EP1080110A2 - Lymphocytes t actives, antigenes specifiques du systeme nerveux et leur utilisation - Google Patents

Lymphocytes t actives, antigenes specifiques du systeme nerveux et leur utilisation

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Publication number
EP1080110A2
EP1080110A2 EP99923190A EP99923190A EP1080110A2 EP 1080110 A2 EP1080110 A2 EP 1080110A2 EP 99923190 A EP99923190 A EP 99923190A EP 99923190 A EP99923190 A EP 99923190A EP 1080110 A2 EP1080110 A2 EP 1080110A2
Authority
EP
European Patent Office
Prior art keywords
cells
injury
antigen
mbp
nervous system
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99923190A
Other languages
German (de)
English (en)
Inventor
Michal Eisenbach-Schwartz
Irun R. Cohen
Pierre Beserman
Alon Mosonego
Gila Moalem
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yeda Research and Development Co Ltd
Original Assignee
Yeda Research and Development Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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
Publication of EP1080110A2 publication Critical patent/EP1080110A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46432—Nervous system antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

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 antiself T cells, an NS-specific antigen or peptide derived therefrom or a nucleotide sequence encoding an NS-specific antigen or peptide derived therefrom can be used to promote nerve regeneration or to 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.
  • the nervous system comprises the central (CNS) and the peripheral (PNS) nervous system.
  • the central nervous system is composed of the brain and spinal cord;
  • the peripheral nervous system 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, 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, and ischemia.
  • the restricted communication between the central nervous system and blood-borne macrophages affects the capacity of axotomized axons to regrow; transplants of activated macrophages can promote central nervous system regrowth (Lazarov Spiegler, O., et al, 1996, FASEB J. 19:1296- 1302; Rapalino, 0. et al . , 1998, Nature Med. 4:814-821).
  • T cells have been shown to enter the central nervous system parenchyma, irrespective of their antigen specificity, but only T cells capable of reacting with a central nervous system antigen seem to persist there (Hickey, W.F. et al., 1991, J. Neurosci . Res. 28:254-260; Werkele, H. , 1993, In The Blood-Brain Barrier, Pardridge, Ed., Raven Press, Ltd. New York, 67-85; Kramer, R. et al . , 1995, Nature Med. 1 (11) :1162-1166) ) .
  • T cells reactive to antigens of central nervous system white matter can induce the paralytic disease experimental autoimmune encephalomyelitis (EAE) within several days of their inoculation into naive recipient rats (Ben Nun, A., et al . , 1981, Eur. J. Immunol. 11:195-199) .
  • Anti-MPB T cells may also be involved in the human disease multiple sclerosis (Ota, K. et al., 1990 Nature 346:183-187; Martin, R. 1997, J. Neural Transm. Suppl . 49:53-67).
  • anti-MBP T cell clones are present in the immune systems of healthy subjects (Burns, J. , et al . 1983, Cell Immunol . 81:435-440; Pette, M. et al . , 1990, Proc. Natl. Acad. Sci. USA 87:7968-7972; Martin, R. et al . , 1990, J. Immunol . 145:540-548; Schiuesener, H.J, et al . , 1985, J. Immunol . 135:3128-3133).
  • Activated T cells which normally patrol the intact central nervous system, transiently accumulate at sites of central nervous system white matter lesions (Hirschberg, D.L., et al . , 1998, . Neuroimmunol . 89:88-96) .
  • a catastrophic consequence of central nervous system 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, A. I., et al . , 1992, Trends Pharmacol. Sci. 13:29-35; Faden, A.I., 1993, Crit . Rev. Neurobiol . 7:175-186; Mclntosh, T.K., 1993, J. Neurotrauma 10:215-261).
  • 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.
  • central nervous system injury Another tragic consequence of central nervous system injury is that neurons in the mammalian central nervous system do not undergo spontaneous regeneration following an injury. Thus, a central nervous system 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.
  • the present invention is directed to methods and compositions for the promotion of nerve regeneration or prevention or inhibition of neuronal degeneration to ameliorate the effects of injury to or disease of the nervous system (NS) .
  • the present invention is based in part on the applicants' unexpected discovery that activated T cells that recognize an antigen of the NS of the patient promote nerve regeneration or confer neuroprotection.
  • nerve regeneration refers to the prevention or inhibition of degenerative effects of injury or disease in the NS .
  • the immune system excluded immune cells from participating in nervous system repair. It was quite surprising to discover that NS-specific activated T cells 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 .
  • Activated T cell includes (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 T cell antigen receptor 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.
  • the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of NS-specific activated T cells and methods for using such compositions to promote nerve regeneration or to prevent or inhibit neuronal degeneration in the CNS or PNS, in an amount which is effective to ameliorate the effects of an injury or disease of the NS .
  • NS-specific activated T cell refers to an activated T cell having specificity for an antigen of the NS of a patient.
  • the antigen used to confer the specificity to the T cells may be a self NS-antigen of the patient, a peptide derived therefrom, or an NS-antigen of another individual or even another species, or a peptide derived therefrom, as long as the activated T cell recognizes an antigen in the NS of the patient.
