WO1999034827A1 - Activated t-cells and their uses - Google Patents

Activated t-cells and their uses Download PDF

Info

Publication number
WO1999034827A1
WO1999034827A1 PCT/US1998/014715 US9814715W WO9934827A1 WO 1999034827 A1 WO1999034827 A1 WO 1999034827A1 US 9814715 W US9814715 W US 9814715W WO 9934827 A1 WO9934827 A1 WO 9934827A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
activated
disease
injury
antigen
Prior art date
Application number
PCT/US1998/014715
Other languages
French (fr)
Inventor
Michal Eisenbach-Schwartz
Irun R. Cohen
David L. Hirschberg
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
Application filed by Yeda Research And Development Co. Ltd. filed Critical Yeda Research And Development Co. Ltd.
Priority to JP2000527274A priority Critical patent/JP2002500199A/en
Priority to IL13415998A priority patent/IL134159A0/en
Priority to PCT/US1998/014715 priority patent/WO1999034827A1/en
Priority to CA002296999A priority patent/CA2296999A1/en
Priority to EP98936862A priority patent/EP1039929A1/en
Priority to AU85717/98A priority patent/AU755872B2/en
Priority to US09/218,277 priority patent/US20030108528A1/en
Priority to PCT/US1999/010953 priority patent/WO1999060021A2/en
Priority to AU40017/99A priority patent/AU768763B2/en
Priority to IL13952599A priority patent/IL139525A0/en
Priority to CA002328612A priority patent/CA2328612A1/en
Priority to EP99923190A priority patent/EP1080110A2/en
Priority to JP2000549639A priority patent/JP2003532618A/en
Publication of WO1999034827A1 publication Critical patent/WO1999034827A1/en
Priority to US09/893,348 priority patent/US20020072493A1/en
Priority to US10/810,653 priority patent/US7560102B2/en
Priority to US11/563,630 priority patent/US20080279869A1/en

Links

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
    • 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
    • A61K39/46—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
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • 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
    • A61K39/46433—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
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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 treatment or diagnosis of injury of the central nervous system (CNS) .
  • activated T-cells are used to deliver (a) a diagnostic substance for detecting a site of injury or disease or (b) a therapeutic substance for ameliorating an effect of a disease or injury, such as, for e.g., promoting axonal regeneration or preventing or inhibiting degeneration caused by injury or disease.
  • the activated T-cells that are used for delivering a substance do not recognize a nervous system (NS) antigen. More preferably, the substance- delivering activated T-cells recognize a non-self antigen (e.g., ovalbumin) .
  • compositions comprising antiself T-cells that recognize an antigen present in a greater concentration in the nervous system (NS) compared to the other organs or circulation are used to prevent or inhibit degeneration of nerves within the CNS.
  • the antiself T-cells of the present invention are not genetically-engineered.
  • pharmaceutical compositions comprising an antigen (or derivative thereof) present in a greater concentration in the NS compared to other organs or the circulation are used to prevent or inhibit degeneration of nerves within the CNS.
  • the activated T-cell compositions of the present invention may be administered alone or in combination with NS-specific antiself T-cells or NS-specific antigen or in combination with NS-specific antiself T-cells and NS-specific antigen.
  • Damage to the CNS 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) and ischemias.
  • 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) and ischemias.
  • PNS peripheral nervous system
  • an invasion of blood derived monocytes as well as activation of microglia within the PNS occurs (Stoll, et al., 1989, Neurosci . , 9_:2327-35; Perry and Gordon, 1991, Int . 5 Rev. Cytol , 125:203-44; Perry and Gordon, 1988, Trends Neurosci .
  • a stimulant e . g . a nerve segment
  • subsequently administered into the CNS of a mammal at or near the site of injury have shown regeneration of the impaired motor or sensory function (PCT Publication WO 97/09885 and Spiegler et al . , 1996, FASEB J. 10:1296).
  • Popovitch et al has shown that CNS trauma such as spinal injury triggers a systemic response to self epitopes 0 such as yelin basic proteins (MBP) (Popovitch et al . , 1996, J. Neurosci. Res., . 4_5:349).
  • Activated T-cells that recognize a self antigen as well as activated T-cells that recognize a non-self antigen have been shown to enter the CNS parenchyma. Only T-cells capable of recognizing a CNS antigen appear to 5 persist in the nervous tissue (Hickey et al . , 1991, J.
  • activated T-cells that recognize a self antigen apparently persist in nervous tissue
  • use of activated T-cells that recognize a non-self antigen for 0 administration to an individual have advantages such as no risk of induction of autoimmune disease.
  • use of non-self activated T-cells eliminates the necessity of activating autologous or syngeneric T-cells; therefore, non- self T-cells may be activated and stored for use in any 5 individual.
  • T-cells reactive to antigens of CNS white matter can induce the paralytic disease experimental autoimmune encephalomyelitis (EAE) within several days of their inoculation into naive recipient 0 rats (Ben Nun et al . , 1981, Eur. J . Immunol . 11, 195-9).
  • EAE experimental autoimmune encephalomyelitis
  • anti-MBP T cell clones are present in the immune systems of healthy subjects (Burns et al . , 1983, Cell . Immunol . 81, 435-40; Schluesener and Wekerle, 1985, J . Immunol . 135, 3128-33). However, little is known about the possible physiological functions of antiself T-cells.
  • the present invention is directed to methods and compositions for the treatment or diagnosis of injury of the central nervous system (CNS) .
  • the present invention provides methods for delivering a therapeutic or detectable substance to a site of injury or disease of the CNS, comprising administering an effective amount of activated T-cells that contain or express a therapeutic or detectable substance to a mammal wherein the amount is effective to detect, diagnose, or monitor a site of injury or disease in the CNS or is effective to ameliorate the effects of an injury or disease of the CNS.
  • the activated T-cells used for delivery of a substance preferably do not recognize a nervous system specific antigen (NS-specific antigen) ; more preferably the activated T-cells recognize a non-self antigen.
  • NS-specific antigen nervous system specific antigen
  • Activated T-cell includes (i) T-cells that have been activated by (a) exposure to a cognate antigen or derivative thereof or (b) exposure to an appropriate mitogen such as a lectin (e.g. concanavalin A (Con A) or phytohemagglutinin (PHA) ) , 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 present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of non-recombinant, NS-specific antiself T-cells and methods of use of such compositions for prevention or inhibition of CNS nerve degeneration in which the amount is effective to ameliorate the effects of an injury or disease of the CNS.
  • NS-specific antiself T-cell refers to an activated T-cell which recognizes a self antigen present in a greater concentration in the nervous system (NS) compared to other organs or the circulation or an antigen that shares an antigenic determinant with an antigen present in a greater concentration in the NS compared to other organs or the circulation.
  • the present invention also provides pharmaceutical compositions and methods of use comprising a therapeutically effective amount of NS-specific antigens (or derivatives thereof) for prevention or inhibition of CNS degeneration in which the amount is effective to activate T-cells in vivo or in vitro wherein the activated T-cells ameliorate the effects of an injury or disease of the CNS.
  • NS-specific antigen refers to an antigen present in the NS or an antigen that shares an antigenic determinant with an antigen present in a greater concentration in the NS compared to other organs or the circulation.
  • therapy for amelioration of effects of injury or disease comprising administration of activated T-cells may optionally be in combination with NS-specific antiself T-cells or a NS- specific antigen (or derivative thereof) or NS-specific antiself T-cells and a NS-specific antigen.
  • FIG. 1 presents low-power epifluorescence micrographs of optic nerve after a controlled crush injury in rats treated with T-cell clones primed with MBP. See text, Section 6, for experimental details.
  • Fig. 2 presents high-power micrographs of the site of optic nerve injury shown in Fig. 1, showing the large concentration of injected cells localized in the site of injury.
  • Fig. 3 presents a serial section through non-injured optic nerve.
  • Fig. 4 is a graphical representation of the number of T- cells at the site of injury of two different T-cell clones primed with antigen MBP or ovalbumin (OVA) (T mp or T 0VA , respectively) after injury, at various time intervals following injury. and T 0VA cells were injected into animals at the time of optic nerve crush, ipsolateral and contralateral nerves were then removed and prepared for microscopy at days 3, 7, 14 and 21. The figure shows that T- cells accumulated at the site of injury, independent of the antigen with which they were primed (each result is an average of 5 different experiments; the bar shows the standard deviation) . See text, Section 6, for experimental details.
  • Fig. 5 shows accumulation of T-cells primed with MBP or OVA (T MBP and T 0VA , respectively) measured immunochemically using antibodies to T-cell receptors.
  • a comparison of the number of accumulated cells in injured optic nerve (ON) and in non-injured optic nerve is illustrated. See text, Section 6, for experimental details.
  • Fig. 6 shows accumulation of T-cells in injured and non- injured optic nerve after various treatment protocols.
  • FIG. 7 shows T-cell accumulation in injured optic nerve 1 week after injury. See text, Section 6, for experimental details.
  • Anti-MBP or anti-OVA or anti-hsp60 T cell lines were raised, maintained, and activated by incubation with MBP from the spinal cords of guinea pigs, or with OVA (Sigma) , or with the 51-70 peptide of MBP, respectively, in the presence of irradiated (2000 rad) syngeneic thymus cells.
  • Activated T-cells (lxlO 7 cells) of the anti-MBP or anti-OVA lines or PBS were injected intraperitoneally into adult Lewis rats immediately after unilateral crush injury of the optic nerve.
