Classifications

• • 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
• • 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
  • A61K39/0008—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
  • A61K40/10—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
  • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • 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
• • 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
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
• • 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
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to compositions, e.g. vaccines, and methods for the treatment of neurodegenerative diseases in which there is accumulation of misfolded and/or aggregated proteins, excluding prion diseases.
• the invention relates to treatment of the neurodegenerative diseases Huntington's disease (HD), Alzheimer's disease (AD) or Parkinson's disease (PD), by administration of an agent selected from the group consisting of Copolymer 1, a Copolymer 1 -related peptide or polypeptide, and T cells activated therewith.
• HD Huntington's disease
• AD Alzheimer's disease
• PD Parkinson's disease
• ABBREVIATIONS A ⁇ o, ⁇ -amyloid peptide 1-40; AD, Alzheimer's disease;
• APC antigen-presenting cell
• CNS central nervous system
• Cop-1 Copolymer 1;
• DAT dopamine transporter
• HD Huntington's disease
• IRPB interphotoreceptor retinoid-binding protein
• MPTP l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine
• OHSC organotypic hippocampal slice culture
• PD Parkinson's disease
• PI propidium iodide
• RGC retinal ganglion cell
• Treg CD4 + CD25 + regulatory T cells
• TTJNEL terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling
• WRH whole retinal homogenate.
• Polyglutamine repeat diseases such as Huntington disease are likewise associated with neuronal cytosolic and intranuclear inclusions (DiFiglia et al., 1997). These inclusions are composed of fibrils that stain similarly to amyloid (Scherzinger et al., 1997). Finally, in Parkinson disease, inclusions known as Lewy bodies, found in the cytoplasm of cells of the basal ganglia, include amyloid-like aggregates of the protein ⁇ -synuclein (Conway et al., 2000; Serpell et al., 2000).
• HD Huntington's disease
• This damage to cells affects cognitive ability (thinking, judgment, memory), movement, and emotional control.
• HD is characterized by uncontrollable, dancelike movements and personality changes.
• HD patients develop slurred speech, an unsteady walk and difficulty in swallowing. There is no effective treatment for HD. After a long illness, individuals with HD die from complications such as choking or infection.
• the mutation that causes HD was identified as an unstable expansion of CAG repeats in the IT15 gene encoding huntingtin, a protein of unknown function (Menalled and Chesselet, 2002).
• AD Alzheimer's disease
• AD Alzheimer's disease
• Parkinson's disease is an idiopathic, slowly progressive, degenerative CNS disorder characterized by slow and decreased movement, muscular rigidity, resting tremor, and postural instability.
• PD Parkinson's disease
• Significant hints into PD pathogenesis have been yielded by the use of l-methyl-4-phenyl-l,2,4,6-tetrahdropyridine (MPTP), a neurotoxin that replicates most of the neuropathological hallmarks of PD in humans, nonhuman primates, and other mammalian species, including mice.
• MPTP l-methyl-4-phenyl-l,2,4,6-tetrahdropyridine
• the MPTP mouse model departs from human PD in a few important ways, it offers a unique means to investigate, in vivo, molecular events underlying the demise of midbrain dopaminergic neurons (Dauer and Przedborski, 2003).
• Acute and/or chronic neuronal loss in the adult CNS results in the irreversible loss of function due to the very poor ability of mature nerve cells to proliferate and compensate for the lost neurons.
• attenuating or reducing neuronal loss is essential for preservation of function.
• the neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and Huntington's disease, the etiology is not clear, hence they are incurable.
• neuroprotective therapy there are some primary and secondary risk factors, which are the target for therapeutic intervention aiming at inhibiting or attenuating progress of neuronal loss, collectively termed as neuroprotective therapy.
• Some of the risk factors are disease-specific but others, like excitatory ammo acids, free radicals and nitric oxide, are common to all the neurodegenerative disorders. These factors are essential self-components in the healthy CNS, but with their accumulation in excess amounts in the degenerative tissue, they become cytotoxic leading to the spread of damage beyond the initial cause of neuron death.
• Glutamate is one of the most common mediators of toxicity in acute and chronic degenerative disorders like status epilepticus, cerebral ischemia, traumatic brain injury, ALS, Huntington's disease, lathyrisms and Alzheimer's disease. Glutamate is a primary excitatory neurotransmitter in the human CNS. L-glutamate is present at a majority of synapses and is capable of displaying dual activity: it plays a pivotal role in normal functioning as an essential neurotransmitter, but becomes toxic when its physiological levels are exceeded. In order to minimize neuronal loss (neuroprotection) several approaches have been adopted, at which the most common is targeting the risk factors in an attempt to neutralize or inhibit their action. Unfortunately, these therapeutic strategies showed marginal efficacy in human subjects with concomitant severe side effects. The failure of agents with discrete singular mechanisms of action argues for a multi- pronged approach.
• T cells that mediate protection are directed not against a particular threatening self-compound but rather against dominant self-antigens that reside at the lesion site (Mizrahi et al., 2002; Schwartz et al., 2003; Bakalash et al., 2002). Further studies by the inventors suggested that T-cell specificity is needed in order to ensure that among the T cells that arrive at the site, those encountering their specific or cross-reactive antigens (presented to them by local microglia acting as APC) will become activated.
• the activated T cells can then provide the necessary cytokines or growth factors that control the activity of the local microglia and the friendliness of the extracellular milieu (Schwartz et al., 2003; Moalem et al., 2000; Kipnis et al, 2000).
• the concept of T cell-dependent "protective autoimmunity" has been formulated by the inventor Prof. Michal Schwartz and her group (Kipnis et al., 2002a; Schwartz and Kipnis, 2002a). According to this concept, an acute or chronic insult to the CNS triggers an autoimmune response directed against proteins residing in the lesion site. T cells homing to the lesion site are activated by cells presenting the relevant antigen.
• Copolymer 1 also called Cop 1
• Copolymer 1 is a random non-pathogenic synthetic copolymer, a heterogeneous mix of polypeptides containing the four amino acids L- glutamic acid (E), L-alanine (A), L-tyrosine (Y) and L-lysine (K) in an approximate ratio of 1.5:4.8:1:3.6, but with no uniform sequence.
• E L- glutamic acid
• A L-alanine
• Y L-tyrosine
• K L-lysine
• Cop 1 clearly helps retard the progression of human multiple sclerosis (MS) and of the related autoimmune condition studied in mice, experimental autoimmune encephalomyelitis (EAE).
• Cop 1 One form of Cop 1, known as glatiramer acetate, has been approved in several countries for the treatment of multiple sclerosis under the trademark Copaxone® (Teva Pharmaceutical Industries Ltd., Petach Tikva, Israel). Vaccination with Cop 1 or with Cop 1 -activated T cells have been shown by the present inventors to boost the protective autoimmunity, after traumatic CNS insult, thereby reducing further injury-induced damage, and can further protect CNS cells from glutamate toxicity. Reference is made to Applicant's previous US Patent Applications Nos. 09/765,301 and 09/765,644 and corresponding published International Application Nos.
• Cop 1 acts as a low-affmity antigen that activates a wide range of self-reacting T cells, resulting in neuroprotective autoimmunity that is effective against both CNS white matter and grey matter degeneration (Kipnis et al., 2002a; Schwartz and Kipnis, 2002a).
• Cop 1 vaccination The neuroprotective effect of Cop 1 vaccination was demonstrated by the inventors in animal models of acute and chronic neurological disorders such as optic nerve injury (Kipnis et al., 2000), head trauma (Kipnis et al., 2003), glaucoma (Schori et al., 2001b), amyotrophic lateral sclerosis (Angelov et al., 2003) and in the applicant's patent applications WO 01/52878, WO 01/93893 and WO 03/047500.
• the use of copolymer for treatment of prion-related diseases is disclosed in WO 01/97785.
• the present invention relates, in one aspect, to a method for treating a neurodegenerative disorder or disease in which there is accumulation of misfolded and/or aggregated proteins, excluding prion-related diseases, said method comprising administering to an individual in need an agent selected from the group consisting of (i) Copolymer 1, (ii) a Copolymer 1 -related peptide, (iii) a Copolymer 1 -related polypeptide, and (iv) T cells activated with (i), (ii) or (iii).
