NS-specific antigen is an immunogenic epitope of the antigen.
  • peptides according to the invention are immunogenic peptides derived from the Nogo protein sequence such as, but not being limited to, the 18-mer p472 Nogo peptide (SEQ ID NO:18) and peptides derived from the Nogo receptor (Fournier et al, 2001) such as the 15-mer peptides of the sequences:
  • the peptide is an analog of a peptide derived from an NS-specific antigen that is immunogenic but not encephalitogenic.
  • the most suitable peptides for this purpose are those in which an encephalitogenic self-peptide is modified at the T-cell receptor (TCR) binding site and not at the MHC binding site(s), so that the immune response is activated but not anergized (Karin et al, 1998; Vergelli et al, 1996).
  • These analogs may be produced by replacement of one or more amino acid residues of the peptide by other amino acid residues, preferably in their TCR binding site. Suitable replacements are those in which charged amino residues like lysine, proline or arginine are replaced by glycine or alanine residues.
  • altered peptides can be produced from peptides p11-30, p51-70, p87-99, p91-110, p131-150, and p151-170 of human MBP, for example from the p87-99 peptide in which the lysine 91 is replaced by glycine and/or the proline 96 is replaced by an alanine residue, thus converting an encephalitogenic peptide in immunogenic but non-encephalitogenic peptide that still recognizes the TCR.
  • altered peptides can be produced from the encephalitogenic p472 Nogo peptide (Nogo p623-640) by replacement of the lys 628 residue and from the Nogo receptor peptides above by replacement of the arg (R) residue by Val or Ala or another similar residue.
  • the analogs also comprise replacement of one or more amino acid residues of the peptide or addition to the peptide of non-natural amino acids including, but not limited to, the D-isomers of the common amino acids, ⁇ -aminoisobutyric acid; 4-aminobutyric acid (Abu); 2-Abu ( ⁇ -Abu); 6-amino hexanoic acid ( ⁇ -Ahx); 2-aminoisobutyric acid (Aib); 3-aminopropionic acid; ornithine; norleucine (Nle); norvaline (Nva); hydroxyproline; sarcosine; citrulline; cysteic acid; t-butylglycine; t-butylalanine; phenylgylcine; cyclohexylalanine; ⁇ -alanine; fluoro-amino acids; designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and
  • the invention also comprises chemical derivatives of the peptides of the invention including, but not being limited to, esters of both carboxylic and hydroxy groups, amides, and the like.
  • the NS-specific antigen peptides of the invention can be chemically synthesized- For example, a peptide corresponding to a portion of an antigen which comprises the desired domain or which mediates the desired activity can be synthesized by use of a peptide synthesizer.
  • NS-specific antigens and peptides derived therefrom and analogs and derivatives thereof can be assayed by various methods known in the art, including, but not limited to, T-cell proliferation assays (Mor and Cohen, 1995) and cytokine production assays.
  • NS-specific antigen or peptide derived therefrom or derivative thereof may be kept in solution or may be provided in a dry form, e.g., as a powder or lyophilizate, to be mixed with appropriate solution prior to use. They may be used both as ingredients of pharmaceutical compositions for neuroprotection and preventing or inhibiting the effects of injury or disease that result in NS degeneration or for promoting nerve regeneration in the NS, particularly in the CNS as well as for in vivo or in vitro activation of T cells.
  • the present invention further provides pharmaceutical compositions comprising a therapeutically effective amount of a nucleotide sequence encoding an NS-specific antigen or a peptide derived therefrom or an analog thereof and methods of use of such compositions to promote nerve regeneration or for neuroprotection and prevention or inhibition of neuronal degeneration in the CNS or PNS in which the amount is effective to ameliorate the effects of an injury or disease of the NS.