  • Fig. 8 illustrates inhibition of secondary degeneration after partial optic nerve crush injury in adult rats. See text, Section 7, for experimental details.
  • Surviving optic nerve fibers were monitored by retrograde labeling of retinal ganglion cells immediately after injury and 2 weeks later. Five days after dye application retinas were excised, and labeled retinal ganglion cells (RGCs) were counted under the fluorescence microscope. Counting was performed in 5 randomly selected fields in each retina (all located at approximately the same distance from the optic disc) .
  • the number of RGCs in each group of injured nerves in rats injected with PBS only or injected with anti-MBP, anti-OVA, or anti-hsp60 T-cells was expressed as percentages of the total numbers of spared neurons following the primary injury (42% of the axons remained viable after the primary injury) .
  • Fig. 9 presents photomicrographs showing retrograde labeled retinas of injured optic nerves of rats injected with (A) PBS, (B) anti-hsp60 T-cells, or (C) anti-MBP T-cells. See text. Section 7, for experimental details.
  • Fig. 10 shows number of surviving RGCs of injured optic nerves of rats injected with anti-MBP T-cells (T ⁇ ) , T-cells generated against a peptide comprising amino acids 51-70 of MBP (T p 51-70), or PBS. See text, Section 7, for experimental details.
  • Fig. 11 presents clinical course of rats injected with anti-MBP T-cells. Results were evaluated according to the neurological paralysis scale (EAE score) . Rats were injected i.p. with lxlO 7 activated anti-MBP T-cells either immediately after optic nerve crush injury (- ⁇ -) or without optic nerve crush injury (—o—) . Each group contained 5 to 9 rats. Data points represents means ⁇ SEM. See text, Section 7, for experimental details.
  • Fig. 12 shows the survival of neurons in uninjured nerves in rats injected i.p. with lxlO 7 activated anti-MBP T- cells or PBS. See text. Section 7, for experimental details.
  • Fig. 13 illustrates inhibition of secondary degeneration after optic nerve crush injury in adult rats. See text, Section 8, for experimental details. Rats were injected intradermally into footpads with a 21-mer peptide based on amino acid residues 35-55 (MOG p35-55) of myelin/oligodendrocyte glycoprotein (chemically synthesized at the Weiz ann Institute, Israel) (50 ⁇ g/animal) or PBS ten days prior to optic nerve crush injury or MOG p35-55 in the absence of crush injury.
  • MOG p35-55 myelin/oligodendrocyte glycoprotein
  • MOG p35-55 was administered with Incomplete Freund's Adjuvant.
  • Surviving optic nerve fiber 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.
  • Fig. 14 illustrates inhibition of secondary degeneration after optic nerve crush injury in adult rats. See text, Section 9, for experimental details.
  • MBP Sigma, Israel
  • MBP (1 mg in 0.5 ml saline) was administered to adult rats by gavage using a blunt needle
  • MBP was administered 5 times, i.e., every third day beginning 2 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
  • compositions comprising activated T-cells are used for delivery of (a) a diagnostic substance or (b) a therapeutic substance to a site of injury or disease of the CNS in a mammal.
  • T-cells of the present invention are T-cells which recognize an antigen not normally present or present in small quantities in the circulation.
  • antigens include but are not limited to NS-specific antigens, cryptic antigens or "non-self" antigens (i.e., antigens not normally present in an individual) .
  • Non-self antigens may be, without limitation, viral, bacterial, etc., including tissue-specific antigens from a different species or individual.
  • T-cells are activated in vitro by exposure to an antigen and administered to a mammal.
  • the present invention provides methods for delivering a therapeutic or detectable substance to a site of injury or disease of the CNS, comprising administering an effective amount of activated T-cells that contain or express a therapeutic or detectable substance to a mammal wherein the amount is effective to detect, diagnose, or monitor a site of injury or disease in the CNS or is effective to ameliorate the effects of an injury or disease of the CNS.
  • compositions comprising NS-specific antiself T-cells and methods of use of such compositions for prevention or inhibition of CNS degeneration are provided.
  • the NS-specific antiself T-cells are non-recombinant.
  • compositions and method of use thereof comprising a NS-specific antigen (or derivative thereof) are used for preventing or inhibiting degeneration of nerves within the CNS.
  • the present invention provides methods for (a) delivery of substances to a site of CNS injury or disease comprising administration of activated T-cells and (b) amelioration of degeneration comprising administration of (i) NS-specific antiself T-cells or (ii) an NS-specific antigen or derivative thereof or both (i) and (ii) .
  • substance-delivering activated T-cells may optionally be administered in combination with (a) NS- specific antiself T-cells or (b) an NS-specific antigen (or derivative thereof) or both (a) and (b) .
  • the methods of the present invention may optionally be combined concurrently with one or more of the following: (a) administration into the CNS of mononuclear phagocytes (preferably cultured monocytes) that have been stimulated to enhance their capacity to promote axonal regeneration; (b) administration into the CNS of a neurotrophic factor such as acidic fibroblast growth factor; and (c) administration of an anti-inflammatory therapeutic substance (i.e., an anti-inflammatory steroid, such as dexamethasone or methylprednisolone, or a non-steroidal anti- inflammatory agent or drug, such as aspirin, indomethacin, ibuprofen, fenoprofen, ketoprofen or haproxen, or an anti- inflammatory peptide, such as Thr-Lys-Pro (TKP) ) .
  • an anti-inflammatory therapeutic substance i.e., an anti-inflammatory steroid, such as dexamethasone or methylprednisolone, or
  • activated T-cells capable of delivering a diagnostic or therapeutic substance to a site of injury or disease in the CNS.
  • the activated T-cells do not recognize a NS-specific antigen; more preferably, the activated T-cells recognize a non-self antigen.
  • such activated T-cells may be used as part of a diagnostic technique for the detection of a site of damage in the CNS caused by injury or disease.
  • the activated T-cells may be used as part of a therapeutic regimen for ameliorating the effects of injury or disease of the CNS by promoting axonal regeneration or inhibiting or preventing CNS degeneration. 5.1.1 DIAGNOSTIC AND THERAPEUTIC COMPOSITIONS
  • Activated T-cells of the present invention can be used for the delivery of various therapeutic and detectable substances to a site of injury or disease within the CNS.
  • the activated T-cells of the present invention are activated by exposure to an antigen that is not NS-specific, more preferably by exposure to a non-self antigen.
  • the detectable substances may be used for detecting, diagnosing or monitoring a site of injury or disease of the CNS.
  • T-cells which preferentially localize to a site of injury or disease in the CNS can be detectably labeled.
  • the T-cells can be detectably labeled with a contrast agent including, without limitation, metals such as gold particles, gadolinium complexes, etc.
  • the T- cells can be labeled detectably with a radioisotope, including but not limited to: 15 Iodine, 131 Iodine, 99m Technecium.
  • the T-cells can also be detectably labeled using a fluorescence emitting metal such as 152 Eu, or others of the lanthanide series.
  • Methods for detectably labeling T-cells may, for example, be such as those described in Harlow and Lane (Harlow, E.
  • Fluorescence emitting metals or radioactive metals can be attached to the T-cells using such metal chelating agents as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) . Labeling of T-cells with a radioisotope can be achieved by incubating cells with a radioactive metabolic precursor.
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • activated T-cells can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include but are not limited to: computed tomography (CT) , whole body scan such as position emission tomography (PET) , magnetic resonance imaging (MRI) , sonography, radiation responsive surgical instrument (Thurston et al . , U.S. Patent 5,441,050), and fluorescence responsive scanning instrument. After labeling, the T-cells of the present invention are activated.
  • CT computed tomography
  • PET position emission tomography
  • MRI magnetic resonance imaging
  • sonography sonography
  • radiation responsive surgical instrument T-cells of the present invention are activated.
  • the T-cells may be activated by exposure of the cells to one or more of a variety of natural and synthetic antigens and epitopes, including but not limited to, lipopolysaccharide (LPS) , myelin basic protein (MBP) , myelin/oligodendrocyte glycoprotein (MOG) , myelin proteolipid protein (PLP) , myelin associated protein (MAG) , S-100, ⁇ - amyloid, Thy-1, neurotransmitter receptors.
  • LPS lipopolysaccharide
  • MBP myelin basic protein
  • MOG myelin/oligodendrocyte glycoprotein
  • PBP myelin proteolipid protein
  • MAG myelin associated protein
  • S-100 ⁇ - amyloid
  • Thy-1 neurotransmitter receptors
  • T- cells are activated by an antigen that is not specific to the NS, more preferably by a non-self antigen.
  • 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 ⁇ (TNF- ⁇ ) , interleukin 2 (IL-2) , and interleukin 4 (IL-4).
  • TNF- ⁇ tumor necrosis factor ⁇
  • IL-2 interleukin 2
  • IL-4 interleukin 4
  • the activated T-cells endogenously produced a substance that ameliorates the effects of injury or disease in the CNS.
  • the activated T-cells endogenously produce a substance that stimulates other cells, including, but not limited to, transforming growth factor- ⁇ (TGF- ⁇ ) , nerve growth factor (NGF) , neurotrophic factor 3 (NT-3) , neurotrophic factor 4/5 (NT-4/5) , brain-derived neurotrophic factor (BDNF) , interferon-5 (IFN-5) , interleukin-6 (IL-6) , wherein the other cells, directly or indirectly, ameliorate the effects of injury or disease.
  • TGF- ⁇ 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-5 interferon-5
  • IL-6 interleukin-6
  • the T-cells may be genetically engineered in vitro to insert therein a nucleotide sequence as described in Kramer et al . , 1995, Nature Medicine, 1 (11) :1162-1166.