• the invention relates to a method for reducing disease progression, and/or protection from neurodegeneration and/or protection from glutamate toxicity in a patient suffering from a neurodegenerative disorder or disease in which there is accumulation of misfolded and/or aggregated proteins, excluding prion-related diseases, which comprises administering to said patient a therapeutically active amount of an agent selected from the group consisting of (i) Copolymer 1, (ii) a Copolymer 1 -related peptide, (iii) a Copolymer 1 -related polypeptide, and (iv) T cells activated with (i), (ii) or (iii).
• an agent selected from the group consisting of (i) Copolymer 1, (ii) a Copolymer 1 -related peptide, (iii) a Copolymer 1 -related polypeptide, and (iv) T cells activated with (i), (ii) or (iii).
• the invention relates to a method for reducing disease progression, and/or protection from neurodegeneration and/or protection from glutamate toxicity in a patient suffering from a neurodegenerative disorder or disease in which there is accumulation of misfolded and/or aggregated proteins, excluding prion-related diseases, which comprises immunizing said patient with an agent selected from the group consisting of (i) Copolymer 1, (ii) a Copolymer 1- related peptide, (iii) a Copolymer 1 -related polypeptide, and (iv) T cells activated with (i), (ii) or (iii).
• the present invention provides a pharmaceutical composition for treatment of a neurodegenerative disorder or disease in which there is accumulation of misfolded and/or aggregated proteins, excluding prion-related diseases, comprising a pharmaceutically acceptable carrier and an active agent selected from the group consisting of (i) Copolymer 1, (ii) a Copolymer 1 -related peptide, (iii) a Copolymer 1 -related polypeptide, and (iv) T cells activated with (i), (ii) or (iii).
• said pharmaceutical composition is a vaccine.
• the present invention relates to the use of an active agent selected from the group consisting of (i) Copolymer 1, (ii) a Copolymer 1 -related peptide, (iii) a Copolymer 1 -related polypeptide, and (iv) T cells activated with (i), (ii) or (iii), for the manufacture of a pharmaceutical composition for treatment of a neurodegenerative disorder or disease in which there is accumulation of misfolded and/or aggregated proteins, excluding prion-related diseases.
• said pharmaceutical composition is a vaccine.
• said neurodegenerative disease or disorder is Huntington's disease.
• said neurodegenerative disease or disorder is Alzheimer's disease.
• said neurodegenerative disease or disorder is Parkinson's disease.
• the invention provides an article of manufacture comprising packaging material and a pharmaceutical composition contained within the packaging material, said pharmaceutical composition comprising an agent selected from the group consisting of Copolymer 1, a Copolymer 1 -related peptide, and a Copolymer 1 -related polypeptide; and said packaging material includes a label that indicates that said agent is therapeutically effective for treating a neurodegenerative disease or disorder selected from Huntington's disease, Alzheimer's disease or Parkinson's disease.
• the active agent is Copolymer 1.
• FIG. 1 shows the neuroprotective effect on retinal ganglion cells (RGCs) of mice by immunization with different doses of Cop 1 (25, 75g or 225 ⁇ g/mouse) injected 7 days before exposure of RGCs to glutamate toxicity.
• RGCs retinal ganglion cells
• the results are presented as mean ⁇ SEM of percentage of RGCs that were protected due to Cop 1 vaccination out of the total RGC death in the non-treated group. * represents statistically significant difference (t-test, p ⁇ 0.05) versus the non-treated group.
• Fig. 2 shows the latency of neuroprotective effect on RGCs of mice by vaccination with 75 ⁇ g Cop 1 injected 7, 14 and 28 days before exposure of RGCs to glutamate toxicity.
• Fig. 3 shows that daily injections of Cop 1 repeated for three days at doses of
• Fig. 4 shows the efficacy of two repeated injections of Cop 1 (75 ⁇ g/mouse), injected at different time intervals (1, 2, 3, 4, 6, 8, weeks).
• the neuroprotective effect of the treatment on RGCs is represented as % of a single injection of Cop 1 (75 ⁇ g/mouse), injected 7 days before induction of glutamate toxicity. This single injection was determined as positive control and performed in each experiment.
• Fig. 5 shows the efficacy of three repeated injections of Cop 1 (75 ⁇ g/mouse) injected at different time intervals (daily, 1, 2, 4, weeks).
• the neuroprotective effect of the treatment on RGCs is represented as % of a single injection of Cop 1 (75 ⁇ g/mouse), injected 7 days before induction of glutamate toxicity. This single injection was determined as positive control and performed in each experiment.
• Fig. 6 shows proliferation of splenocytes from mice following immunization with different doses of Cop 1 (25 ⁇ g, 75 ⁇ g, 225 ⁇ g). The results after 7, 14, 21 and
• Fig. 7 shows INF- ⁇ secretion from stimulated splenocytes 7, 14, 21 or 28 days after immunization with 25 ⁇ g or 75 ⁇ g Cop 1.
• Fig. 8 is a graph showing the rotarod performance of HD R6/2 transgenic mice after vaccination with 75 ⁇ g or 150 ⁇ g Cop 1.
• Fig. 9 shows the rotarod performance of HD R6/2 transgenic mice following vaccination with 150 ⁇ g Cop 1, at different speeds of rotation (2, 5, 15 and 25 rpm).
• TUNEL-positive cells in the RGC layers of C57B1/6J mice 48 h after intravitreal injection of a toxic dose of glutamate. Sections (20 ⁇ m thick) were subjected to TUNEL staining, counterstained with propidium iodide, and viewed by confocal microscopy to detect TUNEL-positive cells. A confocal image of a representative retina is shown. The arrow indicates TUNEL-positive cells in the RGC layer. Scale bar 200 ⁇ m.
• mice C57B1/6J mice were immunized in the flank with 600 ⁇ g of whole retinal homogenate (WRH) emulsified in CFA supplemented with 5 mg/ml of Mycobacterium tuberculosis. Six days later the mice were injected intravitreally with glutamate (400 nmol). One week later surviving RGCs were counted. Significantly more RGCs survived in mice immunized with WRH/CFA than in mice immunized with PBS/CFA.
• WRH retinal homogenate
• mice were immunized in the flank with interphotoreceptor- binding protein (IRBP; 50 ⁇ g) or S-antigen (50 ⁇ g) emulsified in CFA supplemented with 5 mg/ml of Mycobacterium tuberculosis. Control mice were immunized with PBS in CFA.
• IRBP interphotoreceptor- binding protein
• S-antigen 50 ⁇ g
• FIGs. 11A-11F show that susceptibility of retinal ganglion cells to A ⁇ _ 40 toxicity is T cell-dependent.
• A C57BL/6/J mice were injected intravitreally with 5 or 50 ⁇ M A ⁇ 1 _ 40 or were not injected (control), and 1 or 2 weeks later the retinas were excised and the surviving RGCs counted.
• mice were immunized in the flank with interphotoreceptor-binding protein (IRBP; 50 ⁇ g) in CFA, the ⁇ -amyloid peptide (1-40, non-aggregated) (50 ⁇ g) in CFA, or PBS in CFA. In all cases, CFA was supplemented with 5 mg/ml of Mycobacterium tuberculosis. Ten days later the mice were injected intravitreally with a toxic dose of aggregated A ⁇ 1 _ 40 (50 ⁇ M), and after 10 days their retinas were excised and the surviving RGCs counted.
• IRBP interphotoreceptor-binding protein
• T cells specific to IRBP + S-antigen were injected at a dose of 1.2 x 10 T cells in PBS.
• the mice received an intravitreal injection of glutamate (400 nmol), and surviving retinal ganglion cells (RGCs) were counted 1 week later.
• RGCs retinal ganglion cells
• There was no difference between mice that received OVA-specific T cells and naive mice in the numbers of RGCs that survived the glutamate injection (n 4-6 mice per group).
• mice were injected intravenously with 8xl0 6 activated T cells directed either to IRBP or to ⁇ -amyloid peptide (1-40, non-aggregated).
• T-cell transfer One hour after this passive T-cell transfer, the mice were injected with a toxic dose of aggregated A ⁇ i_ 40 .