  • the NS-specific activated T cells are used to promote nerve regeneration or to prevent or inhibit the effects of disease.
  • the disease being treated is an autoimmune disease, in which the autoimmune antigen is an NS antigen
  • 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 present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of an NS-specific antigen or peptide derived therefrom or derivative thereof and methods of use of such compositions to promote nerve regeneration or to prevent or inhibit neuronal degeneration in the CNS or PNS, in which the amount is effective to activate T cells in vivo or in vi tro, wherein the activated T cells inhibit or ameliorate the effects of an injury or disease of the NS .
  • NS-specific antigen refers to an antigen 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 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.
  • the peptide derived from an NS-specific antigen is an immunogenic epitope of the antigen.
  • "Derivatives" of NS-specific antigens or peptides derived therefrom as used herein refers to analogs or chemical derivatives of such antigens or peptides as described below, see Section 5.2.
  • the present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of a nucleotide sequence encoding an NS-specific antigen or peptide derived therefrom or derivative thereof and methods of use of such compositions to promote nerve regeneration or for preventing or inhibiting 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 .
  • therapy for amelioration of effects of injury or disease comprising administration of NS-specific activated T cells may optionally be in combination with an NS-specific antigen or peptide derived therefrom.
  • oral administration of NS-specific antigen or a peptide derived therefrom can be combined with active immunization to build up a critical T cell response immediately after injury.
  • cell banks 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 .
  • 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.
  • autologous stored T cells are used, but, if autologous T cells are not available, then cells should be used which share an MHC type II molecule with the patient, and these would be expected to be operable in that individual.
  • the cells are preferably stored in an activated state after exposure to an NS antigen or peptide derived therefrom.
  • 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.
  • the cells Once the cells are thawed, they are preferably cultured prior to injection in order to eliminate non-viable cells.
  • 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 (prefereably the former) .
  • PHA phytohemagglutinin
  • concanavalin A prefereably the former
  • the few days that it takes to culture the cells should not be detrimental to the patient as the treatment in accordance with the present invention may occur any time up to a week or more after the injury in order to still be effective. Alternatively, if time is of the essence, the stored cells may be administered immediately after thawing.
  • 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 0VA ), anti-p277 (T p277 ) lines, or with PBS, immediately after unilateral crush injury of the optic nerve. Seven days later, both the injured and uninjured optic nerves were removed, cryosectioned and analyzed immunohistochemically for the presence of immunolabeled T cells. T cells were counted at the site of injury and at randomly selected areas in the uninjured optic nerves .
  • 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 of OVA or p277 or hsp60, 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.
  • RGCs 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, oneway 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. Immediately after unilateral crush injury of their optic nerves, rats were injected with PBS (Fig. 3A) or with activated anti-p277 T cells (Fig. 3B) or activated anti-MBP T cells (Fig. 3C) . Two weeks later, 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) RCGs, 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.
  • Fig. 4A 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-10Asp 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.
  • 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 primary injury. Compared with that of PBS treatment, the neuroprotective effects of anti-MBP 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
  • rats were injected with either PBS or activated anti-MBP T cells (T MBP ) .
  • T MBP activated anti-MBP T cells
  • 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.
  • Figs 8(A-C) show retrograde labeling of cell bodies at the red nucleus in rats treated with autoimmune anti-MBP T cells (8A) and in control injured (8B) rats.
  • autoimmune anti-MBP T cells 8A
  • control injured 8B
  • 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 x 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, Section 8, for experimental details. Rats were injected intradermally through the footpads with a 21-mer peptide based on amino acid residues 35-55 (MOG p35-55) of myelin/oligodendrocyte glycoprotein (chemically synthesized at the Weizmann Institute, Israel) (50 ⁇ /animal) 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 Incomplete Freund's Adjuvant. Surviving optic nerve fibers were monitored by retrograde labeling of retinal ganglion cells (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.
  • RGCs retinal ganglion cells
  • Fig. 11 is a graph illustrating inhibition in adult rats of secondary degeneration after optic nerve crush injury by MBP. See text, Section 9, for experimental details.
  • MBP Sigma, Israel
  • MBP 1 mg in 0.5 ml saline
  • 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 retinal ganglion cells (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 show expression of B7 costimulatory molecules in intact and injured rat optic nerve.
  • Optic nerves were excised from adult Lewis rats before (12A, 12B) and three days after injury (12C, 12D, 12E) and analyzed immunohistochemically for expression of the B7 costimulatory 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) (Jackson ImmunoResearch, West Grove, PA) , for one hour at room 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 (12A, 12B) to a star-like shape (12C, 12D) .
  • the B7.2 positive cells were present at a higher density closer to the injury site (12E) .
  • Expression of B7.1 was detectable only from day seven and only at the injured site (12F) .
  • Figs. 13 A-C show immunohistochemical analysis of T cells, macrophages or microglia, and B7.2 costimulatory 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, NJ) (Chen, Y., Kuchroo, V.K.,. Inobe, J. Hafler, D.A. & Weiner, H.L.
  • Regulatory T cell clones induced by oral tolerance suppression of autoimmune encephalomyelitis.