  • the nucleotide sequence is under the control of necessary elements for transcription and translation such that a biologically active protein encoded by the nucleotide sequence can be either expressed continuously or induced to expression as a result of exposure of the T-cells to a microenvironment of a kind present at the site of injury.
  • the presence of the coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the coding sequence or portions or derivatives thereof.
  • the recombinant expression system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.).
  • telomere sequence can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blot using a probe having sequence homology to a coding sequence or transcribed noncoding sequence or particular portions thereof.
  • total nucleic acid of the host cell may be extracted and quantitatively assayed for hybridization to such probes.
  • the levels of a protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like.
  • the T-cells may be stably transfected with said nucleotide sequences or may be transiently transfected. Transient transfection may be applicable for acute one-dose therapeutic regimens.
  • nucleotide sequences may encode various substances including, without limitation, therapeutic substances; enzymes which catalyze a therapeutic substance; a regulatory product which stimulates expression of a therapeutic substance in the T-cells, etc.
  • examples include: nucleotide sequences encoding neurotrophic factors such as NGF; nucleotide sequences encoding enzymes which play a role in CNS nerve regeneration such as the enzyme transglutaminase; nucleotide sequences encoding enzymes which catalyze the production of a neurotransmitter, e.g. enzymes involved in the catalysis of acetylcholine or dopamine, etc.
  • the T-cells can be preserved, e.g. by cryopreservation, either before or after culture.
  • Cryopreservation agents which can be used include but are not limited to dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205) , glycerol, polyvinylpyrrolidone (Rinfret, 1960, Ann. N.Y. Acad. Sci. 8j>:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature 196:548) , albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc.
  • DMSO dimethyl sulfoxide
  • a controlled cooling rate is critical.
  • Different cryoprotective agents (Rapatz et al., 1968, Cryobiology 5 (1) : 18-25) and different cell types have different optimal cooling rates. See, e.g. , Rowe and Rinfret, 1962, Blood 2 ⁇ 0:636; Rowe, 1966, Cryobiology 3 (1) : 12-18; Lewis et al., 1967, Transfusion 7 (1) : 17-32; and Mazur, 1970, Science 168:939-949 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,
  • Frozen cells are preferably thawed quickly (e.g., in a
  • cryoprotective agent if toxic in humans, should be
  • cryoprotective agent 25 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 axonal regeneration as described herein
  • compositions and methods of the present invention comprising activated, substance-delivering T-cells are useful 35 for treating or detecting a site of damage in the CNS caused by injury or disease.
  • Methods for detecting a site of injury or disease of the CNS in a mammal comprise: (a) administering to a mammal an effective amount of labeled activated T-cells; and (b) detecting in the mammal the labeled activated T-cells that have accumulated at said site of injury or disease, in which step (b) is performed after an interval sufficient to permit said labeled activated T-cells administered to step (a) to accumulate at said site of injury or disease.
  • activated T-cells can be used to deliver substances for ameliorating the effects of injury or disease by, for example, promoting axonal regeneration or inhibiting or preventing degeneration of the CNS.
  • substances include, without limitation, growth factors which promote nerve regeneration such as nerve growth factor (NGF) ; substances lacking at the site of injury, e.g. neurotransmitters such as acetylcholine, dopamine; anti-inflammatory substances, etc.
  • NGF nerve growth factor
  • activated T-cells may endogenously produce a substance that has a therapeutic effect on the CNS injury, including, without limitation, interleukins and growth factors.
  • the activated T-cells do not recognize a NS-specific antigen; more preferably, the labeled activated T-cells recognize a non-self antigen.
  • the injury or disease may be situated in any portion of the CNS, including the brain, spinal cord, or optic nerve.
  • trauma including blunt trauma, penetrating trauma, and trauma sustained during a neurosurgical operation or other procedure.
  • stroke including hemorrhagic stroke and ischemic stroke.
  • Other examples of disease are Alzheimer's disease, multiple sclerosis, Huntington's disease, ALS, and Parkinson's disease.
  • optic nerve injury accompanying optic neuropathy or glaucoma.
  • compositions and methods of the present invention are useful for treating CNS injury or disease that results in axonal damage whether or not the subject also suffers from another disease of the central or peripheral nervous system, such as neurological disease of genetic, metabolic, toxic, nutritional, infective or autoimmune origin.
  • the invention also provides methods of preventing or inhibiting CNS degeneration by administering a composition comprising an effective amount of NS-specific antiself T- cells.
  • the NS-specific antiself T- cells are non-recombinant cells.
  • the invention also provides methods of preventing or inhibiting CNS degeneration by administering a composition comprising an effective amount of a NS-specific antigen.
  • the activated, substance-delivering T-cells described, supra , in Section 5.1 may be used alone or in combination with NS-specific antiself T-cells or a NS-specific antigen or NS-specific antiself T-cells and a NS-specific antigen for ameliorating the effects of injury or disease, e.g., for promoting axonal regeneration and preventing or inhibiting CNS degeneration.
  • NS-specific antiself T-cells can be used for ameliorating the effects of injury or disease of the CNS that result in CNS degeneration.
  • the NS-specific antiself T-cells are isolated.
  • 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 known procedures (Burns et al . , Cell Immunol . 81:435 (1983); Pette et al . , Proc . Natl . Acad . Sci. USA 82:7968 (1990); Mortin et al . , J. Immunol . 145:540 (1990); Schluesener et al . , J . Immunol . 135: 3128 (1985); Suruhan- Dires Keneli et al . , Euro . J . Immunol . 2 . 3:530 (1993) which are incorporated herein by reference in their entirety.
  • 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 a 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, a crude NS preparation, or a peptide derived from a NS-antigen, as will be described below.
  • NS-specific ATCs obtained as described above, can be used immediately or may be preserved for later use, e.g. by cryopreservation as described in Section 5.1, supra .
  • NS- specific ATCs 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.
  • compositions comprising a NS-specific antigen are used for ameliorating the effects of injury or disease that result in CNS degeneration. Additionally, NS- specific antigens may be used for in vivo or in vitro activation of antiself T-cells. In an embodiment, the NS- specific antigen is an isolated antigen. In an embodiment, methods of ameliorating the effects of CNS injury or disease comprise administering NS-specific antigen to a mammal wherein the NS-specific Ag activates T-cells in vivo to produce a population of T-cells that accumulate at a site of injury or disease of the CNS.
  • 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.
  • peptides derived from NS- specific, self antigens activate T-cells, but do not induce an autoimmune disease.
  • An example of such a antigen fragment is a peptide comprising amino acids 51-70 of myelin basic protein. SEQ ID NO:l (Kamholz et al . , 1986, Proc. Natl.
  • a NS-specific antigen may be a crude NS- tissue preparation, e.g., derived from tissue obtained at the site of CNS injury. Such a preparation may include cells, both living or dead cells, membrane fractions of such cells or tissue, etc.
  • a NS-specific antigen may be obtained by a NS biopsy from a mammal including, but not limited to, from a site of CNS injury; from cadavers; from cell lines grown in culture. Additionally, a NS-specific antigen may be a protein obtained by genetic engineering, chemically synthesized, etc.
  • the invention also relates to derivatives (e.g., fragments) or analogs of NS- specific antigens which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length NS-specific antigen.
  • functional activities include but are not limited to antigenicity [ability to bind (or compete with a CNS-antigen for binding) to an anti-NS-specific antibody] , immunogenicity (ability to generate antibody which binds to a NS-specific protein) , and ability to interact with T-cells, resulting in activation comparable to that obtained using the corresponding full-length antigen.
  • proteins consisting of or comprising a fragment of a NS-specific antigen consisting of at least 10 (contiguous) amino acids of the CNS-specific antigen is provided.
  • the fragment consists of at least 20 contiguous amino acids or 50 contiguous amino acids of the NS-specific antigen.
  • NS-specific antigen 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 stringent, moderately stringent, or nonstringent conditions.
  • the NS-specific antigen derivatives and analogs of the invention can be produced by various methods known in the art.
  • 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 vitro .
  • the coding nucleic acid sequence can be mutated in vitro or in vivo , to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new 5 restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C. , et al., 1978, J. Biol. Chem
  • Manipulations may also be made at the protein level. Included within the scope of the invention are fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by glycosylation,
  • 25 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.
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, e-Ahx, 6-amino
  • amino acid 35 hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, /3-alanine, fluoro-amino acids, designer amino acids such as /3-methyl amino acids, C ⁇ -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 derivatives thereof can be assayed by various methods known in the art, including, but not limited to T-cell proliferation assays (Mor and Cohen, 1995, J. Immunol. 155:3693-3699) .
  • a NS-specific antigen 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 described in Section 5.2 may be used to prevent or inhibit secondary degeneration which may otherwise follow primary CNS injury, e.g. a cut or a crush in a CNS tissue.
  • secondary degeneration e.g. a cut or a crush in a CNS tissue.
  • such compositions may be used to ameliorate the effects of disease that results in degenerative processes, e.g. degeneration occurring in either grey or white matter (or both) as a result of various diseases or disorders including, without limitation: senile dementias, Alzheimer's disease, Parkinson's Disease, glaucoma, multiple sclerosis, Huntington's disease, ALS, prion diseases such as Creutzfeldt-Jakob disease, etc.
  • 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, gelatine, starch, lactose or lactose monohydrate; 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, gelatine, starch, lactose or lactose monohydrate
  • a disintegrating agent such as alginic acid, maize starch and the
  • Methods of administration include, but are not limited to, parenteral e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, and mucosal e.g., oral, nasal, buccal, vaginal, rectal, intraocular) 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
  • 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.