• Two weeks later their retinas were excised and surviving RGCs counted.
• Neuronal loss in these mice was significantly decreased by transfer of T cells reactive to the IRBP (P ⁇ 0.005, two-tailed Student's t-test), but was not significantly affected by transfer of T cells reactive to non-aggregated ⁇ -amyloid.
• Fig. 14 shows that active immunization with Co ⁇ -1 protects against A ⁇ _ 40 toxicity.
• Figs. 15A-15C show that more neurons survive aggregated A ⁇ j_ 40 intoxication in mice devoid of naturally occurring regulatory CD4+CD25+ T cells than in naive mice.
• BALB/c/OLA nu/nu mice were replenished with 4.5 x 10 7 splenocytes from spleens devoid of Treg or from whole spleens of BALB/c/OLA mice.
• Figs. 16A-16B show death of neural cells in rat organotypic hippocampal slice cultures 24 h after treatment with microglia incubated with aggregated A ⁇ _ 4 o with and without activated T cells.
• OHSCs were obtained from BALB/c/OLA mice. Immediately after sectioning, the slices were co-cultured for 24 h with microglia that had been pre-incubated (12 h) with aggregated A ⁇ _ 40 alone or with a combination of aggregated A ⁇ _ 40 and activated Teff (A).
• Control slices were treated with na ⁇ ve microglia or were left untreated. Twenty-four hours after co- culturing of microglia and brain slices, the slices were stained with propidium iodide (PI) (a fluorescent dye that stains only dead cells) and analyzed by fluorescence microscopy.
• PI propidium iodide
• the methods of the present invention comprise administering to an individual in need an agent selected from the group consisting of (i) Copolymer 1, (ii) a Copolymer 1 -related peptide, (iii) a Copolymer 1 -related polypeptide, and (iv) T cells activated with (i), (ii) or (iii), for the treatment of a neurodegenerative disorder or disease in which there is accumulation of misfolded and/or aggregated proteins, excluding prion-related diseases.
• the neurodegenerative disease or disorder is Huntington's disease.
• the neurodegenerative disease or disorder is Alzheimer's disease.
• the neurodegenerative disease or disorder is Parkinson's disease.
• the treatment with Copolymer 1, Cop 1 -related peptides or polypeptides, ot T cells activated therewith aims to reduce disease progression, to afford protection from neurodegeneration, and/or to afford protection from glutamate toxicity in patients suffering from Huntington's disease, Alzheimer's disease or Parkinson's disease.
• the treatment is performed by immunization.
• therapeutically effective amounts of the selected agent are administered to the patient. The doses and regimen of the two types of treatment may be different.
• a method for treating or preventing neurodegeneration and cognitive decline and dysfunction associated with Huntington's disease, Alzheimer's disease orParkinson's disease comprising administering to an individual in need an agent selected from the group consisting of (i) Copolymer 1, (ii) a Copolymer 1 -related peptide, (iii) a Copolymer 1 -related polypeptide, and (iv) T cells activated with (i), (ii) or (iii).
• an agent selected from the group consisting of (i) Copolymer 1, (ii) a Copolymer 1 -related peptide, (iii) a Copolymer 1 -related polypeptide, and (iv) T cells activated with (i), (ii) or (iii).
• the terms "Cop 1" and "Copolymer 1" are used interchangeably.
• Cop 1 or a Cop 1 -related peptide or polypeptide is intended to include any peptide or polypeptide, including a random copolymer, that cross-reacts functionally with myelin basic protein (MBP) and is able to compete with MBP on the MHC class II in the antigen presentation.
• MBP myelin basic protein
• composition or vaccine of the invention may comprise as active agent a Cop 1 or a Cop 1 -related peptide or polypeptide represented by a random copolymer consisting of a suitable ratio of a positively charged amino acid such as lysine or arginine, in combination with a negatively charged amino acid (preferably in a lesser quantity) such as glutamic acid or aspartic acid, optionally in combination with a non-charged neutral amino acid such as alanine or glycine, serving as a filler, and optionally with an amino acid adapted to confer on the copolymer immunogenic properties, such as an aromatic amino acid like tyrosine or tryptophan.
• a positively charged amino acid such as lysine or arginine
• a negatively charged amino acid preferably in a lesser quantity
• glutamic acid or aspartic acid optionally in combination with a non-charged neutral amino acid such as alanine or glycine, serving as a filler
• compositions may include any of those copolymers disclosed in WO 00/05250, the entire contents of which being hereby incorporated herein by reference. More specifically, the composition for use in the present invention comprises at least one copolymer selected from the group consisting of random copolymers comprising one amino acid selected from each of at least three of the following groups: (a) lysine and arginine; (b) glutamic acid and aspartic acid; (c) alanine and glycine; and (d) tyrosine and tryptophan.
• the copolymers for use in the present invention can be composed of L- or D- amino acids or mixtures thereof. As is known by those of skill in the art, L-amino acids occur in most natural proteins.
• D-amino acids are commercially available and can be substituted for some or all of the amino acids used to make the copolymers used in the present invention.
• the present invention contemplates the use of copolymers containing both D- and L-amino acids, as well as copolymers consisting essentially of either L- or D-amino acids.
• the copolymer contains four different amino acids, each from a different one of the groups (a) to (d).
• the pharmaceutical composition or vaccine of the invention comprises Copolymer 1, a mixture of random polypeptides consisting essentially of the amino acids L-glutamic acid (E), L-alanine (A), L- tyrosine (Y) and L-lysine (K) in an approximate ratio of 1.5:4.8:1:3.6, having a net overall positive electrical charge and of a molecular weight from about 2 KDa to about 40 KDa.
• Copolymer 1 a mixture of random polypeptides consisting essentially of the amino acids L-glutamic acid (E), L-alanine (A), L- tyrosine (Y) and L-lysine (K) in an approximate ratio of 1.5:4.8:1:3.6, having a net overall positive electrical charge and of a molecular weight from about 2 KDa to about 40 KDa.
• the Cop 1 has average molecular weight of about 2 KDa to about 20 KDa, more preferably of about 4,7 KDa to about 13 K Da, still more preferably of about 4 KDa to about 8.6 KDa, of about 5 KDa to 9 KDa, or of about 6.25 KDa to 8.4 KDa. In another preferred embodiment, the Cop 1 has average molecular weight of about 13 KDa to about 20 KDa, more preferably of about 13 KDa to about 16 KDa or of about 15 KDa to about 16 KDa. Other average molecular weights for Cop 1, lower than 40 KDa, are also encompassed by the present invention.
• Copolymer 1 of said molecular weight ranges can be prepared by methods known in the art, for example by the processes described in U.S. Patent No. 5,800,808, the entire contents of which are hereby incorporated by reference in the entirety.
• the Copolymer 1 may be a polypeptide comprising from about 15 to about 100, preferably from about 40 to about 80, amino acids in length.
• the Cop 1 is in the form of its acetate salt known under the generic name glatiramer acetate, that has been approved in several countries for the treatment of multiple sclerosis (MS) under the trade name, Copaxone® (a trademark of Teva Pharmaceuticals Ltd., Petach Tikva, Israel).
• Copolymer 1 for the vaccine disclosed herein is expected to remain if one or more of the following substitutions is made: aspartic acid for glutamic acid, glycine for alanine, arginine for lysine, and tryptophan for tyrosine.
• the Cop 1 -related peptide or polypeptide is a copolymer of three different amino acids each from a different one of three groups of the groups (a) to (d). These copolymers are herein referred to as te olymers.
• the Cop 1 -related peptide or polypeptide is a terpolymer containing tyrosine, alanine, and lysine, hereinafter designated YAK, in which the average molar fraction of the amino acids can vary: tyrosine can be present in a mole fraction of about 0.05-0.250; alanine in a mole fraction of about 0.3 - 0.6; and lysine in a mole fraction of about 0.1-0.5. More preferably, the molar ratios of tyrosine, alanine and lysine are about 0.10:0.54:0.35, respectively.
• the Cop 1-related peptide or polypeptide is a terpolymer containing tyrosine, glutamic acid, and lysine, hereinafter designated YEK, in which the average molar fraction of the amino acids can vary: glutamic acid can be present in a mole fraction of about 0.005 - 0.300, tyrosine can be present in a mole fraction of about 0.005-0.250, and lysine can be present in a mole fraction of about 0.3-0.7.