  • nucleotide sequences encoding NS-specific antigens or peptides derived from an NS-specific antigen include, but are not limited to, nucleotide sequences encoding rat MBP, depicted in FIG. 15 (SEQ ID NO:1); human MBP, depicted in FIG. 16 (SEQ ID NO:2); human PLP, depicted in FIGS. 17 (A-F) (SEQ ID NOs:3-8); human MOG, depicted in FIG. 18 (SEQ ID NO:9); rat PLP and variant, depicted in FIG. 19 (SEQ ID NO:10); rat MAG, depicted in FIG.
  • T cells may be used to promote nerve regeneration or to confer neuroprotection and prevent or inhibit secondary degeneration which may otherwise follow primary NS injury, e.g., spinal cord injury, blunt trauma, penetrating trauma, hemorrhagic stroke, ischemic stroke or damages caused by surgery such as tumor excision.
  • primary NS injury e.g., spinal cord injury, blunt trauma, penetrating trauma, hemorrhagic stroke, ischemic stroke or damages caused by surgery such as tumor excision.
  • compositions may be used to ameliorate the effects of disease that result in a degenerative process, e.g., degeneration occurring in either gray or white matter (or both) as a result of various diseases or disorders, including, without limitation: diabetic neuropathy, senile dementias, Alzheimer's disease, Parkinson's disease, facial nerve (Bell's) palsy, glaucoma, Huntington's chorea, amyotrophic lateral sclerosis (ALS), non-arteritic optic neuropathy, intervertebral disc herniation, vitamin deficiency, prion diseases such as Creutzfeldt-Jakob disease, carpal tunnel syndrome, peripheral neuropathies associated with various diseases, including but not limited to, uremia, porphyria, hypoglycemia, Sjorgren Larsson syndrome, acute sensory neuropathy, chronic ataxic neuropathy, biliary cirrhosis, primary amyloidosis, obstructive lung diseases, acromegaly,
  • the NS-specific activated T cells, the NS-specific antigens, peptides derived therefrom, analogs and derivatives thereof or the nucleotides encoding said antigens, or peptides or any combination thereof of the present invention are used to treat diseases or disorders where promotion of nerve regeneration or prevention or inhibition of secondary neural degeneration is indicated, which are not autoimmune diseases or neoplasias.
  • the compositions of the present invention are administered to a human subject.
  • activated NS-specific T cells may have been used in the prior art in the course of treatment to develop tolerance to autoimmune antigens in the treatment of autoimmune diseases, or in the course of immunotherapy in the treatment of NS neoplasms
  • the present invention can also be used to ameliorate the degenerative process caused by autoimmune diseases or neoplasms as long as it is used in a manner not suggested by such prior art methods.
  • T cells activated by an autoimmune antigen have been suggested for use to create tolerance to the autoimmune antigen and, thus, ameliorate the autoimmune disease.
  • T cells directed to other NS antigens or NS antigens which will not induce tolerance to the autoimmune antigen or T cells which are administered in such a way as to avoid creation of tolerance would not have suggested the use of T cells directed to other NS antigens or NS antigens which will not induce tolerance to the autoimmune antigen or T cells which are administered in such a way as to avoid creation of tolerance.
  • the effects of the present invention can be obtained without using immunotherapy processes suggested in the prior art by, for example, using an NS antigen which does not appear in the neoplasm. T cells activated with such an antigen will still accumulate at the site of neural degeneration and facilitate inhibition of this degeneration, even though it will not serve as immunotherapy for the tumor per se.
  • Nogo protein or a fragment thereof which are active in inhibiting cell proliferation have been disclosed as useful for treatment of a neoplastic disease of the CNS such as glioma, glioblastoma, medulloblastoma, craniopharyngioma, ependyoma, neuroblastoma and retinoblastoma.
  • the present invention does not encompass the use of Nogo or a peptide derived therefrom for treatment of neoplasias in general, and for treatment of a neoplastic disease of the CNS, in particular.