  • compositions for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds 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 multi- dose 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.
  • the compounds 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.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • 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.
  • the 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 are administered with 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.
  • compositions of the invention are administered to a mammal shortly after injury or detection of a degenerative lesion in the CNS.
  • the therapeutic methods of the invention may comprise administration of activated T-cells or NS-specific ATCs or a NS-specific antigen or any combination thereof.
  • the NS-specific ATCs or a NS-specific antigen of the invention are administered in combination with a therapeutic composition which promotes regeneration of axons within the CNS;
  • the latter therapeutic composition may comprise for example, the activated, substance-delivering T- cells of the present invention wherein the said substance promotes nerve regeneration.
  • the therapeutic composition may comprise mononuclear phagocytes as described in PCT Publication No. WO 97/09985, which is incorporated herein by reference in its entirety.
  • mononuclear phagocytes which are cultured ex vivo together with a stimulatory tissue, such as der is or a nerve segment, are administered into the central nervous system of a mammal at or near the site of injury or disease-inflicted lesion.
  • a stimulatory tissue such as der is or a nerve segment
  • the mononuclear phagocytes are allogeneic.
  • the mononuclear phagocytes are autologous .
  • mononuclear phagocyte cells according 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 ATCs or NS-specific antigen.
  • administration of substance-delivering activated T-cells, or NS-specific ATCs, a NS-specific antigen may be administered as a single dose or may be repeated, preferably at 2 week intervals and then 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 activated T-cells or NS-specific antiself T-cells of the invention is proportional to the number of nerve fibers affected by CNS injury or disease at the site being treated.
  • the dose ranges from about 5 x 10 5 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 5 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 affected at the lesion or site of injury being treated.
  • Myelin basic protein was prepared from guinea pig spinal cord as previously described (Ben-Nun et al . , supra (1982)). Chick ovalbumin (OVA) was purchased from Sigma (Israel) . Heat-inactivated Mycobacterium tuberculosis H37RA (M . tuberculosis) and Incomplete Freund's adjuvant (IFA) were purchased from Difco Laboratories (Detroit, MI, USA) .
  • the proliferation medium of the T-cells contained the following: Dulbecco's modified Eagle's medium (DMEM, Dulbecco's modified Eagle's medium (DMEM, Dulbecco's modified Eagle's medium (DMEM, Dulbecco's modified Eagle's medium (DMEM, Dulbecco's modified Eagle's medium
  • Propagation medium contained: DMEM, 2-ME, L-Glu, sodium pyruvate, non-essential amino acids and antibiotics in the same concentration as above and also 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 from the spinal cords of guinea pigs was prepared as described (Hirshfeld, et al . , 1970, FEBS Lett . 7:317). OVA was purchased from Sigma (St. Louis, Missouri) .
  • the p51-70 of the rat 18.5kDa isoform of MBP (sequence: APKRGSGKDSHTRTTHYG) SEQ ID NO: 2 and the p277 of the human hsp60 (sequence: VLGGGCALLRCPALDSLTPANED) SEQ ID NO: 3 (Elias, et al . , 1991, Proc . Natl . Acad . Sci .
  • 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/ l) and emulsified with an equal volume of incomplete Freund's adjuvant (Difco).
  • the cells were washed and activated with the antigen (lO ⁇ g/ l) in proliferation medium containing Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine (2mM) , 2- mercaptoethanol (5xl0 ⁇ 5 M) , sodium pyruvate (ImM) , penicillin (100 IU/ml) , streptomycin (100 ⁇ g/ml) , nonessential amino acids (1 ml/100 ml) and autologous rat serum 1% (volume/volume) .
  • DMEM Dulbecco's modified Eagle's medium
  • 2mM 2- mercaptoethanol
  • ImM sodium pyruvate
  • penicillin 100 IU/ml
  • streptomycin 100 ⁇ g/ml
  • nonessential amino acids 1 ml/100 ml
  • autologous rat serum 1% volume/volume
  • the cells were transferred to propagation medium which additionally contained 10% fetal calf serum (FCS) (volume/volume) and 10% T cell growth factor derived from the supernatant of concanavalin A-stimulated spleen cells.
  • FCS fetal calf serum
  • T cell growth factor derived from the supernatant of concanavalin A-stimulated spleen cells.
  • Cells were grown in propagation medium for 4- 10 days before being re-exposed to antigen (lO ⁇ g/ml) in the presence of irradiated (2000 rad) thymus cells (10 7 cells/ml) in proliferation medium.
  • the T-cell lines were expanded by repeated re-exposure and propagation.
  • T-cells were washed and suspended in 10.7 ⁇ m Hoechst 33342 Stain (Molecular Probes, USA) for 10 minutes at 37°C. The cells were washed twice with 50 ml volumes of PBS and then resuspended at 5 x 10 ⁇ cells/ml on ice until injection.
  • Crush injuries were performed as previously described (Hirschberg et al . , 1994, J. Neuroimmunol. 50:9-16). Briefly, rats were deeply anesthetized by i.p. injection of xylazine (10 mg/kg; Rompun) and ketamine (50 mg/kg; Velalar) . Under 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.
  • rats were euthanized by over- anesthesia with ether and their optic nerves were surgically removed, immersed in Tissue-Tek (Miles Inc., USA), and frozen in liquid nitrogen cooled in iso-pentane (BDH, UK) . The nerves were then transferred to dry ice and stored at -70 °C until sectioning. Longitudinal cryostat nerve sections (20 ⁇ m thick) were picked up onto gelatin-coated glass slides (four sections per slide) and frozen at -20°C until viewed or prepared for fluorescence staining.
  • Nerves excised at various time periods after injury were prepared and sectioned. Hoechst-labeled nuclei or immunostained cells in each section were counted using the fluorescence microscope. For each time point five sections were counted, and the numbers were averaged.
  • T-cells were then incubated overnight at 4°C with a mouse monoclonal antibody directed against rat macrophages (EDI; 1:400; Serotec, UK) and antibody against rat glial fibrillary acidic protein (GFAP; 1:100; BioMakor) , all diluted in PBS containing 3% FCS. Staining of T-cells was accomplished by incubating nerve sections for 1 hr at room temperature with a mouse monoclonal antibody directed against rat T-cell receptor (TCR) (1:100, Hunig et al . , J . Exp . Med . , 169:73. 1989) , in PBS containing 3% FCS and 2% BSA.
  • TCR mouse monoclonal antibody directed against rat T-cell receptor
  • T-cell clones primed to MBP were activated with MBP for 2 days before being labeled with Hoechst stain and injected into animals i.p. at the time of injury.
  • the nerves were excised, cryosectioned and analyzed microscopically for the presence of labeled T-cells.
  • T mp cells were detected in the injured optic nerves at day 3 and accumulated until a peak at day 14 (Fig. 1) . Large clusters of cells were observed at the injury site and fewer individual cells were seen proximal and distal (Fig. 2) .
  • labeled T-cells were still detectable in the degenerating optic nerves.
  • T-cells were found in the non-injured optic nerves (Fig. 3) , non-injured sciatic nerve or injured sciatic nerve at any time after injury. Labeled T-cells were occasionally found in capillaries and in connective tissue but were not concentrated or localized into any specific areas. T-cells that were not prestimulated with antigen did not accumulate in any of the nerves, including damaged nerves.
  • T mp cells The accumulation of T mp cells in injured CNS, but not in injured PNS, suggests that there might be some specific interaction between the primed T-cells and the CNS tissue from which the MBP antigen was originally derived.
  • T 0VA chick ovalbumin
  • Rats were injected with a labeled T 0VA clone prestimulated with ovalbumin (OVA) using the same protocol as with the cells.
  • OVA ovalbumin
  • T 0VA and cells were counted in longitudinal sections of optic nerve prepared, 3, 7, 14 and 21 days after injury. No significant different was observed in numbers of Tr ⁇ p and T 0VA cells in injured optic nerve (Fig. 4) , indicating that antigen specificity has little to do with the accumulation of T-cells in CNS injury sites. cells were detectable slightly earlier than T 0VA cells in the optic nerve injury site, and antigen specificity may play a role in this but is not sufficient to explain the large accumulation of T 0VA cells in the site of injury.
  • Fig. 5 shows accumulation of T-cells measured immunocytochemically using antibodies to T-cell receptors. This detection technique rules out the possibility that the observed labeling is due to phagocytic cells which had phagocytized the pre-labeled T-cells shown in Fig. 1.
  • the graph shows a striking elevation in T-cell accumulation following injury, regardless of whether the systematically injected T-cells are specific to a self-epitope (MBP) or to a non-self epitope (OVA) .
  • MBP self-epitope
  • OVA non-self epitope
  • Fig. 6 shows that accumulation of T-cells is dependent on the lesion and not the breakdown of the blood-brain barrier.
  • T-cells specific to either MBP or OVA were injected 2 weeks after injury and their accumulation analyzed a week later, namely 21 days following the primary lesion. Their accumulation was compared to that of T-cells injected immediately after injury and detected either 7 or 21 days later. It appears that the time elapsed between the injury and the injection of T-cells, which is a factor in the sealing of the blood-brain barrier, is not a factor in the T- cell accumulation. 7.
  • T-cells in nerve sections are described in Section 6, supra.
  • MBP and OVA were dissolved in PBS (1 mg/ml) and emulsified with an equal volume of Incomplete Freund's Adjuvant (IFA) supplemented with 4 mg/ml M . tuberculosis .