• the molar ratios of glutamic acid, tyrosine, and lysine are about 0.26:0.16:0.58, respectively. It is possible to substitute aspartic acid for glutamic acid, arginine for lysine, and/or tryptophan for tyrosine.
• the Cop 1 -related peptide or polypeptide is a terpolymer containing lysine, glutamic acid, and alanine, hereinafter designated KEA, in which the average molar fraction of the amino acids can vary: glutamic acid can be present in a mole fraction of about 0.005-0.300, alanine in a mole fraction of about 0.005-0.600, and lysine can be present in a mole fraction of about 0.2 - 0.7. More preferably, the molar ratios of glutamic acid, alanine and lysine are about 0.15:0.48:0.36, respectively.
• the Cop 1 -related peptide or polypeptide is a terpolymer containing tyrosine, glutamic acid, and alanine, hereinafter designated YEA, in which the average molar fraction of the amino acids can vary: tyrosine can be present in a mole fraction of about 0.005-0.250, glutamic acid in a mole fraction of about 0.005-0.300, and alanine in a mole fraction of about 0.005-0.800.
• the molar ratios of glutamic acid, alanine, and tyrosine are about 0.21: 0.65:0.14, respectively. It is possible to substitute tryptophan for tyrosine, aspartic acid for glutamic acid, and/or glycine for alanine.
• the average molecular weight of the terpolymers YAK, YEK, KEA and YEA can vary between about 2 KDa to 40 KDa, preferably between about 3 KDa to 35 KDa, more preferably between about 5 KDa to 25 KDa.
• Copolymer 1 and related peptides and polypeptides may be prepared by methods known in the art, for example, under condensation conditions using the desired molar ratio of amino acids in solution, or by solid phase synthetic procedures.
• Condensation conditions include the proper temperature, pH, and solvent conditions for condensing the carboxyl group of one amino acid with the amino group of another amino acid to form a peptide bond.
• Condensing agents for example dicyclohexylcarbodiimide, can be used to facilitate the formation of the peptide bond.
• Blocking groups can be used to protect functional groups, such as the side chain moieties and some of the amino or carboxyl groups against undesired side reactions.
• the copolymers can be prepared by the process disclosed in
• the process can be adjusted to make peptides and polypeptides containing the desired amino acids, that is, three of the four amino acids in Copolymer 1, by selectively eliminating the reactions that relate to any one of glutamic acid, alanine, tyrosine, or lysine.
• the molecular weight of the copolymers can be adjusted during polypeptide synthesis or after the copolymers have been made. To adjust the molecular weight during polypeptide synthesis, the synthetic conditions or the amounts of amino acids are adjusted so that synthesis stops when the polypeptide reaches the approximate length which is desired.
• polypeptides with the desired molecular weight can be obtained by any available size selection procedure, such as chromatography of the polypeptides on a molecular weight sizing column or gel, and collection of the molecular weight ranges desired.
• the copolymers can also be partially hydrolyzed to remove high molecular weight species, for example, by acid or enzymatic hydrolysis, and then purified to remove the acid or enzymes.
• the copolymers with a desired molecular weight may be prepared by a process, which includes reacting a protected polypeptide with hydrobromic acid to form a trifluoroacetyl-polypeptide having the desired molecular weight profile.
• the reaction is performed for a time and at a temperature which is predetermined by one or more test reactions.
• the time and temperature are varied and the molecular weight range of a given batch of test polypeptides is determined.
• the test conditions which provide the optimal molecular weight range for that batch of polypeptides are used for the batch.
• a trifluoroacetyl-polypepti e having the desired molecular weight profile can be produced by a process, which includes reacting the protected polypeptide with hydrobromic acid for a time and at a temperature predetermined by test reaction.
• the trifluoroacetyl-polypeptide with the desired molecular weight profile is then further treated with an aqueous piperidine solution to form a low toxicity polypeptide having the desired molecular weight.
• a test sample of protected polypeptide from a given batch is reacted with hydrobromic acid for about 10-50 hours at a temperature of about 20-28°C.
• the best conditions for that batch are determined by running several test reactions.
• the protected polypeptide is reacted with hydrobromic acid for about 17 hours at a temperature of about 26°C.
• Cop 1 As binding motifs of Cop 1 to MS-associated HLA-DR molecules are known (Fridkis-Hareli et al, 1999), polypeptides derived from Cop 1 having a defined sequence can readily be prepared and tested for binding to the peptide binding groove of the HLA-DR molecules as described in the Fridkis-Hareli et al (1999) publication. Examples of such peptides are those disclosed in WO 00/05249 and WO 00/05250, the entire contents of which are hereby incorporated herein by reference, and include the peptides of SEQ ID NOs. 1-32 hereinbelow.
• Such peptides and other similar peptides derived from Cop 1 would be expected to have similar activity as Cop 1.
• Such peptides, and other similar peptides are also considered to be within the definition of Cop 1 -related peptides or polypeptides and their use is considered to be part of the present invention.
• Cop 1 -related peptide or polypeptide is meant to encompass other synthetic amino acid copolymers such as the random four-amino acid copolymers described by Fridkis-Hareli et al., 2002 (as candidates for treatment of multiple sclerosis), namely copolymers (14-, 35- and 50- mers) containing the amino acids phenylalanine, glutamic acid, alanine and lysine (poly FEAK), or tyrosine, phenylalanine, alanine and lysine (poly YFAK), and any other similar copolymer to be discovered that can be considered a universal antigen similar to Cop 1.
• the present invention relates to the treatment of a neurodegenerative disease or disorder selected from Huntington's disease,
• T cells which have been activated preferably in the presence of Cop 1, or by a Cop 1 -related peptide or polypeptide.
• 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 for this purpose are described in USSN 09/756,301 and USSN 09/765,644, corresponding to WO 01/93893, each and all of them hereby incorporated by reference in its entirety as if fully disclosed herein.
• the dosage of Cop 1 to be administered will be determined by the physician according to the age of the patient and stage of the disease and may be chosen from a range of 1-80 mg, preferably 20 mg, although any other suitable dosage is encompassed by the invention.
• the treatment should be preferably carried out by administration of repeated doses at suitable time intervals, preferably every 1, 4 or 6 weeks, but any other suitable interval between the immunizations is envisaged by the invention according to the neurodegenerative disease to be treated, the age and condition of the patient.
• Pharmaceutical 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.
• the composition comprising Copolymer 1 or a Copolymer 1 -related peptide or polypeptide is administered in a regimen that confers protective autoimmunity and is sometimes referred to herein as a vaccine for neuroprotective vaccination.
• a vaccine if desired, may contain Copolymer 1 emulsified in an adjuvant suitable for human clinical use.
• the active agent may be administered without any adjuvant or it may be emulsified in an adjuvant suitable for human clinical use.
• the adjuvant is selected from aluminum hydroxide, aluminum hydroxide gel, and aluminum hydroxyphosphate, or any other adjuvant that is found to be suitable for human clinical use.
• the vaccine adjuvant is amorphous aluminum hydroxyphosphate having an acidic isoelectric point and an A1:P ratio of 1:1 (herein referred to as Alum-phos). It is clear that this is given by way of example only, and that the vaccine can be varied both with respect to the constituents and relative proportions of the constituents.
• 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.
• Cop 1 or a Cop 1 -related peptide or polypeptide may be used as a sole therapy or in combination with one or more drugs for the treatment of Alzheimer's, Huntington's or Parkinson's disease.
• the additional drug or drugs is/are administered at the same day of vaccination, and daily or at any other interval thereafter, according to the manufacturer's instructions, with no association to the vaccine regimen.
• HD R6/2 transgenic mice model was selected as the in vivo test system in the present invention. These mice overexpress exon 1 of the human Huntington's disease gene with an increased CAG repeat length that encodes huntingtin (Mangiarini et al., 1996). HD R6/2 transgenic mice show behavioral-motor deficits at as early as 5-6 weeks of age.