  • 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) .
  • Retrograde labelling of RGCs by the dye gives a reliable indication of the number of still-functioning neurons, as only intact axons can transport the dye to their cell bodies in the retina.
  • the retina was detached from the eye, prepared as a flattened whole mount in 4% paraformaldehyde solution, and examined for labelled ganglion cells by fluorescence microscopy.
  • RGCs were counted from three different regions in the retina. The results are expressed as normalized percentage of each retina to untreated injured animal mean of the same experiment . The median of each group is shown as a bar (Control vs. MBP OTx4 ** P ⁇ 0.01; Control vs. MBP OT ** P, 0.01; Control vs. OVA OT ns P>0.05.
  • Fig. 15 shows the nucleotide sequence of rat myelin basic protein gene, SEQ ID NO:l, Genbank accession number M25889 (Schaich et al . , Biol. Chem. 367:825-834, 1986).
  • Fig. 16 shows the nucleotide sequence of human myelin basic protein gene, SEQ ID NO: 2, Genbank accession number M13577 (Kamholz et al . , Proc. Natl. Acad. Sci. U.S.A. 83(13): 4962-4966, 1986) .
  • Figs 17 show the nucleotide sequences of human myelin proteolipid protein gene exons 1-7, SEQ ID NOs: 3-8, respectively, Genbank accession number M15026-M15032 respectively (Diehl et al . , Proc. Natl. Acad Sci. U.S.A. 83 (24) :9807-9811, 1986; published erratum appears in Proc Natl Acad Sci U.S.A. 86(6):617-8. 1991).
  • Fig. 18 shows the nucleotide sequence of human myelin oligodendrocyte glycoprotein gene, SEQ ID NO: 9, Genbank accession number Z48051 (Roth et al . , submitted (17-Jan-1995) Roth, CNRS UPR 8291, CIGH, CHU Purpan,ière, France, 31300; Gonzalez et al . , Mol . Phylogent . Evol . 6:63-71, 1996).
  • Fig. 19 shows the nucleotide sequence of rat proteolipid protein and variant, SEQ ID NO: 10, Genbank accession number M16471 (Nave et al, Proc. Natl. Acad. Sci . U.S.A 84:600-604. 1987).
  • Fig. 20 shows the nucleotide sequence of rat myelin- associated glycoprotein, SEQ ID NO: 11, Genbank accession number M14871 (Arquint et al, Proc. Natl. Acad. Sci. USA 84:600-604, 1987) .
  • Fig. 21 shows the amino acid sequence of human myelin basic protein, SEQ ID NO: 12, Genbank accession number 307160 (Kamholz et al . , 1986, Proc. Natl. Acad. Sci. U.S.A. 83 (13) :4962-4966, 1986).
  • Fig. 22 shows the amino acid sequence of human proteolipid protein, SEQ ID NO: 13, Genbank accession number 387028.
  • Fig. 23 shows the amino acid sequence of human myelin oligodendrocyte glycoprotein, SEQ ID NO: 14, Genbank accession number 793839 (Roth et al . , Genomics 28 (2) : 241-250 , 1995; Roth Submitted (17-JAN-1995) Roth CNRS UPR 8291, CIGH, CHU Purpan,ière, France, 31300; Gonzalez et al . , Mol. Phyloqent . Evol. 6:63-71, 1996) .
  • NS-specific activated T cells (2) NS-specific antigens, peptides derived therefrom and derivatives thereof; (3) nucleotide sequences encoding NS- specific antigens and peptides derived therefrom; (4) therapeutic uses of non-recombinan , NS-specific activated T cells, NS-specific antigens, peptides derived therefrom and derivatives thereof, and nucleotide sequences encoding NS- specific antigens and peptides derived therefrom; and (5) formulations and modes of administration of nonrecombinant , NS- specific activated T cells, NS-specific antigens, peptides derived therefrom and derivatives thereof, and nucleotide sequences encoding NS-specific antigens and peptides derived therefrom.
  • NS-specific activated T cells can be used 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.
  • the NS-specific activated T cells are preferably autologous, most preferably of the CD4 and/or CD8 phenotypes, but they may also be allogeneic T cells from related donors, e.g., siblings, parents, children, or HLA-matched or partially matched, semi-allogeneic or fully allogeneic donors.
  • 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 central nervous system and in need of T cell neuroprotection.
  • semi-allogeneic T cells are based on the fact that T cells can recognize a specific antigen epitope presented by foreign antigen presenting cells (APC) , provided that the APC express 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.
  • APC foreign antigen presenting cells
  • a semi-allogeneic population of T cells that can recognize at least one allelic product of the subject's MHC molecules, preferably an HLA-DR or an 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 neuroprotective 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- irrdiation, e.g., 1.5-10.0 Rads (Ben-Nun, A., Wekerle, H. and Cohen, I.R., Nature 292:60-61 (1981); Ben-Nun, A. and Cohen, I.R., J. Immunol. 129:303-308 (1982)); and/or by pressure treatment, for example as described in U.S. Patent No. 4,996,194 (Cohen et al .