  • Rats were immunized subcutaneously in the hind footpads with 0.1 ml of the emulsion.
  • IFA Incomplete Freund's Adjuvant
  • animals were euthanized and draining lymph nodes were surgically removed and dissociated under sterile conditions.
  • the cells were washed and placed in proliferation medium with irradiated thy ocytes (2000 rads) and either 10 ⁇ g/ml of MBP, OVA or M . tuberculosis for 3 days.
  • T-cell lines were expanded by re-exposure and propagation and tested for specificity in an antigen specific T cell proliferation assay. Lines were expanded and stocks were frozen in liquid nitrogen. The cells were thawed and stimulated once before being used in experiments.
  • T-cell lines were activated by restimulation in vitro with their own antigen (10 ⁇ g/ml) in proliferation medium. After incubation for 48-72 hrs at 37 °C 90% relative humidity and 7.5% C0 2 , the cells were washed. Viable cells were isolated on Percoll and suspended in PBS. Animals were injected with 10 x 10 6 cells/ml i.p. Control animals were injected with 1 ml PBS i.p. 7.1.3 CRUSH INJURY OF RAT SCIATIC NERVE
  • the optic nerve was exposed, without damaging the retinal blood supply.
  • Solid crystals of the dye 4- (4- (didecylamino) styryl) -n-methyl-pyridinium iodide (4-Di-10- Asp) (Molecular Probes, Europe BV) , were deposited 1-2 mm from the distal border of the injury site.
  • Non-injured optic nerves were similarly labeled at approximately the same distance from the globe.
  • Five days after dye application the retinas were excised under deep anesthesia, flat mounted in 4% paraformaldehyde solution, and labeled retinal ganglion cells (RGCs) were counted by fluorescence microscopy.
  • the effect of injected T-cells on the numbers of surviving optic nerve fibers was monitored by retrograde labeling of RGCs (see above) immediately after injury in order to assess primary degeneration and two weeks later in order to assess secondary degeneration.
  • Five days after dye (4-Di-10-Asp) application the retinas were excised, whole mounted and their RGCs were counted. The counting was done in five randomly selected fields in each retina (all located at approximately the same distance from the optic disc) . In all cases the dye was applied 2 ml distally to the site of the prior insert. Using this lengthening approach, only those RGCs whose axons were still viable could be labeled.
  • 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, moribound state.
  • the injured optic nerve was analyzed for T-cell accumulation. As shown in Fig. 7, in the uninjured optic nerves of control rats injected with phosphate-buffered saline (PBS) no T-cells could be detected. Small but significant numbers of T-cells were observed in the uninjured optic nerves of rats injected with anti-MBP T-cells (primed against a peptide comprising amino acids 51-70 of MBP ⁇ P52- 70 " known to be capable of inducing experimental autoimmune encephalomyelitis (EAE) under these experimental conditions) , but not of rats injected with anti-OVA T-cells.
  • PBS phosphate-buffered saline
  • Crush injury of the optic nerve was accompanied by a small but significant accumulation of endogenous T-cells, possibly reflecting a response to self antigens triggered by the injury.
  • T-cell accumulation was significantly increased by 5- to 6-fold) in rats injected with anti-OVA, anti-hsp60, or anti-MBP T-cells.
  • Fig. 9 represents micrographs of retrogradeably labeled retinas of injured optic nerves of rats injected with PBS, anti-hsp60 T- cells, or anti-MBP T-cells. Since only the anti-MBP T-cells showed a neuroprotective effect, and as hsp60, like MBP, is a self antigen which is expressed in injured tissues including EAE lesions, it was interesting to find out whether the protective effect of the anti-MBP T-cells is a function of their aggressiveness in causing an autoimmune disease.
  • T-cells activated with a fragment of a NS- specific antigen that does not cause autoimmune disease were substantially as effective in inhibiting secondary degeneration as T-cells activated with full-length NS- specific antigen that does cause autoimmune disease.
  • 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. 5
  • the number of labeled ganglion cells was about 12.5 fold greater in animals injected with MOG p35-55 compared to animals 0 receiving PBS.
  • Bovine MBP (Sigma, Israel) (1 mg/dose) was administered to rats by gavage using a blunt needle. MBP was administered 0 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. 5
  • the number of labeled RGCs was about 1.3 fold greater in animals treated with MBP compared to untreated animals.
  • 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 a NS-specific self-antigen as illustrated by MBP.

Abstract

The present invention relates to compositions and methods for the treatment or diagnosis of injury of the central nervous system (CNS). In particular, the invention relates to compositions comprising activated T-cells which are used to deliver (a) a diagnostic substance or (b) a therapeutic substance to a site of damage of the CNS caused by injury or disease. The invention also relates to pharmaceutical compositions comprising antiself T-cells that recognize antigens present in a greater concentration in the CNS compared to the circulation and methods of use thereof to prevent or inhibit degeneration of nerves within the CNS. The invention also relates to pharmaceutical compositions comprising an antigen (or derivative thereof) present in a greater concentration in the CNS compared to the circulation (NS-specific antigen or derivative) and methods of use thereof to prevent or inhibit degeneration of nerves within the CNS. The substance-delivering activated T-cell composition of the present invention may be administered alone or in combination with NS-specific T-cells or NS-specific antigen or in combination with NS-specific T-cells and NS-specific antigen.

Description

ACTIVATED T-CELLS AND THEIR USES
1. FIELD OF THE INVENTION
The present invention relates to compositions and methods for the treatment or diagnosis of injury of the central nervous system (CNS) . In an embodiment, activated T-cells are used to deliver (a) a diagnostic substance for detecting a site of injury or disease or (b) a therapeutic substance for ameliorating an effect of a disease or injury, such as, for e.g., promoting axonal regeneration or preventing or inhibiting degeneration caused by injury or disease. In a preferred embodiment, the activated T-cells that are used for delivering a substance do not recognize a nervous system (NS) antigen. More preferably, the substance- delivering activated T-cells recognize a non-self antigen (e.g., ovalbumin) . In another embodiment, pharmaceutical compositions comprising antiself T-cells that recognize an antigen present in a greater concentration in the nervous system (NS) compared to the other organs or circulation are used to prevent or inhibit degeneration of nerves within the CNS. In a preferred embodiment the antiself T-cells of the present invention are not genetically-engineered. In another embodiment, pharmaceutical compositions comprising an antigen (or derivative thereof) present in a greater concentration in the NS compared to other organs or the circulation (NS- specific antigen or derivative) are used to prevent or inhibit degeneration of nerves within the CNS. The activated T-cell compositions of the present invention may be administered alone or in combination with NS-specific antiself T-cells or NS-specific antigen or in combination with NS-specific antiself T-cells and NS-specific antigen.
2. BACKGROUND OF THE INVENTION
Damage to the CNS 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) and ischemias. Following traumatic injuries in the peripheral nervous system (PNS) , an invasion of blood derived monocytes as well as activation of microglia within the PNS occurs (Stoll, et al., 1989, Neurosci . , 9_:2327-35; Perry and Gordon, 1991, Int . 5 Rev. Cytol , 125:203-44; Perry and Gordon, 1988, Trends Neurosci . , 1J,: 273-277; Jordan and Thomas, 1988, Brain macrophages: Questions or origin and Interrelationships , 11:165-178; Griffin, et al., 1990, Ann . Neurol . , 27:8; Giulian, et al., 1989, J. Neurosci . , £: 4416-29; Giulian,
10 1987, J . Neurosci . Res . , 18.: 155-171; de Groot, et al. , 1989, 179: 314-27; and Bauer, et al., J. Neurosci . Res . , 38:365-75) . By contrast, the invasion of blood derived monocytes is delayed and more limited in its scope in traumatic injuries to the CNS (Perry and Gordon, 1991, Int . Rev . Cytol , 125:203-
15 44; Andersson, et al., 1991, Immunol . Lett . , J3(): 177-81; and Perry et al., 1987, J. Exp . Med . , 165:1218-1223) . In addition, the duration of events associated with the acute phase of the injury, though less pronounced, is prolonged in the CNS as compared to the PNS. For example, several weeks
20 after injury, numerous activated macrophages and microglia are found in the CNS, while only a few are detectable in PNS nerves at such time after injury (Perry et al., 1987, J . Exp. Med . , 165:1218-1223; Lunn et al . , 1990, Neuroscience 35:157- 165) .
25 Neurons in mammalian CNS do not undergo spontaneous regeneration following an injury. Thus, a CNS injury causes permanent impairment of motor and sensory functions. In contrast, neurons in the PNS have a far greater capacity to regenerate. Studies using allogenic macrophages incubated
30 with a stimulant (e . g . a nerve segment) and subsequently administered into the CNS of a mammal at or near the site of injury have shown regeneration of the impaired motor or sensory function (PCT Publication WO 97/09885 and Spiegler et al . , 1996, FASEB J. 10:1296).
35 Another tragic consequence of CNS injury is that the primary injury is often compounded by a degenerative process which results in a secondary loss, over time, of adjacent neurons that were not damaged by the initial injury. It has been suggested that the secondary degeneration results from diffusion of toxic chemicals produced by damaged neurons (Mclntosh, 1993, J . Neurotrauma 1JD:215; Lynch and Dawson, 5 1994, Curr. Opin. Neurol. 1:510; Smith et al . , 1995, New
Horiz. 3_:562; Faden, 1996, Pharmacol. Toxicol., J7_8:12; Faden, 1996, JAMA, 276:569).