  • the present invention also provides pharmaceutical compositions useful in methods to promote nerve regeneration or to confer neuroprotection and prevent or inhibit neuronal degeneration in the CNS or PNS, comprising a therapeutically effective amount of at least one ingredient selected from the group consisting of:
  • compositions comprising ingredients (b) and/or (c) above are also effective to activate T cells in vitro, wherein the activated T cells inhibit or ameliorate the effects of an injury or disease of the NS.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • the carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monochydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate or sodium lauryl sulphate; a glidant, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; and/or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lacto
  • Methods of administration include, but are not limited to, parenteral, e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical and intradermal routes. Administration can be systemic or local.
  • parenteral e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical and intradermal routes.
  • parenteral e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical and intradermal routes.
  • mucosal e.g., oral, intranasal, buccal
  • the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, or fractionated vegetable oils
  • preservatives e.g
  • the pharmaceutical compositions may take the form of, for example, tablets, lozenges or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.
  • Preparations for oral administration may be also suitably formulated to give controlled release of the active compound.
  • compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • compositions may also be formulated as rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
  • compositions comprising NS-specific activated T cells, an NS-specific antigen or peptide derived therefrom, or derivative thereof, or a nucleotide sequence encoding such antigen or peptide, are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous or intraperitoneal administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.
  • compositions comprising NS-specific antigen or peptide derived therefrom or derivative thereof may optionally be administered with an adjuvant.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • the pharmaceutical compositions of the invention are administered to a mammal, preferably a human, shortly after injury or detection of a degenerative lesion in the NS.
  • the therapeutic methods of the invention may comprise administration of an NS-specific activated T cell or an NS-specific antigen or peptide derived therefrom or derivative thereof, or a nucleotide sequence encoding such antigen or peptide, or any combination thereof.
  • the NS-specific antigen may be administered before, concurrently or after administration of NS-specific activated T cells, a peptide derived from an NS-specific antigen or derivative thereof or a nucleotide sequence encoding such antigen or peptide.
  • compositions of the invention are administered in combination with one or more of the following: (a) mononuclear phagocytes, preferably cultured monocytes (as described in PCT publication No. WO 97/09985, which is incorporated herein by reference in its entirety), that have been stimulated to enhance their capacity to promote neuronal regeneration; (b) a neurotrophic factor such as acidic fibroblast growth factor; and (c) an anti-inflammatory therapeutic substance, e.g., an anti-inflammatory steroid, such as dexamethasone or methyl-prednisolone, or a non-steroidal anti-inflammatory peptide, such as Thr-Lys-Pro (TKP)).
  • mononuclear phagocytes preferably cultured monocytes (as described in PCT publication No. WO 97/09985, which is incorporated herein by reference in its entirety)
  • a neurotrophic factor such as acidic fibroblast growth factor
  • an anti-inflammatory therapeutic substance e.g.,
  • mononuclear phagocyte cells according to PCT Publication No. WO 97/09985 and U.S. patent application Ser. No. 09/041,280, filed Mar. 11, 1998, are injected into the site of injury or lesion within the CNS, either concurrently, prior to, or following parenteral administration of NS-specific activated T cells, an NS-specific antigen or peptide derived therefrom or derivative thereof, or a nucleotide sequence encoding such antigen or peptide
  • administration of NS-specific activated T cells, NS-specific antigen or peptide sequence encoding such antigen or peptide may be administered as a single dose or may be repeated, preferably at 2-week intervals and then at successively longer intervals once a month, once a quarter, once every six months, etc.
  • the course of treatment may last several months, several years or occasionally also through the life-time of the individual, depending on the condition or disease which is being treated.
  • the treatment may range between several days to months or even years, until the condition has stabilized and there is no or only a limited risk of development of secondary degeneration.
  • the therapeutic treatment in accordance with the invention may be for life.
  • the therapeutic effect depends at times on the condition or disease to be treated, on the individual's age and health condition, on other physical parameters (e.g., gender, weight, etc.) of the individual, as well as on various other factors, e.g., whether the individual is taking other drugs, etc.