• mice Behavioral anomalies do not appear until 8 weeks, followed by the development of a progressive severe neurological phenotype with low weight, clasping, tremor and convulsions, and an early death at 10-14 weeks (Carter et al., 1999). Based on the glutamate toxicity model, an optimal neuroprotective effect in mice was established by a regimen of repeated injections of 75 ⁇ g Cop 1 at 4 weeks interval. The same regimen of treatment was found beneficial to HD R6/2 transgenic mice and reduced the rate of motor function deterioration, as shown by a significant preservation of the rotarod performance and prolonged life span of the animals.
• the designated point of injection was at a depth of 2 mm from the brain surface, 2.92 mm behind the bregma in the anteroposterior axis and 0.5 mm lateral to the midline.
• a window was drilled in the scalp above the designated coordinates in the right and left hemispheres.
• the neurotracer dye FluoroGold (5% solution in saline; Fluorochrome, Denver, CO) was then applied (1 ⁇ l, at a rate of 0.5 ⁇ l/min in each hemisphere) using a Hamilton syringe, and the skin over the wound was sutured. Retrograde uptake of the dye provides a marker of the living cells.
• mice were given a lethal dose of pentobarbitone (170 mg/kg). Their eyes were enucleated and the retinas were detached and prepared as flattened whole mounts in paraformaldehyde (4% in PBS). Labeled cells from 4-6 selected fields of identical size (0.5 mm 2 ) were counted. The selected fields were located at approximately the same distance from the optic disk (0.3 mm) to overcome the variation in RGC density as a function of distance from the optic disk. Fields were counted under the fluorescence microscope (magnification x900) by observers blinded to the treatment received by the mouse. The average number of RGCs per field in each retina was calculated. The effectiveness of the different vaccine formulations in protecting neurons is measured by counting the surviving RGCs.
• Glutamate toxicity an in vivo model for selection of dose and regimen of Cop 1 vaccination Glutamate is an amino acid normally present at low concentrations in the
• Glutamate toxicity is assessed by intraocular injection of glutamate into the eyes of C57B1/6J mice and then measuring the subsequent death of RGCs, the neurons that carry visual signals to the brain.
• Cop 1 dose determination To study the effect of the dose of Cop 1 vaccination on glutamate-induced RGC death, Cop 1 emulsified in complete Freund's adjuvant (CFA; 25, 75 or 225 ⁇ g Cop 1 in total volume of 100 ⁇ l) was injected subcutaneously at one site in the flank of C57BL/6J mice, and seven days later glutamate (200 nmol) was injected into the vitreal body of the mice. After seven days, the surviving RGCs were counted. The amount of RGCs death following glutamate toxicity without any prior immunization was taken as 100% of protectable cells. The results, presented in Fig. 1, show that effective vaccination was obtained by treatment with either 25 ⁇ g or 75 ⁇ g Cop 1. Latency of neuroprotective effect was determined by vaccination with 75 ⁇ g
• Cop 1 seven, fourteen and twenty-eight days prior to glutamate injection.
• the neuroprotective effect of a single injection of Cop 1 is optimal at 7 days post-immunization (reduction of RGC death by > 40%).
• the neuroprotective effect was reduced 14 and 28 days after vaccination.
• Cop 1 was originally developed as a therapy for multiple sclerosis (MS), an autoimmune disease characterized by unregulated T-cell activity against self- peptides of the CNS. Cop 1 is given to MS patients once a day at a dosage of 20 mg per patient by subcutaneous injections. We examined if daily injections of Cop 1 repeated for several days can maintain the neuroprotective effect on RGCs.
• MS multiple sclerosis
• mice were immunized with Cop 1 daily for two or three days (Cop 1, 25 ⁇ g/mouse and 75 ⁇ g/mouse).
• the results, presented in Fig. 3, show that daily injections of Cop 1 repeated for two days, give neuroprotection on RGCs and better protection is achieved with 75 ⁇ g Cop 1, while immunization during three consecutive days cause loss of the neuroprotective effect on RGCs.
• mice received three repeated 75 ⁇ g Cop 1 injections daily or at intervals of 1, 2, and 4 weeks.
• the results are shown in Figs. 4 and 5, respectively.
• the neuroprotective effect of the treatment is represented as % of a single injection of Cop 1 (75 ⁇ g/mouse) injected 7 days before glutamate toxicity was induced. This single injection was determined as positive control and was performed in each experiment.
• a 4-week interval between Cop 1 injections (75 ⁇ g/mouse) had the highest neuroprotective efficacy. It is striking that daily administration of Cop 1, the regimen used as therapy for multiple sclerosis, provides poor neuroprotection.
• Example 2 Correlation between the cellular immune response to Cop 1 vaccination and the neuroprotective effect Two ex vivo markers correlate with the efficacy - the T cell stimulation index and interferoh- ⁇ ( ⁇ FN- ⁇ ).
• the stimulation index indicates the extent to which
• Cop-1 -responsive T cells are present in the lymphocyte population.
• IFN- ⁇ secretion is characteristic of T cells of the "Thl" subtype. These markers thus provide a means of profiling the cellular immune response.
• the correlation between the neuroprotective effect and the cellular immune response to Cop 1 vaccination was thus determined by in vitro evaluation of T-cell proliferation and the level-profile of cytokine secretion.
• Cop 1 vaccination was examined by isolating splenic lymphocytes from mice immunized with different doses of Cop 1 (25, 75 and 225 ⁇ g/mouse), 7, 14, 21 and 28 days after immunization, and measuring the proliferative response of the splenocytes to Cop 1 by [ 3 H]thymidine incorporation, and the induction of cytokine production (IFN- ⁇ ) by ELISA assay.
• Uptake of labeled thymidine by splenocytes represents proliferation of specific T-cells to Cop 1, following Cop 1 vaccination.
• SI stimulation index
• Cop 1 the mean cpm of cells incubated in vitro with the antigen (Cop 1) divided by the mean cpm of cells incubated in vitro without the antigen (Cop 1).
• a positive response was defined as SI>2.
• a single injection of Cop 1 resulted in increased SI after 7 days for the three doses. After 14 days, only marginal proliferation of T-cells was seen for injection of 25 and 225 ⁇ g Cop 1 and less proliferation for injection of 75 ⁇ g Cop 1.
• the SI decreased after 21 and 28 days, meaning that the splenocytes proliferation in response to Cop 1 had abated.
• Cop-1 should not be expected to confer neuroprotection and is not the regimen of choice for Huntington's disease; vaccinations spaced at wider intervals are more likely to prove effective.
• Example 3 In vivo animal test system for Huntington's disease The beneficial effect of Cop 1 vaccination was examined for exertion of neuroprotective effects using the HD R6/2 transgenic mice test system.
• R6/2 transgenic mice over express the mutated human huntingtin gene that includes the insertion of multiple CAG repeats (Mangiarini et al., 1996). These mice show progressive behavioral-motor deficits starting as early as 5-6 weeks of age, and leading to premature death at 10-13 weeks. The symptoms include low body weight, clasping, tremor and convulsions (Carter et al., 1999). Two different doses of Cop 1 vaccination were tested, 75 ⁇ g Cop 1/mouse
• mice were placed on a rod rotating at 2 rpm: the time until the mouse falls off the rotating rod (best of three attempts, up to 180 sec for each trial), is used as the measure of animal motor-function.
• Fig. 8 Each mouse was tested twice weekly and the two scores averaged. The results are shown in Fig. 8. Each point on the graphs represents the average group score for each-week (SEM indicated by error bar). " The arrows on the x-axis represent the timing of Cop 1 (or PBS) injections. The results show that vaccination with Cop 1, either 75 ⁇ g/mouse or 150 ⁇ g/mouse, starting on day 45 of age, produced a significant improvement in motor performance during the follow- up period of 8 to 14 weeks. However, vaccination with 75 ⁇ g Cop 1/mouse starting on day 60 of age had no significant effect (data not shown).
• Control and Cop 1 vaccinated HD R6/2 transgenic mice were subjected to rotarod performance test on day 45 using four different speeds: 2. 5, 15 and 25 rpm.
• Fig. 9 shows that the improvement in rotarod performance following Cop 1 vaccination is dependent on the speed of rotation. Significant better performance of the twelve- week old vaccinated HD R6/2 mice compared to non-treated HD R6/2 control mice was most clearly apparent using 5 rpm rotarod speed.