  • the 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, J. Immunol. 155:3693-3699). For an illustrative example, see Section 6.1.
  • Circulating T cells of a subject which recognize myelin basic protein or another NS antigen, such as the amyloid precursor protein, are isolated and expanded using known procedures.
  • T cells are isolated and the NS-specific ATCs are then expanded by a known procedure (Burns et al . , Cell Immunol. 81:435, 1983; Pette et al . , Proc. Natl. Acad. Sci. USA 87:7968, 1990; Mortin et al . , J . Immunol . 145:540, 1990; Schluesener et al . , J. Immunol . 135:3128, 1985; Suruhan-Dires Keneli et al . , Euro . . Immunol . 23:530, 1993, which are incorporated herein by reference in their entirety) .
  • 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, including but 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 and neurotransmitter receptors.
  • MBP myelin basic protein
  • MOG myelin oligodendrocyte glycoprotein
  • PGP proteolipid protein
  • MAG myelin-associated glycoprotein
  • S-100 ⁇ -amyloid
  • Thy-1 Thy-1
  • P0 p2
  • P2 neurotransmitter receptors
  • the isolated T cells are activated by one or more cryptic epitopes, including but limited to the following MBP peptides: pll-30, p51-70, p
  • the T cells may be activated by culturing them in medium to which at least one suitable growth promoting factor has been added.
  • Growth promoting factors suitable for this purpose include, without limitation, cytokines, for instance tumor necrosis factor a (TNF- ⁇ ) , interleukin 2 (IL-2) , and interleukin 4 (IL-4) .
  • the activated T cells endogenously produce a substance that ameliorates the effects of injury or disease in the NS .
  • the activated T cells endogenously produce a substance that stimulates other cells, including, but not limited to, transforming growth factor-/? (TGF- / 8) , nerve growth factor (NGF) , neurotrophic factor 3 (NT- 3) , neurotrophic factor 4/5 (NT-4/5) , brain derived neurotrophic factor (BDNF) ; interferon- ⁇ (IFN- 7 ), and interleukin-6 (IL-6) , wherein the other cells, directly or indirectly, ameliorate the effects of injury or disease.
  • TGF- / 8 transforming growth factor-/?
  • NGF nerve growth factor
  • NT- 3 neurotrophic factor 3
  • NT-4/5 neurotrophic factor 4/5
  • BDNF brain derived neurotrophic factor
  • IFN- 7 interferon- ⁇
  • IL-6 interleukin-6
  • the T cells are administered to a mammalian subject.
  • the T cells are administered to 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 et al . , J. Immunol. 129:303, 1982) .
  • the NS-specific antigen may be a purified antigen or a crude NS preparation, as will be described below.
  • 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 ATCs.
  • 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, Nature 183:1394-1395, 1959; Ashwood-Smith, Nature 190:1204-1205, 1961), glycerol, polyvinylpyrrolidone (Rinfret, Ann. N.Y. Acad. Sci. 85:576, 1960), polyethylene glycol (Sloviter and Ravdin, Nature 196:548, 1962), albumin, dextran, sucrose, ethylene glycol, i-erythritol , D-ribitol, D-mannitol (Rowe et al . , Fed. Proc.
  • DMSO dimethyl sulfoxide
  • a controlled cooling rate is critical.
  • Different cryoprotective agents (Rapatz et al . , Cryobiology 5(l):18-25, 1968) and different cell types have different optimal cooling rates. See, e.g., Rowe and Rinfret, Blood 20:636 (1962); Rowe, Crvobiology 3(1):12-18 (1966); Lewis et al . , Transfusion 7(l):17-32 (1967); and Mazur, Science 168:939-949 (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.
  • -196°C liquid nitrogen
  • cryopreservation of viable cells or modifications thereof, are available and envisioned for use, e.g., cold metal-mirror techniques. See Livesey and Linner, Nature 327:255 (1987); Linner et al . , J. Histochem. Cvtochem. 34(9) :1123-1135 (1986); see also U.S. Patent No. 4,199,022 by Senken et al . , U.S. Patent No. 3,753,357 by Schwartz, U.S. Patent No. 4,559,298 by Fahy.
  • 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 . , Cancer 45:3075-3085, 1980), low molecular weight dextran and citrate, citrate, hydroxyethyl starch (Stiff et al., Cryobiology 20:17-24, 1983), or acid citrate dextrose (Zaroulis and Senseman, Cryobiology 17:311-317, 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 neuronal regeneration as described herein with respect to non-frozen T cells.
  • the T cells may be used immediately, assuming that they were activated prior to freezing.
  • the thawed cells are cultured before injection to the patient in order to eliminate non-viable cells.
  • 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.
  • 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 neuroregenerative and neuroprotective effect .
  • compositions comprising an NS-specific antigen or peptide derived therefrom or derivative thereof can be used for 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. Additionally, NS-specific antigens or peptides derived therefrom or derivatives thereof may be used for in vivo or in vi tro activation of T cells. In one embodiment, the NS-specific antigen is an isolated or purified antigen.