  • the optimal dose of the therapeutic compositions comprising NS-specific activated T cells of the invention is proportional to the number of nerve fibers affected by NS injury or disease at the site being treated.
  • the dose ranges from about 5 ⁇ 10 6 to about 10 7 for treating a lesion affecting about 10 nerve fibers, such as a complete transection of a rat optic nerve, and ranges from about 10 7 to about 10 8 for treating a lesion affecting about 10 6 -10 7 nerve fibers, such as a complete transection of a human optic nerve.
  • the dose of T cells can be scaled up or down in proportion to the number of nerve fibers thought to be affected at the lesion or site of injury being treated.
  • Female Lewis rats were supplied by the Animal Breeding Center of the Weizmann Institute of Science (Rehovot, Israel), matched for age (8-12 weeks) and housed four to a cage in a light and temperature-controlled room.
  • the T-cell proliferation medium contained the following: Dulbecco's modified Eagle's medium (DMEM, Biological Industries, Israel) supplemented with 2 mM L-glutamine (L-Glu, Sigma, USA), 5 ⁇ 10 ⁇ 5 M 2-mercaptoethanol (2-ME, Sigma), penicillin (100 IU/ml; Biological Industries), streptomycin (100 ⁇ /ml; Biological Industries), sodium pyruvate (1 mM; Biological Industries), non-essential amino acids (1 ml/100 ml; Biological Industries) and autologous rat serum 1% (vol/vol) (Mor et al, 1990).
  • DMEM Dulbecco's modified Eagle's medium
  • L-Glu L-glutamine
  • 2-ME 2-mercaptoethanol
  • penicillin 100 IU/ml
  • streptomycin 100 ⁇ /ml
  • sodium pyruvate 1 mM
  • non-essential amino acids 1 ml/100 ml; Biological Industries
  • Propagation medium contained: DMEM, 2-ME, L-Glu, sodium pyruvate, non-essential amino acids and antibiotics in the same concentration as above with the addition of 10% fetal calf serum (FCS), and 10% T cell growth factor (TCGF) obtained from the supernatant of concanavalin A-stimulated spleen cells (Mor et al, 1990).
  • FCS fetal calf serum
  • TCGF T cell growth factor
  • MBP from the spinal cords of guinea pigs was prepared as described (Hirshfeld, et al, 1970). OVA was purchased from Sigma (St. Louis, Mo.).
  • the p51-70 of the rat 18.5 kDa isoform of MBP (sequence: APKRGSGKDSHTRTTHYG) (SEQ ID NO:15) and the p277 peptide of the human hsp60 (sequence: VLGGGCALLRCPALDSLTPANED) (SEQ ID NO:16) (Elias et al, 1991) were synthesized using the 9-fluorenylmethoxycarbonyl (Fmoc) technique with an automatic multiple peptide synthesizer (AMS 422, ABIMED, Langenfeld, Germany). The purity of the peptides was analyzed by HPLC and amino acid composition.
  • T-cell lines were generated from draining lymph node cells obtained from Lewis rats immunized with an antigen (described above in Antigens).
  • the antigen was dissolved in PBS (1 mg/ml) and emulsified with an equal volume of IFA (Difco Laboratories, Detroit, Mich.) supplemented with 4 mg/ml Mycobacterium tuberculosis (Difco 15 Laboratories, Detroit, Mich.).
  • the emulsion (0.1 ml) was injected into hind foot pads of the rats. Ten days after the antigen was injected, the rats were killed and draining lymph nodes were surgically removed and dissociated.
  • the cells were washed and activated with the antigen (10 ⁇ g/ml) in proliferation medium (described above in Media). After incubation for 72 h at 37° C., 90% relative humidity and 7% CO 2 , the cells were transferred to propagation medium (described above in Media). Cells were grown in propagation medium for 4-10 days before being re-exposed to antigen (10 ⁇ g/ml) in the presence of irradiated (2000 rad) thymus cells (10 7 cells/ml) in proliferation medium. The T cell lines were expanded by repeated re-exposure and propagation.