• the effect of Cop 1 vaccination on weight loss of HD R6/2 transgenic mice was tested on the three groups. Mice were weighed twice a week at the same time during the day.
• Example 1 shows that Cop 1 vaccination attenuates neuronal cell death induced by exposure to elevated levels of the excitotoxic neurotransmitter glutamate, and that the neuroprotective effect is dependent upon activation and proliferation of T-cells specific to Cop 1 that secrete LNF- ⁇ (Thl).
• the neuroprotective effect is short-lived, unless maintained by a boosting regime - it is build up by 7 days post immunization, and is then reduced due to activation of regulatory cells which terminate the response.
• the Cop 1 dose found to be the most active in the animal models was 75 ⁇ g Cop 1/mouse, that translate to a human adult dose of 20 mg on a mg/m basis.
• neuroprotective Cop 1 vaccination should be administered in doses spaced at least one month apart, preferably 4-6 weeks apart, more preferably every 5 or 6 weeks.
• Example 4 Human clinical trials for Huntington's disease
• the primary objective of the human study is to evaluate the tolerability, safety and immunological response of the serial administration of 20 mg or 2x20 mg dose of Cop 1 (Copaxone® or another Cop 1 formulation) versus placebo, in patients suffering from Huntington's disease.
• the secondary objective of the study is to evaluate the neurological course of patients with HD disease following administration of Cop 1, by measuring the following neurological clinical parameters: Unified Huntington's Disease Rating Scale (UHDRS) and Total Motor Scale (TMS).
• UHDRS Unified Huntington's Disease Rating Scale
• TMS Total Motor Scale
• Eligible patients female and male, 18-70 years old, symptomatic patients with clinically diagnosed HD and a confirmatory family history of HD will receive one administration of placebo (40 mg mannitol/i ⁇ jection) and three administrations of Copaxone® (20 mg/ml subcutaneously or 2x20 mg/ml subcutaneously, 1 in each arm) at 6 weeks intervals between administrations.
• Blood samples for immunological profile analysis will be taken ⁇ af screening and prior to first injection.
• Each administration of Copolymer 1 will be followed by a series of blood sampling to determine the immunological profile on days 7, 14, 28 and just prior to next injection and termination.
• UHDRS is a research tool that has been developed by the Huntington Study Group (HSG).
• the purpose of the scale is to allow the researchers to grade the symptoms of HD in a way that allows them to make accurate comparisons between individual patients, and to better chart the course of the disease in patients.
• the scale is divided into a number of different subscales, including the Total Motor Score 4 (TMS-4).
• TMS-4 Total Motor Score 4
• a primary end-point is the change over a period of time, e.g. one-year period, in the TMS-4 subscale of the UHDRS, the standard rating scale for trials in HD.
• the pre-determined and end-points of the trial are compared for the patients on Copaxone and the one may assume the possibility that the drug can be said to have had some kind of impact on Huntington's disease.
• Neurodegenerative diseases differ in etiology but are propagated similarly.
• neuronal loss caused by intraocular injection of aggregated ⁇ -amyloid was significantly greater in immunodeficient mice than in normal mice.
• the neurodegeneration was attenuated or augmented by elimination or addition, respectively, of naturally occurring CD4 + CD25 + regulatory T cells (Treg).
• Treg CD4 + CD25 + regulatory T cells
• Vaccination with retina-derived antigens or with Copolymer- 1, but not with ⁇ -amyloid reduced the ocular neuronal loss.
• microglia encountering activated T cells overcame the cytotoxicity of aggregated ⁇ - amyloid.
• the destructive effect could be attenuated either by elimination of naturally occurring CD4 + CD25 + regulatory T cells (Treg) or by evoking an immune response directed against antigens derived from the tissue's own constitutively expressed proteins (rather than against the threatening compound itself).
• the therapeutic effect could be reproduced by passive transfer of T cells directed against the same self-antigens.
• mice were handled according to the Association for Research in Vision and Ophthalmology (ARVO) resolution on the use of animals in research.
• the mice were housed in a light- and temperature-controlled room and matched for age in each experiment. Mice were anesthetized by i.p.
• ketamine 80 mg/kg; Ketaset, Fort Dodge, IA
• xylazine 16 mg/kg; Vitamed, Ramat-Gan, Israel.
• pentobarbitone 170 mg/kg; C.T.S., Kiryat Malachi, Israel.
• IRBP Bovine interphotoreceptor retinoid-binding protein
• Bovine S-antigen (arrestin) was prepared from the Con A column flowthrough by the method of Buczylko and Palczewski (Palczewski et al., 1994) as modified by Puig et al. (1995).
• Whole retinal homogenate (WRH) was prepared from syngeneic -retinas ' homogenized ⁇ in BS ⁇ Ovalbumin (OVA), Con A, and ⁇ -amyloid peptide 1-40 (A ⁇ 1-- o) ) were purchased from Sigma-Aldrich, St. Louis, MO.
• a ⁇ (1 _ 4 o ) peptide was dissolved in endotoxin-free water, and ⁇ -amyloid aggregates were formed by incubation of A ⁇ (1 _ 40) , as described (Ishii et al., 2000).
• Glatiramer acetate (Copaxone®; Cop-1) as purchased from Teva Pharmaceuticals Ltd. (Petach Tikva, Israel). (ix) Immunization.
• mice were immunized with IRBP (50 ⁇ g), S- antigen (50 ⁇ g), A ⁇ _ 4 o (50 ⁇ g), WRH (600 ⁇ g), or Cop-1 (75 ⁇ g), each emulsified in an equal volume of CFA (Difco, Detroit, MI) containing Mycobacterium tuberculosis (5 mg/ml; Difco). The emulsion (total volume 0.15 ml) was injected s.c. at one site in the flank. Control mice were injected with PBS in CFA or with PBS only. (x) Labeling of RGCs in mice. Labeling was carried out as described in Materials and Methods, Section I (v).
• mice were given a lethal dose of pentobarbitone (170 mg/kg). Their eyes were enucleated and the retinas were detached, prepared as flattened whole mounts in 4% paraformaldehyde in PBS, and labeled cells from four to six fields of identical size (0.076 mm ) were counted (Schori et al., 2001b). The average number of RGCs per field was calculated for each retina. The number of RGCs in the contralateral (uninjured) eye was also counted, and served as an internal control. (xiii) In-situ detection of cell death by terminal deoxynucleotidyl transf erase DNA (TUNEL).
• TUNEL terminal deoxynucleotidyl transf erase DNA
• mice were killed 48 h after intraocular glutamate injection and their eyes were removed and processed for cryosectioning. Frozen sections were fixed in 3.7% formalin for 10 min at room temperature and washed twice with PBS. The sections were transferred to 100%) methanol for 15 min at -20°C, washed twice for 5 min in ethanol 100%, 95% and 70% successively, and then incubated for 10 min with PBS. For permeabilization, proteases were digested with proteinase K for 20 min at room temperature. The open ends of the DNA fragments were labeled using an in-situ apoptosis detection kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
• an in-situ apoptosis detection kit R&D Systems, Minneapolis, MN
• splenocytes were subjected to AutoMacs (Miltenyi Biotec, Bergisch Gladbach, Germany) with the "deplete sensitive" program. Recovered populations were analyzed by FACSsort (Becton Dickinson, Franklin Lakes, NJ) (Kipnis et al., 2002a).
• FACSsort Becton Dickinson, Franklin Lakes, NJ
• spleens were harvested and mashed. T cells were purified (enriched by negative selection) on T cell columns (R&D Systems).
• the enriched T cells were incubated with anti-CD8 microbeads (Miltenyi Biotec), and negatively selected CD4 + T cells were incubated with PE- conjugated anti-CD25 antibodies (30 ⁇ g/10 8 cells) in PBS/2% fetal calf serum. They were then washed and incubated with anti-PE microbeads (Miltenyi Biotec) and subjected to magnetic separation with AutoMACS. The retained cells were eluted from the column as purified CD4 + CD25 + cells (Treg).