  • methods of promoting nerve regeneration or of preventing or inhibiting the effects of CNS or PNS injury or disease comprise administering NS-specific antigen or a peptide derived therefrom or derivative thereof to a mammal wherein the NS-specific antigen or peptide derived therefrom or derivative thereof activates T cells in vivo to produce a population of T cells that accumulate at a site of injury or disease of the CNS or PNS.
  • the NS-specific antigen may be an antigen obtained from NS tissue, preferably from tissue at a site of CNS injury or disease.
  • 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.
  • natural or synthetic NS-specific antigens or epitopes include, but are not limited to, MBP, MOG, PLP, MAG, S-100, ⁇ -amyloid, Thy-1, P0, P2 and a neurotransmitter receptor.
  • NS-specific antigens include but are not limited to, human MBP, depicted in Fig. 21, (SEQ ID NO:12); human proteolipid protein, depicted in Fig. 22 (SEQ ID NO:13); and human oligodendrocyte glycoprotein, depicted in Fig. 23 (SEQ ID NO: 14) .
  • peptides derived from NS- specific, self-antigens or derivatives of NS-specific antigens activate T cells, but do not induce an autoimmune disease.
  • An example of such peptide is a peptide comprising amino acids 51- 70 of myelin basic protein (residues 51-70 of SEQ ID NO:12) .
  • an NS-specific antigen may be a crude NS-tissue preparation, e.g., derived from NS tissue obtained from mammalian NS . Such a preparation may include cells, both living or dead cells, membrane fractions of such cells or tissue, etc.
  • an NS-specific antigen may be obtained by an NS biopsy or necropsy from a mammal including, but not limited to, from a site of CNS injury; from cadavers; from cell lines grown in culture.
  • an NS-specific antigen may be a protein obtained by genetic engineering, chemically synthesized, etc.
  • the invention also relates to peptides derived from NS-specific antigens or derivatives including chemical derivatives and analogs of NS- specific antigens which are functionally active, i.e., they are capable of displaying one or more know 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 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.
  • a peptide derived from a CNS-specific or PNS-specific antigen preferably has a sequence comprised within the antigen sequence and is either: (1) an immunogenic peptide, i.e., a peptide that can elicit a human T cell response detected by a T cell proliferation or by cytokine (e.g.
  • interferon interferon
  • IL interleukin
  • IL-4 interleukin-4
  • IL-10 interferon- ⁇
  • IL-10 interleukin-2, IL-4 or IL-10 production
  • a "cryptic epitope” also designated herein as “immunosilent " or “nonimmunodominant” epitope
  • 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., Nature Med. 5(1), 1999
  • Cryptic epitopes for use in the present invention include, but are not limited to, peptides of the myelin basic protein sequence: peptide pll-30, p51-70, p91-110, pl31-150, and pl51-170. Other peptides can be identified by their capacity to elicit a human T cell response detected by T cell proliferation or by cytokine (e.g. IFN- ⁇ , IL-2, IL-4, or IL- 10) production. Such cryptic epitopes are particularly preferred as T cells activated thereby will accumulate at the injury site, in accordance with the present invention, but are particularly weak in autoimmunity. Thus, they would be expected to have fewer side effects.
  • cytokine e.g. IFN- ⁇ , IL-2, IL-4, or IL- 10
  • peptides consisting of or comprising a fragment of an NS-specific antigen consisting of at least 10 (contiguous) amino acids of the NS-specific antigen are provided.
  • the fragment consists of at least 20 contiguous amino acids or 50 contiguous amino acids of the NS-specific antigen.
  • NS-specific antigen also include but are not limited to those molecules comprising regions that are substantially homologous to the full-length antigen or fragments thereof (e.g., in various embodiments, 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, Proc. Natl. Acad. Sci. USA 85(8) :2444-8, 1988; Altschul et al . , J. Mol. Biol. 215 (3) : 40310, 1990; Thompson, et al . , Nucleic Acids Res. 22 (22) :4673-80, 1994; Higgins, et al . , Methods Enzymol 266:383- 402, 1996; Altschul, et al . , 1990, J. Mol. Biol. 215(3) :403- 410, 1990) .
  • the NS-specific antigen derivatives 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, T., 1990, Molecular Cloning, A Laboratory Manual, 2d ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) .
  • the sequence can be cleaved at appropriate sites with restriction endonuclease (s) , followed by further enzymatic modification if desired, isolated, and ligated in vi tro .
  • the coding nucleic acid sequence can be mutated in vi tro 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 vi tro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vi tro site-directed mutagenesis (Hutchinson, C, et al . , J. Biol. Chem 253:6551, 1978), etc.
  • Manipulations may also be made at the protein level. Included within the scope of the invention are 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. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 ; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
  • derivatives of an NS-specific antigen can be chemically synthesized.
  • 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.
  • nonclassical amino acids or chemical amino acids analogs can be introduced as a substitution or addition into the amino acid sequence.