  • Crush injury of the optic nerve was performed as previously described (Duvdevani et al, 1990). Briefly, rats were deeply anesthetized by i.p. injection of Rompum (xylazine, 10 mg/kg; Vitamed, Israel) and Vetaler (ketamine, 50 mg/kg; Fort Dodge Laboratories, Fort Dodge, Iowa). Using a binocular operating microscope, a lateral canthotomy was performed in the right eye and the conjunctiva was incised lateral to the cornea. After separation of the retractor bulbi muscles, the optic nerve was exposed intraorbitally by blunt dissection. Using calibrated cross-action forceps, a moderate crush injury was inflicted on the optic nerve, 2 mm form the eye (Duvdevani et al, 1990). The contralateral nerve was left undisturbed and was used as a control.
  • FIG. 1 shows accumulation of T cells measured immunohistochemically.
  • the number of T cells was considerably higher in injured nerves rats injected with anti-MBP, anti-OVA or anti-p277 cells; statistical analysis (one-way ANOVA) showed significant differences between T cell numbers in injured optic nerves of rats injected with anti-MBP, anti-OVA, or anti-p277 T cells and in injured optic nerves of rats injected with PBS (P ⁇ 0.001); and between injured optic nerves and uninjured optic nerves of rats injected with anti-MBP, anti-OVA, or anti-p277 T cells (P ⁇ 0.001).
  • Nerves were excised and their compound action potentials (CAPs) were recorded in vitro using a suction electrode experimental set-up (Yoles et al, 1996). At different times after injury and injection of T cells or PBS, rats were killed by intraperitoneal injection of pentobarbitone (170 mg/kg) (CTS Chemical Industries, Israel).
  • CAPs compound action potentials
  • Both optic nerves were removed while still attached to the optic chiasma, and were immediately transferred to a vial containing a fresh salt solution consisting of 126 mM NaCl, 3 mM KCl, 1.25 mM NaH 2 PO 2 26 mM NaHCO 3 2 mM MgSO 4 , 2 mM CaCl 2 and 10 mM D-glucose, aerated with 95% O 2 and 5% CO 2 at room temperature. After 1 hour, electrophysiological recordings were made. In the injured nerve, recordings were made in a segment distal to the injury site.
  • This segment contains axons of viable RGCs that have escaped both primary and secondary damage, as well as the distal stumps of non-viable RGCs that have not yet undergone Wallerian degeneration.
  • the nerve ends were connected to two suction Ag—AgCl electrodes immersed in the bathing solution at 37° C. A stimulating pulse was applied through the electrode, and the CAP was recorded by the distal electrode.
  • a stimulator SD9; Grass Medical Instruments, Quincy, Mass.
  • the measured signal was transmitted to a microelectrode AC amplifier (model 1800; A-M Systems, Everett, Wash.).
  • the data were processed using the LabView 2.1.1 data acquisition and management system (National Instruments, Austin, Tex.). For each nerve, the difference between the peak amplitude and the mean plateau of eight CAPs was computed and was considered as proportional to the number of propagating axons in the optic nerve. The experiments were done by experimenters “blinded”, to sample identity. In each experiment the data were normalized relative to the mean CAP of the uninjured nerves from PBS-injected rats.
  • Clinical disease was scored every 1 to 2 days according to the following neurological scale: 0, no abnormality; 1, tail atony; 2, hind limb paralysis; 3, paralysis extending to thoracic spine; 4, front limb paralysis; 5, moribund state.