• effector T cells consisting of CD4 + CD25 ⁇ T cells
• Teff effector T cells
• mrIL-2 mouse recombinant IL-2
• the washed lymphocytes (2xl0 6 cells/ml) were activated with the relevant antigens (IRBPi- 20 or aggregated A ⁇ _ 40 , each at 10 ⁇ g/ml) in stimulation medium containing RPMI supplemented with L-glutamine (2 mM), 2-mercaptoethanol (5xl0 -5 M), penicillin (100 IU/ml), streptomycin (100 IU/ml), and autologous mouse serum 1% (vol/vol).
• Microglial cultures Microglia were purified from the cerebral cortices of newborn (day 0) BALB/c/OLA mice, as described (Butovsky et al., 2001).
• IFN- ⁇ (20 ng/ml; R&D Systems), ⁇ -amyloid (Sigma- Aldrich; aggregated A ⁇ 1 _ 40 25 ⁇ M), or activated T cells (1.5 l0 5 per well) were added to the culture medium for 12 h. After treatment, microglia were washed three times with PBS and prepared for application on hippocampal slices.
• (xviii) In-vitro model of hippocampal slices. BALB/c/OLA mice, aged 8-10 days, were decapitated and their brains were rapidly removed under sterile conditions and placed in ice-cold preparation medium consisting of minimum essential medium (MEM; Gibco, Carlsbad, CA) with 1% L-glutamine (Gibco) at pH 7.35.
• MEM minimum essential medium
• Gibco Gibco, Carlsbad, CA
• the frontal pole was removed and the brains were cut into 350- ⁇ m horizontal slices on a vibratome (Pelco, Redding, Germany), beginning at the ventral surface. Slices containing the hippocampi were cultured on Falcon cell culture inserts, pore size 0.4 ⁇ m (Becton Dickinson), in 6-well plates.
• the cultivation medium contained 50% MEM, 25% Hanks balanced salt solution (Gibco), 25% normal horse serum, 2% glutamine, 10 ⁇ g/ml insulin- transferrin-sodium selenite supplement (Boehringer Mannheim, Mannheim, Germany), 2.64 mg/ml glucose (Braun, Melsungen, Germany), 0.1 mg/ml streptomycin, 100 U/ml penicillin, and 0.8 ⁇ g/ml vitamin C (all from Sigma- Aldrich).
• the organotypic hippocampal slice cultures (OHSCs) were incubated at 35°C in a humidified atmosphere with 5% C0 2 for 24 h, during which time the slices were either left untreated or treated with 4xl0 5 microglia per well.
• Tissue loss was assessed by addition of propidium iodide (PI) (5 ⁇ g/ml; Sigma) to the medium for 30 min at the end of the incubation period. Excess PI was then washed away with cultivation medium, and the slices were prepared for microscopy and visualized. To quantify neural cell death in the OHSCs, PI intensity in each slice was assessed by use of Image-Pro software (Media Cybernetics, Carlsbad, CA). PI staining intensity for a specific treatment was compared to that of the untreated control, using a two-tailed Student's t-test.
• PI propidium iodide
• EXAMPLE 5 Retinal proteins can evoke a protective T cell-based response to glutamate intoxication.
• mice of different genetic backgrounds differ in their ability to resist injurious conditions (Schori et al., 2001b; Kipnis et al., 2001; Schori et al., 2002). The differences were attributed, at least in part, to strain- related variations in the ability to manifest a T cell-dependent protective response (Kipnis et al., 2001).
• Glutamate 400 nmol was injected into the right eyes of C57BL/6J mice, and 48 h later we examined retinal cryosections subjected to terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). Apoptotic cell death was observed in the RGC layer (Fig.
• results in the figure are presented as the loss of neurons expressed as a percentage of the number of RGCs in strain-matched normal retinas.
• the retinal self-proteins IRBP and S-antigen both of which are capable of causing uveitis in susceptible mice (Caspi et al., 1990a; Caspi et al., 1990b) but were used here for purposes of protection, are not intended for development as a therapeutic vaccination; this is purely an experimental paradigm, used here for proof of concept, that supports our previous contention that the same T cells can be both protective and destructive, and that
• EXAMPLE 6 Ability to withstand the toxicity of ⁇ -amyloid is T cell- dependent. Having shown that the physiologically relevant antigen for protection against neurotoxicity is not the toxic compound itself (glutamate) but a self-antigen that resides in the site of damage, we then examined whether the same vaccination might be beneficial against different toxic self-compounds provided that the toxicity is restricted to the same site.
• Fig. 11 A shows the ⁇ -amyloid-induced neuronal loss expressed as a percentage of the average number of RGCs in normal wild-type retina.
• 11B and 11C show representative photomicrographs of whole-mounted retinas excised from mice after intraocular injection of PBS and aggregated A ⁇ 1 _ 4 o, respectively.
• Fig. 11D shows neuronal loss as a percentage of the number of neurons in normal retinas.
• Figs. HE and HF show representative micrographs of retinas from wild-type and nude mice, respectively, after intraocular injection of aggregated A ⁇ _ 40 .
• mice that received OVA-activated T cells did not differ significantly from that in na ⁇ ve mice injected with glutamate (1652.6 ⁇ 56 and 1535.6 ⁇ 74, respectively; not shown). Results are expressed as the percentage increase in neuronal survival relative to survival in control mice (Fig. 13A).
• T cells specific to IRBP or to non-aggregated A ⁇ o obtained from immunized mice were prepared and activated ex vivo, and then passively transferred into C57BL/6J mice exposed to toxicity of aggregated A ⁇ 1 _ 40 .
• T cells directed to the aggregated A ⁇ _ 40 itself to confer protection is in line with observations in our laboratory that microglia, upon encountering aggregated A ⁇ 1 _ 40 , fail to express MHC class II (MHC-II). Consequently, such microglia fail to present ⁇ -amyloid to the T cells, with the result that even if ⁇ - amyloid-specific T cells home to the CNS they will not be locally activated (Butovsky- et-al., unpublished observations). -To- fight - ⁇ -amyloid toxicity, a * more appropriate choice would therefore be antigens that reside in the site and can be presented to homing T cells.
• MHC-II MHC class II
• EXAMPLE 7 Naturally occurring regulatory CD4 + CD25 + T cells restrict the body's ability to withstand ⁇ -amyloid toxicity in the retina.
• nude mice replenished with the same number of splenocytes, which were obtained from whole spleens of wild-type mice and therefore contained both Treg and effector T cells (Teff).
• Treg and effector T cells Teff
• the recipient mice were injected with a toxic dose of aggregated A ⁇ o, and surviving RGCs were counted 2 weeks later.
• T cells prevent microglia from developing an inflammatory cytotoxic phenotype.
• organotypic hippocampal slice cultures (OHSCs) our group showed that after rat microglia are pretreated with aggregated A ⁇ i_ 4 o they become cytotoxic to neural tissue and their ability to express MHC-II is suppressed (Butovsky et al., unpublished observations). We therefore carried out an in-vitro experiment to determine whether murine microglia exposed to aggregated A ⁇ !
• Fig. 16A Representative micrographs of variously treated OHSCs and untreated controls are shown in Fig. 16B (1-4).
• EXAMPLE 9 In vivo animal test system for Alzheimer's disease The beneficial effect of Cop 1 vaccination can be examined for exertion of neuroprotective effects using the transgenic mice test system. There are no spontaneous animal mutations with sufficient similarities to AD to be useful as experimental models. Various transgenic animal models for testing potential treatments for Alzheimer's disease are known.
• Models are known that are based on the ability to control expression of one or more of the three major forms of the human ⁇ -amyloid precursor protein (APP), APP695, APP751, and APP770, or subfragments thereof, as well as various point mutations based on naturally occurring mutations, such as the familial Alzheimer's disease (FAD) mutations at amino acid 717, and predicted mutations in the APP gene, as described in US 6,717,031 and Johnson- Wood et al. (1997).
• a suitable model is, for example, the transgenic hAPP770/FAD717 mouse model.
• a suitable formulation comprising Cop 1 is administered to the offspring of the transgenic mice or cells derived therefrom, and detecting or measuring an Alzheimer's disease marker in the transgenic mouse, or in cells derived from the transgenic mouse.
• the Alzheimer's disease marker is a behavior and the observed difference is a change in the behavior observed in the transgenic mouse to which the compound has been administered.