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, a- amino isobutyric acid; 4-aminobutyric acid, Abu; 2-amino butyric acid, ⁇ -Abu; e -Ahx, 6-amino hexanoic acid; Aib, 2- amino isobutyric acid; 3 -amino propionic acid; ornithine; norleucine; novaline; hydroxyproline; sarcosine; citrulline; cysteic acid; t-butylglycine; t-butylalanine; phenylgylcine; cyclohexylalanine; 0 -alanine; fluoro-amino acids; designer amino acids such as ⁇ -methyl amino acids, Cc ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
  • the amino acid can be D (dextrorotary) or L (levorotary)
  • NS-specific antigens and peptides derived therefrom 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, J. Immunol . 155:3693-3699, 1995) .
  • An 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.
  • compositions comprising a nucleotide sequence encoding an NS-specific antigen or peptide derived therefrom can be used for preventing or inhibiting the effects of injury or disease that result in CNS or PNS degeneration or for promoting nerve regeneration in the CNS or PNS.
  • Specific illustrative examples of useful nucleotide sequences encoding NS-specific antigens or peptides derived from an NS-specific antigen include but are not limited to nucleotide sequences encoding rat myelin basic protein (MBP) peptides, depicted in Fig. 15 (SEQ ID NO:l); human MBP, depicted in Fig.
  • MBP myelin basic protein
  • compositions described in Sections 5.1 through 5.3 may be used to promote nerve regeneration or to prevent or inhibit secondary degeneration which may otherwise follow primary NS injury, e.g., blunt trauma, penetrating trauma, hemorrhagic stroke, ischemic stroke or damages caused by surgery such as tumor excision.
  • primary NS injury e.g., 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 grey 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, malab
  • the NS-specific activated T cells, the NS-specific antigens, peptides derived therefrom, 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 subj ect .
  • While 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 .
  • 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, lac
  • 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.
  • 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
  • the pharmaceutical compositions may take the form of, for example, tablets 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 suitably formulated to give controlled release of the active compound.
  • compositions may take the form of tablets or lozenges formulated in conventional manner .
  • 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 in 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
  • 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, such as Incomplete Freund ' s Adjuvant .
  • an adjuvant such as Incomplete Freund ' s 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 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 (i.e., an anti-inflammatory steroid, such as dexamethasone or methylprednisolone, 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 i.e
  • mononuclear phagocyte cells according to PCT Publication No. WO 97/09985 and U.S. patent application Serial No. 09/041,280, filed March 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 x 10 6 to about 10 7 for treating a lesion affecting about 10 5 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.
  • 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 can be established with 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 and then sensitized to a NS antigen. 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 treated neurodegenerative conditions such as Alzheimer's disease or Parkinson's disease.
  • semi-allogeneic or allogeneic T cells can be stored frozen in banks for use by any individual who shares one MHC type II molecule with the source of the T cells.
  • Female Lewis rats were supplied by the Animal Breeding Center of the Weizmann Institute of Science (Rehovot, IL) , 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 15 Industries, Israel) supplemented with 2mM L-glutamine (L- Glu, Sigma, USA), 5 x 10" 5 M 2-mercaptoethanol (2-ME, Sigma), penicillin (100 IU/ l ; 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., Clin. Invest. 85:1594, 1990) .
  • DMEM Dulbecco's modified Eagle's medium
  • L- Glu L-glutamine
  • Sigma 5 x 10" 5 M 2-mercaptoethanol
  • penicillin 100 IU/ l
  • streptomycin 100 ⁇ /ml
  • sodium pyruvate (1
  • 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 . , supra, 1990) .
  • FCS fetal calf serum
  • TCGF T cell growth factor
  • MBP Myelin basic protein
  • 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
  • the antigen was dissolved in PBS (lmg/ml) and emulsified with an equal volume of incomplete Freund's adjuvant (Difco Laboratories, Detroit, Michigan) supplemented with 4 mg/ml Mycojacteriu ⁇ i tuberculosis (Difco 15 Laboratories, Detroit, Michigan) .
  • the emulsion (0.1 ml) was injected into hind foot pads of the rats.
  • the cells were washed and activated with the antigen (10 ⁇ g/ml) in proliferation medium (described above in Section 6.1.2).
  • Crush injury of the optic nerve was performed as previously described (Duvdevani et al . , Neurol . Neurosci . 2:31-38, 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.
  • 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 ant-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) .
  • the neurotracer dye distal to the site of the primary crush after two weeks ensures that only axons that survived both the primary damage and the secondary degeneration will be counted.
  • This approach makes it possible to differentiate between neurons that are still functionally intact and neurons in which the axons are injured but the cell bodies are still viable, as only those neurons whose fibers are morphologically intact can take up dye applied distally to the site of injury and transport it to their cell bodies.
  • the number of labeled ganglion cells reliably reflects the number of still-functioning neurons. Labeling and measurement were done by exposing the right optic nerve for a second time, again without damaging the retinal blood supply.
  • Nerves were excised and their compound action potentials (CAPs) were recorded in vi tro using a suction electrode experimental set-up (Yoles et al., J. Neurotrauma 13:49-57, 1996).
  • CAPs compound action potentials
  • rats were killed by intraperitoneal injection of pentobarbitone (170 mg/kg) (CTS Chemical Industries, Israel) .