  • secondary degeneration was quantified by injecting the dye immediately or 2 weeks after the primary injury, and calculating the additional loss of RGCs between the first and the second injections of the dye. The percentage of RGCs that had survived secondary degeneration was then calculated. The percentage of labeled RGCs (reflecting still-viable neurons) was significantly greater in the retinas of the rats injected with anti-MBP T cells than in the retinas of the PBS-injected control rats (FIG. 2). In contrast, the percentage of labeled 30 RGCs in the retinas of the rats injected with anti-OVA or anti-p277 T cells was not significantly greater than that in the control retinas.
  • the observed neuroprotective effect could reflect the rescue of spared neurons, or a delay of Wallerian degeneration of the injured neurons (which normally occurs in the distal stump), or both.
  • No effect of the injection of anti-MBP T cells on the mean CAP amplitudes of uninjured nerves was observed (FIG. 6B, Table 2). It is unlikely that the neuroprotective effect observed on day 14 could have been due to the regrowth of nerve fibers, as the time period was too short for this.
  • the strong neuroprotective effect of the anti-MBP T cells seen on day 14 was associated with a significantly decreased CAP amplitude recorded on day 7 (Table 2).
  • the anti-MBP T cells manifested no substantial effect on the uninjured nerve on day 7, indicating that the reduction in electrophysiological activity observed in the injured nerve on day 7 might reflect the larger number of T cells present at the injury site relative to the uninjured nerve (FIG. 1).
  • the observed reduction in CAP amplitude in the injured nerve on day 7 reflected a transient resting state in the injured nerve. This transient effect has not only disappeared, but was even reversed by day 14 (Table 2).
  • Bladder expression was done at least twice a day (particularly during the first 48 h after injury, when it was done 3 times a day) until the end of the second week, by which time the rats had developed autonomous bladder voidance. Approximately twice a week, locomotor activity (of the trunk, tail and hind limbs) in an open field was evaluated by placing the rat for 4 min in the middle of a circular enclosure made of molded plastic with a smooth, non-slip floor (90 cm diameter, 7 cm wall height).
  • the above-described increase in motor activity seen after treatment with the anti-MBP T cells could result from much higher percentage of spared tissue based on a linear regression curve on which the behavioral score is correlated with the amount of neural spinal cord tissue (for example, a difference between 11 and 7 on the locomotion score would be read as a difference between 30% and less than 10% of spared tissue).
  • FIG. 9 shows the diffusion anisotropy in axial sections along the contused cord of a rat treated with autoimmune T cells, as compared with that of PBS-treated control rat.
  • the images show anisotropy in the white matter surrounding the gray matter in the center of the cord.
  • Sections taken from the lesion sites of PBS-treated control rats show limited areas of anisotropy, which were significantly smaller than those seen at comparable sites in the cords of the rats treated with the anti-MBP T cells. Quantitative analysis of the anisotropy, reflecting the number of spared fibers, is shown in FIG. 9. The imaging results show unequivocally that, as a result of the treatment with the autoimmune anti-MBP T cells, some spinal cord tracts had escaped the degeneration that would otherwise have occurred.
  • T cell preparation that can produce EAE in the undamaged CNS was found to be neuroprotective in the damaged spinal cord, suggesting that the context of the tissue plays an important part in determining the outcome of its interaction with T cells. It would seem that the tissue deploys specific signals to elicit particular T cell behaviors. Among such signals are co-stimulatory molecules, particularly members of the B7 family (Lenchow et al, 1996).
  • the injured rat optic nerve transiently expresses elevated levels of the co-stimulatory molecule B7.2, which is constitutively expressed at low levels in the rat CNS white matter and which is thought to be associated with regulation of the cytokine profile of the responding T cells (Weiner, 1997).
  • B7.2 co-stimulatory molecule
  • anti-MBP T cells which cause a monophasic autoimmune disease upon interacting with a healthy CNS nerve, might implement a maintenance program when they interact with damaged CNS tissue expressing increased amounts of B7.2 and probably other co-stimulatory molecules.