• This behavior may be behavior using working memory, behavior using reference memory, locomotor activity, emotional reactivity to a novel environment or to novel objects, and object recognition.
• the behavior of the Cop 1 -treated Alzheimer transgenic mouse model can be tested using the Morris water maze, as described (Postina et al, 2004). It is expected that the treated animals will exhibit an improvement in their behavior.
• the present results support the contention that the ⁇ - amyloid peptide in its aggregated form (found in senile plaques) has a toxic effect in the CNS, not only because it is directly toxic to neurons (Jen et al., 1998; Carter and Lippa, 2001) but also because it apparently induces microglia to adopt a cytotoxic phenotype.
• the failure of ⁇ -amyloid vaccination to protect against ⁇ - amyloid-induced stress in the eye is in line with observations from our laboratory that cell-surface MHC-II expression is impaired in microglia encountering aggregated ⁇ -amyloid (Butovsky et al., unpublished observations).
• T cells that can be locally activated can trasform the adjacent microglial population from an enemy into a friend.
• strain-related differences in the ability of mice to withstand the toxicity of aggregated A ⁇ _ 4 o- The present results are also in line with our contention that naturally occurring CD4 + CD25 + regulatory T cells constitutively control the ability to withstand neurodegenerative conditions.
• Treg In rats or mice devoid of Treg, the susceptibility to autoimmune disease development is increased, despite the benefit in terms of protection against injurious conditions Therefore, one of the aims of neuroprotective therapy is to weaken Treg.
• pharmacological intervention with a compound that mimics the physiological weakening (but not blocking) of Treg might provide a way to boost the T cell-based self-defense. It was shown by our group that the same autoimmune T cells can be both supportive and destructive (Kipnis et al., 2002b). Accordingly, in animals that are inherently susceptible to autoimmune disease the protocol used for eliciting the T cell response critically affects the outcome.
• autoimmune response whose benefit is offset by its persistence or intensity
• autoimmune response to CNS might not be expressed early enough to be accommodated within the therapeutic window, or it might fail to meet other requirements, such as timely shut-off (Shaked et al., 2004a).
• susceptible strains devoid of immune cells (SCID) and thus- lacking a -T cell-based regulatory mechanism passive transfer of encephalitogenic T cells causes EAE, but is not sufficient for conferring any neuroprotection (Kipnis et al., 2002b).
• CD4 + CD25 + regulatory T cells are passively transferred into SCID mice (Kipnis et al., 2004b), they can have a protective effect similar to that of encephalitogenic T cells passively transferred into the wild type (Hauben et al., 2000a, 2000b; Moalem et al., 1999a).
• encephalitogenic T cells passively transferred into the wild type (Hauben et al., 2000a, 2000b; Moalem et al., 1999a).
• the likelihood that the spontaneously evoked response to a CNS injury will be destructive is very low; on the other hand, it might be too weak to be beneficial and need boosting.
• whether or not autoimmunity will be beneficial under severe conditions in susceptible strain is determined by both regulation and context.
• the antigen selected for vaccination should not be the disease-specific protein such as the aggregated A ⁇ i_ 4 o in Alzheimer's disease, Lewy bodies in Parkinson's disease, or prion protein (PrP) in prion disease (Dodart et al., 2003; White et al., 2003), but a peptide derived from an immunodominant self-protein that resides at the site of CNS damage, a cryptic self-peptide, or an altered self-peptide, but preferably a non-self peptide that cross-reacts weakly with self such as Copolymer 1 and Copolymer 1-related peptides and polypeptides.
• the disease-specific protein such as the aggregated A ⁇ i_ 4 o in Alzheimer's disease, Lewy bodies in Parkinson's disease, or prion protein (PrP) in prion disease (Dodart et al., 2003; White et al., 2003)
• PrP prion protein
• mice protected mice from " the neuro ' de ' geherative effects " of existing aggregated A ⁇ _ 40 .
• the proposed strategy does not argue against the possible benefit of antibodies specific to A ⁇ - amyloid (Dodart et al., 2003; Furlan et al., 2003; Mohajeri, et al., 2002) as long as the peptide used for vaccination is not encephalitogenic.
• the two approaches rather than being mutually antagonistic, might complement one another.
• PD Parkinson's disease
• MPTP l-methyl-4-phenyl-l,2,3,6-tetrahydro ⁇ yridine
• the beneficial effect of Cop 1 immunization is examined for exertion of neuroprotective effects using the MPTP mice test system or another suitable model for Parkinson's disease.
• Neuroprotective therapy for PD with Cop 1 can attenuate the neurodegenerative effects and the rate of disease progression.
• EXAMPLE 10 Effect of Cop 1 in the Parkinson MPTP rodent model
• Male C57BL/6J mice are injected i.p. with 20 mg/kg MPTP.HCl in PBS, four times, at 2-h intervals.
• Cop 1 (75 ⁇ g or 150 ⁇ g Cop 1/mouse in PBS) or PBS (control group) is administered to the MPTP-treated animals 12 h after the last MPTP administration.
• the motor dysfunction in PD is due to a profound reduction in striatal dopamine content caused by the loss of dopaminergic nerve fibers in the striatum.
• Motor performance of MPTP-treated mice immunized with Cop 1 or injected with PBS is measured on a Rotarod, as previously described (Hunot et al., 2004).
• the rotarod performance test assesses the capacity of the mice to stay on a rotating rod. It can be expected that immunization with Cop 1 will display improvement of motor functions on the Rotarod (increased Rotarod time) compared to control mice.
• Other PD parameters related to neuroprotection can be carried out one week or more after immunization such as stereological quantification of dopamine neuron number and optical density measurement of dopamine fiber loss using immunostaining for dopamine transporter (DAT) and tyrosine hydroxylase (TH).
• DAT dopamine transporter
• TH tyrosine hydroxylase
• Kipnis J Yoles E, Mizrahi T, Ben-Nun A, Schwartz M. (2002b) Myelin specific Thl cells are necessary for post-traumatic protective autoimmunity. J. Neuroimmunol. 130:78-85. Kipnis J, Nevo U, Panikashvili D, Alexandrovich A, Yoles E, Akselrod S, Shohami E, Schwartz M. (2003) Therapeutic vaccination for closed head injury. J. Neurotrauma 20(6):559-569.
• 2004a Low-dose gamma-irradiation promotes survival of injured neurons in the central nervous system via homeostasis-driven proliferation of T cells. Eur. J. Neurosci. 19: 1191-1198.
• 2004b Dual effect of CD4+CD25+ regulatory T cells in neurodegeneration: Pro- and anti-inflammatory cytokines determine microglial activity. Proc Natl Acad Sci U S A. 101 Suppl 2: 14663-14669.
• Moalem G Leibowitz-Amit R, Yoles E, Mor F, Cohen IR, Schwartz M (1999a) Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat Med 5:49-55. Moalem G, Monsonego A, Shani Y, Cohen IR, Schwartz M. (1999b) Differential T cell response in central and peripheral nerve injury: connection with immune privilege. Faseb J. 13: 1207-1217. Moalem G, Gdalyahu A, Shani Y, Otten U, Lazarovici P, Cohen IR, Schwartz M. (2000) Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity. J. Autoimmun.
• Pepperberg DR Okajima TL, Ripps H, Chader GJ, Wiggert B. (1991) Functional properties of interphotoreceptor retinoid-binding protein. Photochem. Photobiol. 54: 1057-1060. Perlmutter DH, (2002) The cellular response to aggregated proteins associated with human disease. J. Clin. Invest. 110: 1219-1220.
• Puig J, Arendt A Tomson FL, Abdulaeva G, Miller R, Hargrave PA, McDowell JH. (1995) Synthetic phosphopeptide from rhodopsin sequence induces retinal arrestin binding to photoactivated unphosphorylated rhodopsin. FEBS Lett. 362:185-188.
• Hasenbank R Bates GP, Davies SW, Lehrach H, Wanker EE. (1997) Huntingtin- encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell. 90, 549 -558.
• Schori H Yoles E, Schwartz M (2001a) T-cell-based immunity counteracts the potential toxicity of glutamate in the central nervous system. J Neuroimmunol 119:199-204.

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