Popovitch et al . has shown that CNS trauma such as spinal injury triggers a systemic response to self epitopes 0 such as yelin basic proteins (MBP) (Popovitch et al . , 1996, J. Neurosci. Res., .4_5:349). Activated T-cells that recognize a self antigen as well as activated T-cells that recognize a non-self antigen have been shown to enter the CNS parenchyma. Only T-cells capable of recognizing a CNS antigen appear to 5 persist in the nervous tissue (Hickey et al . , 1991, J.
Neurosci . Res . 28, 254-60). Although activated T-cells that recognize a self antigen (antiself T-cells) apparently persist in nervous tissue, use of activated T-cells that recognize a non-self antigen (non-self T-cells) for 0 administration to an individual have advantages such as no risk of induction of autoimmune disease. Further, use of non-self activated T-cells eliminates the necessity of activating autologous or syngeneric T-cells; therefore, non- self T-cells may be activated and stored for use in any 5 individual.
T-cells reactive to antigens of CNS white matter, such as myelin basic protein (MBP) , can induce the paralytic disease experimental autoimmune encephalomyelitis (EAE) within several days of their inoculation into naive recipient 0 rats (Ben Nun et al . , 1981, Eur. J . Immunol . 11, 195-9). Studies have suggested a role for anti-MBP T-cells in the human disease multiple sclerosis (Ota et al . , 1990, Nature 346, 183-7; Martin, 1997, J. Neural . Transm . Suppl . 49, 53- 67; Sun, 1993, Acta Neurol. Scand. Suppl. 142:1-56). Despite 5 their pathogenic potential, anti-MBP T cell clones are present in the immune systems of healthy subjects (Burns et al . , 1983, Cell . Immunol . 81, 435-40; Schluesener and Wekerle, 1985, J . Immunol . 135, 3128-33). However, little is known about the possible physiological functions of antiself T-cells.
Citation or identification of any reference shall not be construed as an admission that such reference is available as prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention is directed to methods and compositions for the treatment or diagnosis of injury of the central nervous system (CNS) . The present invention provides methods for delivering a therapeutic or detectable substance to a site of injury or disease of the CNS, comprising administering an effective amount of activated T-cells that contain or express a therapeutic or detectable substance to a mammal wherein the amount is effective to detect, diagnose, or monitor a site of injury or disease in the CNS or is effective to ameliorate the effects of an injury or disease of the CNS. The activated T-cells used for delivery of a substance preferably do not recognize a nervous system specific antigen (NS-specific antigen) ; more preferably the activated T-cells recognize a non-self antigen. "Activated T-cell" as used herein includes (i) T-cells that have been activated by (a) exposure to a cognate antigen or derivative thereof or (b) exposure to an appropriate mitogen such as a lectin (e.g. concanavalin A (Con A) or phytohemagglutinin (PHA) ) , and (ii) progeny of such activated T-cells. As used herein, 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. As used herein, a derivative of an antigen is a fragment or amino acid variant (e.g., an insertion, substitution and/or deletion derivative) of the corresponding full-length antigen so long as the fragment or amino acid variant is capable of displaying one or more functional activities of the corresponding full-length antigen. Such functional activities include but are not limited to antigenicity [ability to bind (or compete with the antigen for binding) to an anti-antigen-specific antibody] , immunogenicity (ability to generate antibody which binds to the antigen) , and ability to interact with T-cells resulting in activation comparable to that obtained using the corresponding full-length antigen. The present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of non-recombinant, NS-specific antiself T-cells and methods of use of such compositions for prevention or inhibition of CNS nerve degeneration in which the amount is effective to ameliorate the effects of an injury or disease of the CNS. "NS-specific antiself T-cell" as used herein refers to an activated T-cell which recognizes a self antigen present in a greater concentration in the nervous system (NS) compared to other organs or the circulation or an antigen that shares an antigenic determinant with an antigen present in a greater concentration in the NS compared to other organs or the circulation.
The present invention also provides pharmaceutical compositions and methods of use comprising a therapeutically effective amount of NS-specific antigens (or derivatives thereof) for prevention or inhibition of CNS degeneration in which the amount is effective to activate T-cells in vivo or in vitro wherein the activated T-cells ameliorate the effects of an injury or disease of the CNS. "NS-specific antigen" as used herein refers to an antigen present in the NS or an antigen that shares an antigenic determinant with an antigen present in a greater concentration in the NS compared to other organs or the circulation. In the practice of the invention, therapy for amelioration of effects of injury or disease comprising administration of activated T-cells may optionally be in combination with NS-specific antiself T-cells or a NS- specific antigen (or derivative thereof) or NS-specific antiself T-cells and a NS-specific antigen.
4. BRIEF DESCRIPTION OF THE FIGURES Fig. 1 presents low-power epifluorescence micrographs of optic nerve after a controlled crush injury in rats treated with T-cell clones primed with MBP. See text, Section 6, for experimental details. Fig. 2 presents high-power micrographs of the site of optic nerve injury shown in Fig. 1, showing the large concentration of injected cells localized in the site of injury.
Fig. 3 presents a serial section through non-injured optic nerve.
Fig. 4 is a graphical representation of the number of T- cells at the site of injury of two different T-cell clones primed with antigen MBP or ovalbumin (OVA) (Tmp or T0VA, respectively) after injury, at various time intervals following injury.
Figure imgf000008_0001
and T0VA cells were injected into animals at the time of optic nerve crush, ipsolateral and contralateral nerves were then removed and prepared for microscopy at days 3, 7, 14 and 21. The figure shows that T- cells accumulated at the site of injury, independent of the antigen with which they were primed (each result is an average of 5 different experiments; the bar shows the standard deviation) . See text, Section 6, for experimental details.
Fig. 5 shows accumulation of T-cells primed with MBP or OVA (TMBP and T0VA, respectively) measured immunochemically using antibodies to T-cell receptors. A comparison of the number of accumulated cells in injured optic nerve (ON) and in non-injured optic nerve is illustrated. See text, Section 6, for experimental details. Fig. 6 shows accumulation of T-cells in injured and non- injured optic nerve after various treatment protocols. T- cells specific to MBP
Figure imgf000008_0002
were injected either immediately after nerve injury
Figure imgf000008_0003
Cell Injection = 0) or 14 days after injury
Figure imgf000008_0004
Cell Injection = 14) . Their accumulation at the optic nerve was analyzed either 7 days (nerve excision - day 7) or 21 days (nerve excision = day 21) after injury. Fig. 7 shows T-cell accumulation in injured optic nerve 1 week after injury. See text, Section 6, for experimental details. Anti-MBP or anti-OVA or anti-hsp60 T cell lines were raised, maintained, and activated by incubation with MBP from the spinal cords of guinea pigs, or with OVA (Sigma) , or with the 51-70 peptide of MBP, respectively, in the presence of irradiated (2000 rad) syngeneic thymus cells. See text, Section 7, for experimental details. Activated T-cells (lxlO7 cells) of the anti-MBP or anti-OVA lines or PBS were injected intraperitoneally into adult Lewis rats immediately after unilateral crush injury of the optic nerve. Seven days after injury both optic nerves were removed, cryosectioned, and analyzed immunohistochemically for the presence of labeled T- cells. Bars shows the mean total numbers of T-cells counted in 2 or 3 sections of each nerve. Each group contained 3 or 4 rats .
Fig. 8 illustrates inhibition of secondary degeneration after partial optic nerve crush injury in adult rats. See text, Section 7, for experimental details. Surviving optic nerve fibers were monitored by retrograde labeling of retinal ganglion cells immediately after injury and 2 weeks later. Five days after dye application retinas were excised, and labeled retinal ganglion cells (RGCs) were counted under the fluorescence microscope. Counting was performed in 5 randomly selected fields in each retina (all located at approximately the same distance from the optic disc) . The number of RGCs in each group of injured nerves in rats injected with PBS only or injected with anti-MBP, anti-OVA, or anti-hsp60 T-cells was expressed as percentages of the total numbers of spared neurons following the primary injury (42% of the axons remained viable after the primary injury) .
Fig. 9 presents photomicrographs showing retrograde labeled retinas of injured optic nerves of rats injected with (A) PBS, (B) anti-hsp60 T-cells, or (C) anti-MBP T-cells. See text. Section 7, for experimental details.
Fig. 10 shows number of surviving RGCs of injured optic nerves of rats injected with anti-MBP T-cells (T^) , T-cells generated against a peptide comprising amino acids 51-70 of MBP (Tp 51-70), or PBS. See text, Section 7, for experimental details.
Fig. 11 presents clinical course of rats injected with anti-MBP T-cells. Results were evaluated according to the neurological paralysis scale (EAE score) . Rats were injected i.p. with lxlO7 activated anti-MBP T-cells either immediately after optic nerve crush injury (-■-) or without optic nerve crush injury (—o—) . Each group contained 5 to 9 rats. Data points represents means ± SEM. See text, Section 7, for experimental details.
Fig. 12 shows the survival of neurons in uninjured nerves in rats injected i.p. with lxlO7 activated anti-MBP T- cells or PBS. See text. Section 7, for experimental details. Fig. 13 illustrates inhibition of secondary degeneration after optic nerve crush injury in adult rats. See text, Section 8, for experimental details. Rats were injected intradermally into footpads with a 21-mer peptide based on amino acid residues 35-55 (MOG p35-55) of myelin/oligodendrocyte glycoprotein (chemically synthesized at the Weiz ann Institute, Israel) (50 μg/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 fiber 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. Fig. 14 illustrates inhibition of secondary degeneration after optic nerve crush injury in adult rats. See text, Section 9, for experimental details. MBP (Sigma, Israel) (1 mg in 0.5 ml saline) was administered to adult rats by gavage using a blunt needle MBP was administered 5 times, i.e., every third day beginning 2 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.