  • 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 P0 2 26 mM NaHC0 3 2 mM MgS0 4 , 2 mM CaCl 2 and 10 mM D-glucose, aerated with 95% 0 2 and 5% C0 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 retinal ganglion cells that have escaped both primary and secondary damage, as well as the distal stumps of non-viable retinal ganglion cells 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, Massachusetts) was used for supramaximal electrical stimulation at a rate of 1 pps to ensure stimulation of all propagating axons in the nerve.
  • the measured signal was transmitted to a microelectrode AC amplifier (model 1800; A-M Systems, Everett, Washington) .
  • the data were processed using the LabView 2.1.1 data acquisition and management system (National Instruments, Austin, Texas) .
  • LabView 2.1.1 data acquisition and management system National Instruments, Austin, Texas
  • 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 experimentors "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.
  • Rats were injected intraperitoneally immediately after optic nerve injury with PBS or with 1 x 10 7 activated T cells of the various cell lines.
  • the degree of primary damage to the optic nerve axons and their attached RGCs was measured by injecting the dye 4-Di-10-Asp distal to the site of the lesion immediately after the injury.
  • a time lapse of 2 weeks between a moderate crush injury and dye application is optimal for demonstrating the number of still viable labeled neurons as a measure of secondary degeneration, and as the response of secondary degeneration to treatment .
  • 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.
  • T cells reactive to a "cryptic" epitope of MBP the peptide 51-70 (p51-70) was examined.
  • “Cryptic" epitopes activate specific T cells after an animal is immunized with the particular peptide, but not with the whole antigen (Mor et al . , J . Immunol . 155:3693-3699. 1995).
  • the T cell line reactive to the whole MBP and the T cell line reactive to the cryptic epitope p51-70 were compared for the severity of the EAE they induced, and for their effects on secondary degeneration.
  • the percentage of RGCs surviving secondary degeneration in the retinas of rats injected with either of the lines was significantly higher than in the retinas of the PBS-injected rats.
  • the neuroprotective effect of the autoimmune T cells there was no correlation between the neuroprotective effect of the autoimmune T cells and their virulence. It is possible that the anti-p51-70 T cells encounter little antigen in the intact CNS, and therefore cause only mild EAE. Their target antigen may however become more available after injury, enabling these T cells to exert a neuroprotective effect.
  • the rats were injected intraperitoneally with PBS or with 1 x 10 7 activated anti-MBP or anti-OVA T cells.
  • the optic nerves were excised 7, 11 or 14 days later and the compound action potentials (CAPs) , a measure of nerve conduction, were recorded from the injured nerves.
  • CAPs compound action potentials
  • the mean CAP amplitudes of the distal segments recorded from the injured nerves obtained from the PBS-injected control rats were 33% to 50% of those recorded from the rats injected with the anti-MBP T cells (Fig. 6A, Table 2) .
  • 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.
  • 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) .
  • Ratios were calculated for uninjured nerves as (mean CAP of uninjured nerves from T cell-injected rats/mean CAP of uninjured nerves from PBS-injected rats) x 100, or for injured nerves as (mean CAP of injured nerves from T cell-injected rats/mean CAP of injured nerves from PBS-injected rats) x 100.
  • Bladder expression was done at least twice a day (particularly during the first 48h 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 .
  • 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
  • Control rats were similarly injured but received either no T cells or T cells specific to the non-self antigen ovalbumin (OVA) .
  • OVA ovalbumin
  • Recovery of the rats was assessed every 3 to 4 days in terms of their behavior in an open-field locomotion test, in which scores range form 0 (complete paraplegia) to 21 (normal mobility) .
  • the locomotor performance of the rats was judged by observers blinded to the identity of the treatment received by the rats. Included in the study was a group of uninjured, sham-operated (laminectomized but not contused) rats which were injected with anti-MBP T cells to verify the activity of the T cells.
  • EAE clinical experimental autoimmune encephalomyelitis
  • the average score of the rats that had been treated with the anti-MBP T cells was 10.2 + 0.8, and in some rats the value was high as 13. All the rats in the treated group could support their body weight and some could frequently walk in a coordinated fashion.
  • the recovery curve based on locomotor activity is nonlinear.
  • the rats were subjected to a more severe insult, resulting in a functional score of 1.9 + 0.8 (mean + SEM) in the untreated group and 7.7 + 1.4 in the treated group (Fig. 7B) .
  • This difference of more than 3 fold in behavioral scores was manifested by the almost total lack of motor activity in the control rats as compared with the ability of the autoimmune T cell-treated rats to move all their joints.
  • the beneficial effect was specific to treatment with anti-MBP T cells; no effect was observed after treatment with T cells specific to the non-self antigen OVA (data not shown) .
  • the positive effect of the autoimmune T cells seems to be expressed in the preservation of CNS tissue that escaped the initial lesion, i.e., in neuroprotection.
  • Sections of red nuclei from injured rats treated with anti-MBP T cells contained 5-fold more labeled cells than sections taken from the untreated injured rats.
  • Photomicrographs of red nuclei taken from rats treated with anti-MBP T cells (with an open field score of 10) and from PBS- treated rats (with a score of 6) are shown in Fig. 8.