  • the neuroprotective effects of the T cells may be mediated, at least in part, by antigen-dependent regulation of specific cytokines or neurotrophic factors (Kerschensteiner et al, 1999) produced locally at the site of injury.
  • the present invention is also directed to manipulating B7.2 co-stimulatory molecule to prevent or inhibit neuronal degeneration and ameliorate the effects of injury to or disease of the nervous system.
  • B7.2 molecule can be up-regulated for this purpose, using drugs or by genetic manipulation, without undue experimentation.
  • autoimmune response can be advantageous suggests that natural autoimmune T cells may have undergone positive selection during ontogeny, as proposed by the theory of the immunological homunculus (Cohen, 1992), and are not merely a default resulting from the escape from negative selection of T cells that recognize self antigens (Janeway, 1992). Such a response could then be considered as a mechanism of potential physiological CNS self-maintenance, which is, however, not sufficient for the purpose because of the immune-privileged character of the CNS.
  • Rats were injected intradermally in the footpads with MOG p35-55 (50 ⁇ g/animal) and IFA, or PBS, ten days prior to optic nerve crush injury. RGCs were assessed two weeks after injury using retrograde labeling as described above. The number of RGCs in rats injected with PBS or MOG p35-55 was expressed as a percentage of the total number of neurons in rats injected with MOG p35-55 in the absence of crush injury.
  • the number of labeled RGCs was about 12.5 fold greater in animals injected with MOG p35-55 compared to animals receiving PBS.
  • Bovine MBP (Sigma, Israel) (1 mg/dose) was administered to rats by gavage using a blunt needle. MBP was administered 5 times, every third day, beginning 2 weeks prior to optic nerve crush injury. The number of RGCs in treated animals was expressed as a percentage of the total number of neurons in animals subjected to optic nerve crush injury but which did not receive MBP.
  • the number of labeled RGCs was about 1.3 fold greater in animals treated with MBP compared to untreated animals.
  • Neuronal injury in the CNS causes degeneration of directly damaged fibers as well as of fibers that escaped the primary insult. It also triggers a systemic response of autoimmune T cells to MBP that might affect the course of degeneration of the injured nerve. Whether the effect of these T cells on the nerve is detrimental or beneficial may depend, in part, on the nature and level of the co-stimulatory molecules expressed by the damaged tissue.
  • CD40 appears to be dominant during cell differentiation in the lymph nodes and B7 during activation of T cells in the target organ (Grewal et al, 1996).
  • the B7 co-stimulatory molecule is a member of the immunoglobulin superfamily that interacts with CD28 and CTLA-4 on T H cells.
  • B7.1 and B7.2 Two related forms of B7 (B7.1 and B7.2). Both molecules have a similar organization of extracellular domains but markedly different cytosolic domains. Both B7 molecules are expressed on antigen-presenting cells (APCs) such as dendritic cells, activated macrophages and activated B cells as B7.1 or B7.2., which might preferentially support activation of the Th1 or the Th2 type of immune response, respectively (Kuchroo et al, 1995; Karandikar et al, 1998). We were therefore interested in determining the identity of B7 molecule subtype expressed in intact and injured CNS white matter, and its possible influence on the course of the response to the injury.
  • APCs antigen-presenting cells
  • B7.2 The co-stimulatory molecule expressed constitutively in the intact optic nerves of adult Lewis rats was identified as B7.2.
  • FIGGS. 12A, 12B To examine the effects of neurotrauma on the expression of B7 co-stimulatory molecules, we inflicted a mild crush injury on the optic nerves of Lewis rats and assessed the neural expression of B7 by immunohistochemical analysis. The most striking effect of the injury was seen on B7.2 expression manifested on post-injury day 3 by its elevation at the margins of the injury site (FIGS. 12C, D, E). In contrast, expression of B7.1 was not detected in the optic nerve either before or 3 days after injury. On day 7, however, B7.1 was detectable at the site of injury, having pattern reminiscent of that seen for macrophages or microglia (FIG. 12F).