5. DETAILED DESCRIPTION OF THE INVENTION
In the practice of the invention, compositions comprising activated T-cells are used for delivery of (a) a diagnostic substance or (b) a therapeutic substance to a site of injury or disease of the CNS in a mammal. Generally, T-cells of the present invention are T-cells which recognize an antigen not normally present or present in small quantities in the circulation. Such antigens include but are not limited to NS-specific antigens, cryptic antigens or "non-self" antigens (i.e., antigens not normally present in an individual) . Non-self antigens may be, without limitation, viral, bacterial, etc., including tissue-specific antigens from a different species or individual.
In an embodiment, T-cells are activated in vitro by exposure to an antigen and administered to a mammal. The present invention provides methods for delivering a therapeutic or detectable substance to a site of injury or disease of the CNS, comprising administering an effective amount of activated T-cells that contain or express a therapeutic or detectable substance to a mammal wherein the amount is effective to detect, diagnose, or monitor a site of injury or disease in the CNS or is effective to ameliorate the effects of an injury or disease of the CNS.
Pharmaceutical compositions comprising NS-specific antiself T-cells and methods of use of such compositions for prevention or inhibition of CNS degeneration are provided. In a preferred embodiment, the NS-specific antiself T-cells are non-recombinant.
Pharmaceutical compositions and method of use thereof comprising a NS-specific antigen (or derivative thereof) are used for preventing or inhibiting degeneration of nerves within the CNS. The present invention provides methods for (a) delivery of substances to a site of CNS injury or disease comprising administration of activated T-cells and (b) amelioration of degeneration comprising administration of (i) NS-specific antiself T-cells or (ii) an NS-specific antigen or derivative thereof or both (i) and (ii) . In the practice of the invention, substance-delivering activated T-cells may optionally be administered in combination with (a) NS- specific antiself T-cells or (b) an NS-specific antigen (or derivative thereof) or both (a) and (b) .
If desired, the methods of the present invention may optionally be combined concurrently with one or more of the following: (a) administration into the CNS of mononuclear phagocytes (preferably cultured monocytes) that have been stimulated to enhance their capacity to promote axonal regeneration; (b) administration into the CNS of a neurotrophic factor such as acidic fibroblast growth factor; and (c) administration of an anti-inflammatory therapeutic substance (i.e., an anti-inflammatory steroid, such as dexamethasone or methylprednisolone, or a non-steroidal anti- inflammatory agent or drug, such as aspirin, indomethacin, ibuprofen, fenoprofen, ketoprofen or haproxen, or an anti- inflammatory peptide, such as Thr-Lys-Pro (TKP) ) .
5.1 DELIVERY OF SUBSTANCES
Described herein are activated T-cells capable of delivering a diagnostic or therapeutic substance to a site of injury or disease in the CNS. In a preferred embodiment the activated T-cells do not recognize a NS-specific antigen; more preferably, the activated T-cells recognize a non-self antigen. In one embodiment, such activated T-cells may be used as part of a diagnostic technique for the detection of a site of damage in the CNS caused by injury or disease. In another embodiment, the activated T-cells may be used as part of a therapeutic regimen for ameliorating the effects of injury or disease of the CNS by promoting axonal regeneration or inhibiting or preventing CNS degeneration. 5.1.1 DIAGNOSTIC AND THERAPEUTIC COMPOSITIONS
Activated T-cells of the present invention can be used for the delivery of various therapeutic and detectable substances to a site of injury or disease within the CNS. In a preferred embodiment, the activated T-cells of the present invention are activated by exposure to an antigen that is not NS-specific, more preferably by exposure to a non-self antigen. The detectable substances may be used for detecting, diagnosing or monitoring a site of injury or disease of the CNS.
In an embodiment, the T-cells are allogeneic T-cells, e.g. a pooled T-cell preparation obtained from a blood bank. The use of allogeneic T-cells is applicable for various treatments comprising limited administrations, including but not limited to, delivery of T-cells to a site of CNS injury for diagnostic purposes; for an acute single administration or one-dose therapy, etc. In another embodiment, the T-cells are syngeneic T-cells, preferably autologous T-cells (i.e., from the same individual) . 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.
For use in the diagnostic methods of the invention, T-cells which preferentially localize to a site of injury or disease in the CNS can be detectably labeled.
The T-cells can be detectably labeled with a contrast agent including, without limitation, metals such as gold particles, gadolinium complexes, etc. Alternatively, the T- cells can be labeled detectably with a radioisotope, including but not limited to: 15Iodine, 131Iodine, 99mTechnecium. The T-cells can also be detectably labeled using a fluorescence emitting metal such as 152Eu, or others of the lanthanide series. Methods for detectably labeling T-cells may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) and Current Protocols in Immunology (Current Protocols in Immunology, 1997, Eds., Coligan et al., John Wiley & Sons, Inc., NIH) which are incorporated herein by reference in their entirety. Labeling of T-cells with metal particles may be achieved by incubating cells in a suspension comprising the metal particles wherein the T-cells spontaneously internalize such particles into the cell's cytosol. Such substances may also be introduced into the cells by a variety of electroporetic techniques (Current Protocols in Immunology, 1997, Eds. Coligan et al., John Wiley & Sons, Inc., NIH). Fluorescence emitting metals or radioactive metals can be attached to the T-cells using such metal chelating agents as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) . Labeling of T-cells with a radioisotope can be achieved by incubating cells with a radioactive metabolic precursor.
Presence of labeled, activated T-cells can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include but are not limited to: computed tomography (CT) , whole body scan such as position emission tomography (PET) , magnetic resonance imaging (MRI) , sonography, radiation responsive surgical instrument (Thurston et al . , U.S. Patent 5,441,050), and fluorescence responsive scanning instrument. After labeling, the T-cells of the present invention are activated. The T-cells may be activated by exposure of the cells to one or more of a variety of natural and synthetic antigens and epitopes, including but not limited to, lipopolysaccharide (LPS) , myelin basic protein (MBP) , myelin/oligodendrocyte glycoprotein (MOG) , myelin proteolipid protein (PLP) , myelin associated protein (MAG) , S-100, β- amyloid, Thy-1, neurotransmitter receptors. Preferably, T- cells are activated by an antigen that is not specific to the NS, more preferably by a non-self antigen.
During ex vivo activation of the T-cells, 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 α (TNF-α) , interleukin 2 (IL-2) , and interleukin 4 (IL-4). In an embodiment, the activated T-cells endogenously produced a substance that ameliorates the effects of injury or disease in the CNS.
In another embodiment, the activated T-cells endogenously produce a substance that stimulates other cells, including, but not limited to, transforming growth factor-β (TGF-β) , nerve growth factor (NGF) , neurotrophic factor 3 (NT-3) , neurotrophic factor 4/5 (NT-4/5) , brain-derived neurotrophic factor (BDNF) , interferon-5 (IFN-5) , interleukin-6 (IL-6) , wherein the other cells, directly or indirectly, ameliorate the effects of injury or disease.
In another embodiment, the T-cells may be genetically engineered in vitro to insert therein a nucleotide sequence as described in Kramer et al . , 1995, Nature Medicine, 1 (11) :1162-1166. The nucleotide sequence is under the control of necessary elements for transcription and translation such that a biologically active protein encoded by the nucleotide sequence can be either expressed continuously or induced to expression as a result of exposure of the T-cells to a microenvironment of a kind present at the site of injury.
Due to the inherent degeneracy of the genetic code, other nucleotide sequences that encode substantially the same or a functionally equivalent amino acid sequence of a protein, are within the scope of the invention. Preferably, the expression product of said nucleotide sequence is a secretory protein. The recombinant T-cells which contain a coding sequence and which express a biologically active gene product may be identified by at least four general approaches: (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expression of mRNA transcripts in the cell; and (d) detection of the product encoded by the nucleotide sequence as measured by immunoassay or by its biological activity. In the first approach, the presence of the coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the coding sequence or portions or derivatives thereof. In the second approach, the recombinant expression system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the coding sequence is inserted within a marker gene sequence of a vector, recombinant cells containing the coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence under the control of the same or different promoter used to control the expression of the coding sequence. Expression of the marker in response to induction or selection indicates expression of the coding sequence. In the third approach, transcriptional activity of a nucleotide sequence can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blot using a probe having sequence homology to a coding sequence or transcribed noncoding sequence or particular portions thereof. Alternatively, total nucleic acid of the host cell may be extracted and quantitatively assayed for hybridization to such probes. In the fourth approach, the levels of a protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like. The T-cells may be stably transfected with said nucleotide sequences or may be transiently transfected. Transient transfection may be applicable for acute one-dose therapeutic regimens.
Such nucleotide sequences may encode various substances including, without limitation, therapeutic substances; enzymes which catalyze a therapeutic substance; a regulatory product which stimulates expression of a therapeutic substance in the T-cells, etc. Examples include: nucleotide sequences encoding neurotrophic factors such as NGF; nucleotide sequences encoding enzymes which play a role in CNS nerve regeneration such as the enzyme transglutaminase; nucleotide sequences encoding enzymes which catalyze the production of a neurotransmitter, e.g. enzymes involved in the catalysis of acetylcholine or dopamine, etc. As a result, T-cells which localize at the site of CNS injury or disease produce and secrete the needed substances at the site.