  • 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 grey 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. 7.3.3 DISCUSSION OF RESULTS
  • the same 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 costimulatory molecules, particularly members of the B7 family (Lenchow et al., Annu. Rev. Immunol. 14:233-258, 1996).
  • the injured rat optic nerve transiently expresses elevated levels of the costimulatory 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 (H. L. Weiner, Annu . Rev. Med . 48:341-51, 1997).
  • B7.2 costimulatory 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 costimulatory 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 (M. Kerschensteiner et al., J . Exp . Med . 189:865-870, 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 (I. R. Cohen, Immunol . Today 13, 490-494 (1992), and are not merely a default resulting from the escape from negative selection of T cells that recognize self antigens (C. A. Janeway, Jr., Immunol . Today 13:11-6, 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.
  • a single injection of autoimmune T cells lasted for at least 100 days.
  • this procedure offers a form of self- maintenance .
  • This specific autoimmune response when properly controlled, is useful as part of a self-derived remedy for spinal cord injury.
  • 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. Retinal ganglion cells 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 retinal ganglion cells 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.
  • Autoimmune T cells can under under certain conditions be beneficial to traumatized CNS axons.
  • the effect of such T cells on the damaged tissue might be influenced by the nature and amount of the costimulatory molecules it expresses.
  • the B7.2 costimulatory molecule is constitutively expressed in the intact rat optic nerve, and after injury is up-regulated at the margins of the injury site.
  • Pre-injury induction of oral tolerance to MBP resulted in a further post- injury increase in B7.2 at the margins and at the injury site itself, as well as a better preservation of the traumatized nerve.
  • B7.2 expression in the brain and its up-regulated after trauma seem to be directly related to post-traumatic maintenance displayed by autoimmune T cells.
  • CD40 appears to be dominant during cell differentiation in the lymph nodes and B7 during activation of T cells in the target organ
  • B7 costimulatory molecules are expressed on antigen-presenting cells (APCs) as B7.1 or B7.2., which might preferentially support activation of the Thl or the Th2 type of immune response, respectively (Kuchroo et al . , "B7-1 and B7-2 costimulatory molecules activate differentially the Thl/Th2 developmental pathways: application to autoimmune disease therapy", Cell 80:707-718, 1995; and Karandikar et al . ,
  • the costimulatory molecule expressed constitutively in the intact optic nerves of adult Lewis rats was identified as B7.2. (Figs. 12A, 12B) .
  • B7 costimulatory molecules expressed constitutively in the intact optic nerves of adult Lewis rats.
  • FIGs. 12A, 12B To examine the effects of neurotrauma on the expression of B7 costimulatory 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) .
  • 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) .
  • T cells which, based on antigen recognition, secrete TGF as the dominant cytokine and thus favor an immune response of Th2/3 type (Chen, Y., "Regulatory T Cell Clones Induced by Oral Tolerance: Suppression of Autoimmune Encephalomyelitis", Science 265: 1237-1240, 1994) .
  • 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 .
  • the results of experiments described in Section 7 demonstrate that the beneficial effects of T cells in ameliorating damage due to injury in the CNS are associated with an NS-specific self-antigen as illustrated by MBP. More specifically, the administration of non-recombinant T cells which were activated by exposure to an antigen which can cause autoimmune disease (T MBP ), rather than aggravating the injury, led to a significant degree of protection from secondary degeneration. Thus, activating T cells by exposure to a fragment of an NS-specific antigen was beneficial in limiting the spread of injury in the CNS.
  • the present findings show that secondary degeneration can be inhibited by the transfer into the individual on non-recombinant T cells which recognize an NS-specific self antigen which is present at a site of injury.
  • the T cells may recognize cryptic or non-pathogenic epitopes of NS-self antigens.

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Abstract

L'invention concerne des compositions et des procédés destinés à traiter des lésions ou des maladies des systèmes nerveux central et périphérique. Dans un mode de réalisation, le traitement est effectué au moyen de lymphocytes T activés qui reconnaissent un antigène du système nerveux ou un peptide dérivé à partir de celui-là ou un dérivé de ce dernier, et ce afin de favoriser la régénération des nerfs ou d'empêcher ou d'inhiber la dégénérescence neuronale à l'intérieur du système nerveux. Le traitement consiste à administrer un antigène spécifique du système nerveux ou un peptide dérivé à partir de celui-ci, ou une séquence de nucléotides codant pour cet antigène ou peptide; à favoriser la régénération des nerfs ou à empêcher ou inhiber la dégénérescence neuronale à l'intérieur du système nerveux, que ce soit dans le système nerveux central ou dans celui périphérique. Les lymphocytes T activés spécifiques du système nerveux peuvent être administrés isolément ou en combinaison avec un peptide ou un antigène spécifique du système nerveux dérivé de ceux-ci, ou avec un dérivé de ces derniers ou une séquence de nucléotides codant pour cet antigène ou peptide, ou avec n'importe quelle combinaison de ces éléments.
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CA2328612A1 (fr) 1999-11-25
WO1999060021A3 (fr) 2000-06-15
WO1999060021A2 (fr) 1999-11-25
JP2003532618A (ja) 2003-11-05

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