  • NI-250 This activity was later related to a high molecular weight membrane protein, designated NI-250, with a smaller component, NI-35, in rat.
  • the bovine homologue of rat NI-250, bNI-220 was recently purified (Chen et al, 2000; PCT Publication WO 00/31235).
  • the cloning of nogo A, the rat cDNA encoding NI-220/250 was recently reported (see FIG. 1 a of Chen et al, 2000; and PCT Publication WO 00/31235, the entire contents of both of which being hereby incorporated herein by reference).
  • the rat nogo gene (SEQ ID NO: 17) encodes at least three major protein products: Nogo-A (SEQ ID NO:18) (1,163 amino acids; database accession number AJ242961), Nogo-B (SEQ ID NO:20) (360 amino acids; AJ242962) and Nogo-C (SEQ ID NO:21) (199 amino acids; AJ242963).
  • Nogo-A SEQ ID NO:18
  • Nogo-B SEQ ID NO:20
  • Nogo-C SEQ ID NO:21
  • the sequence of the amino acid p472 (SEQ ID NO:19) containing the residues 623-640 of rat Nogo-A is shown in the box in FIG. 1 a of Chen et al, 2000.
  • the cloning of the corresponding human cDNA and protein is reported in Prinjha et al, 2000. See also WO 00/60083 and WO 01/36631.
  • PCT Publication WO 00/31235 describes methods for the production of recombinant Nogo proteins, fragments, derivatives and analogs thereof, and DNA molecules coding therefor.
  • This publication further describes the use of a Nogo protein or fragment thereof for the treatment of neoplastic diseases of the CNS such as glioma, glioblastoma, retinoblastoma, and the like; and further describes the use of a ribozyme or an antisense Nogo nucleic acid for treatment of a subject with damage to the CNS and/or for inducing regeneration of neurons, wherein said ribozyme or antisense Nogo nucleic acid acts by inhibiting the production of Nogo in the subject. It is thus unexpected that immunization with Nogo or fragments thereof and/or administration of T cells activated therewith can promote nerve regeneration or prevent or inhibit neuronal degeneration in the NS, as shown according to the present invention.
  • Acute incomplete spinal cord injury at the low thoracic levels causes an immediate loss of hindlimb motor activity that spontaneously recovers within the first 12 days post-injury and stabilizes on deficient movement abilities.
  • the amount of motor function restoration is the sum up effect of the positive recovery from spinal shock and the negative effect of longitudinal and ventral spread of damage.
  • a therapeutic approach aiming at reducing the spread of damage through neuroprotection will result in a better recovery in terms of hindlimb motor activity.
  • the hind limb motor skills of the animals were scored using the BBB scoring method developed by Basso et al, 1996, following the kinetics and amount of hindlimb motor activity in the two experimental groups.
  • T cells which accumulated at the site of injury included both T cells which are activated by exposure to an antigen present at the site of injury as well as T cells which are activated by an antigen not normally present in the individual.

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PCT/IL2002/000518 WO2003002602A2 (fr) 2001-06-28 2002-06-27 Peptides nogo et derivees du recepteur nogo pour la neuroprotection induite par les lymphocytes t
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Cited By (14)

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US20030091578A1 (en) * 2001-09-14 2003-05-15 Jingwu Zhang Autologous T-cell vaccines materials and methods
US20030120061A1 (en) * 1999-02-23 2003-06-26 Baylor College Of Medicine T cell receptor Vbeta-Dbeta-Jbeta sequence and methods for its detection
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WO2006119352A3 (fr) * 2005-05-03 2007-01-11 Univ South Florida Procede pour traiter le declin cognitif et la perte synaptique lies a la maladie d'alzheimer
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US20070026463A1 (en) * 2005-07-28 2007-02-01 Wyeth Compositions and methods of mutant Nogo-66 domain proteins
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