As will be evident to those skilled in the art, the T-cells can be preserved, e.g. by cryopreservation, either before or after culture.
Cryopreservation agents which can be used include but are not limited to dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205) , glycerol, polyvinylpyrrolidone (Rinfret, 1960, Ann. N.Y. Acad. Sci. 8j>:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature 196:548) , albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc. 21:157) , D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al., 1960, J. Appl. Physiol. 15:520), amino acids (Phan The Tran and Bender, 1960, Exp. Cell Res. 2jp_:651), methanol, acetamide, glycerol monoacetate (Lovelock, 1954, Bioche . J. 5^:265), inorganic salts (Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan The Tran and Bender, 1961, in Radiobiology, Proceedings of the Third Australian Conference on Radiobiology, Ilbery, P.L.T., ed. , Butterworth, London, p. 59) , and DMSO combined with hydroxyethel starch and human serum albumin (Zaroulis and Leiderman, 1980, Cryobiology 17:311-317) .
A controlled cooling rate is critical. Different cryoprotective agents (Rapatz et al., 1968, Cryobiology 5 (1) : 18-25) and different cell types have different optimal cooling rates. See, e.g. , Rowe and Rinfret, 1962, Blood 2^0:636; Rowe, 1966, Cryobiology 3 (1) : 12-18; Lewis et al., 1967, Transfusion 7 (1) : 17-32; and Mazur, 1970, Science 168:939-949 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.
After thorough freezing, cells can be rapidly transferred to a long-term cryogenic storage vessel. In one embodiment, samples can be cryogenically stored in mechanical freezers, such as freezers that maintain a temperature of about -80°C or about -20°C. In a preferred embodiment, samples can be cryogenically stored in liquid nitrogen (-196°C) or its vapor. Such storage is greatly facilitated by the availability of highly efficient liquid nitrogen refrigerators, which resemble large Thermos containers with an extremely low vacuum and internal super insulation, such that heat leakage and nitrogen losses are kept to an absolute minimum.
Considerations and procedures for the manipulation, cryopreservation, and long term storage of T-cells can be found, for example, in the following references, incorporated by reference herein: Gorin, 1986, Clinics in Haematology 15 (1) : 19-48; Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, July 22-26, 5 1968, International Atomic Energy Agency, Vienna, pp. 107- 186.
Other methods of cryopreservation of viable cells, or modifications thereof, are available and envisioned for use, e.g., cold metal-mirror techniques. See Livesey and Linner,
10 1987, Nature 327:255; Linner et al., 1986, J. Histochem.
Cytochem. 34 (9) :1123-1135; 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
15 water bath maintained at 37-41°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
20 (Spitzer et al. , 1980, Cancer 45.: 3075-3085) , low molecular weight dextran and citrate, hydroxyethyl starch (Stiff et al., 1983, Cryobiology 0:17-24), or acid citrate dextrose (Zaroulis and Leiderman, 1980, Cryobiology 17:311-317), etc. The cryoprotective agent, if toxic in humans, should be
25 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.
Once frozen T-cells have been thawed and recovered, they are used to promote axonal regeneration as described herein
30 with respect to non-frozen T-cells.
5.1.2 USES
The compositions and methods of the present invention comprising activated, substance-delivering T-cells are useful 35 for treating or detecting a site of damage in the CNS caused by injury or disease. Methods for detecting a site of injury or disease of the CNS in a mammal comprise: (a) administering to a mammal an effective amount of labeled activated T-cells; and (b) detecting in the mammal the labeled activated T-cells that have accumulated at said site of injury or disease, in which step (b) is performed after an interval sufficient to permit said labeled activated T-cells administered to step (a) to accumulate at said site of injury or disease.
For use in the therapeutic methods of the invention, activated T-cells can be used to deliver substances for ameliorating the effects of injury or disease by, for example, promoting axonal regeneration or inhibiting or preventing degeneration of the CNS. Such substances include, without limitation, growth factors which promote nerve regeneration such as nerve growth factor (NGF) ; substances lacking at the site of injury, e.g. neurotransmitters such as acetylcholine, dopamine; anti-inflammatory substances, etc. Further, activated T-cells may endogenously produce a substance that has a therapeutic effect on the CNS injury, including, without limitation, interleukins and growth factors.
In a preferred embodiment, the activated T-cells do not recognize a NS-specific antigen; more preferably, the labeled activated T-cells recognize a non-self antigen. The injury or disease may be situated in any portion of the CNS, including the brain, spinal cord, or optic nerve. One example of such injury or disease is trauma, including blunt trauma, penetrating trauma, and trauma sustained during a neurosurgical operation or other procedure. Another example of such injury or disease is stroke, including hemorrhagic stroke and ischemic stroke. Other examples of disease are Alzheimer's disease, multiple sclerosis, Huntington's disease, ALS, and Parkinson's disease. Yet another example of such injury or disease is optic nerve injury accompanying optic neuropathy or glaucoma. Still further examples of CNS injury or disease will be evident to those skilled in the art from this description and are encompassed by the present invention. The compositions and methods of the present invention are useful for treating CNS injury or disease that results in axonal damage whether or not the subject also suffers from another disease of the central or peripheral nervous system, such as neurological disease of genetic, metabolic, toxic, nutritional, infective or autoimmune origin.
5.2 AMELIORATION OF CNS DAMAGE 5.2.1 THERAPEUTIC COMPOSITIONS AND USES
The invention also provides methods of preventing or inhibiting CNS degeneration by administering a composition comprising an effective amount of NS-specific antiself T- cells. In a preferred embodiment the NS-specific antiself T- cells are non-recombinant cells.
The invention also provides methods of preventing or inhibiting CNS degeneration by administering a composition comprising an effective amount of a NS-specific antigen.
The activated, substance-delivering T-cells described, supra , in Section 5.1 may be used alone or in combination with NS-specific antiself T-cells or a NS-specific antigen or NS-specific antiself T-cells and a NS-specific antigen for ameliorating the effects of injury or disease, e.g., for promoting axonal regeneration and preventing or inhibiting CNS degeneration.
5.2.1.1 NS-SPECIFIC ANTISELF T-CELLS
NS-specific antiself T-cells (ATCs) can be used for ameliorating the effects of injury or disease of the CNS that result in CNS degeneration. In a preferred embodiment the NS-specific antiself T-cells are isolated.
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. In order to obtain NS-specific antiself T-cells, T-cells are isolated and the NS-specific ATCs are then expanded by known procedures (Burns et al . , Cell Immunol . 81:435 (1983); Pette et al . , Proc . Natl . Acad . Sci. USA 82:7968 (1990); Mortin et al . , J. Immunol . 145:540 (1990); Schluesener et al . , J . Immunol . 135: 3128 (1985); Suruhan- Dires Keneli et al . , Euro . J . Immunol . 2.3:530 (1993) which are incorporated herein by reference in their entirety.
Following their proliferation in vitro , the T-cells are administered to a mammalian subject. In a preferred embodiment, 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 a 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, a crude NS preparation, or a peptide derived from a NS-antigen, as will be described below.
NS-specific ATCs, obtained as described above, can be used immediately or may be preserved for later use, e.g. by cryopreservation as described in Section 5.1, supra . NS- specific ATCs 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.
5.2.1.2 NS-SPECIFIC ANTIGENS
Pharmaceutical compositions comprising a NS-specific antigen are used for ameliorating the effects of injury or disease that result in CNS degeneration. Additionally, NS- specific antigens may be used for in vivo or in vitro activation of antiself T-cells. In an embodiment, the NS- specific antigen is an isolated antigen. In an embodiment, methods of ameliorating the effects of CNS injury or disease comprise administering NS-specific antigen to a mammal wherein the NS-specific Ag activates T-cells in vivo to produce a population of T-cells that accumulate at a site of injury or disease of the CNS.
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.
In a preferred embodiment, peptides derived from NS- specific, self antigens activate T-cells, but do not induce an autoimmune disease. An example of such a antigen fragment is a peptide comprising amino acids 51-70 of myelin basic protein. SEQ ID NO:l (Kamholz et al . , 1986, Proc. Natl.
Acad. Sci. USA 83:4962-4966, GenBank accession number M13577; Roth et al . , 1987, J. Neurosci. Res. 17 (4) : 321-328 , GenBank accession number M30516) .
In addition, a NS-specific antigen may be a crude NS- tissue preparation, e.g., derived from tissue obtained at the site of CNS injury. Such a preparation may include cells, both living or dead cells, membrane fractions of such cells or tissue, etc.
A NS-specific antigen may be obtained by a NS biopsy from a mammal including, but not limited to, from a site of CNS injury; from cadavers; from cell lines grown in culture. Additionally, a NS-specific antigen may be a protein obtained by genetic engineering, chemically synthesized, etc.
In addition to purified antigen, the invention also relates to derivatives (e.g., fragments) or analogs of NS- specific antigens which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length NS-specific antigen. Such functional activities include but are not limited to antigenicity [ability to bind (or compete with a CNS-antigen for binding) to an anti-NS-specific antibody] , immunogenicity (ability to generate antibody which binds to a NS-specific protein) , and ability to interact with T-cells, resulting in activation comparable to that obtained using the corresponding full-length antigen.
In a specific embodiment of the invention, proteins consisting of or comprising a fragment of a NS-specific antigen consisting of at least 10 (contiguous) amino acids of the CNS-specific antigen is provided. In other embodiments, the fragment consists of at least 20 contiguous amino acids or 50 contiguous amino acids of the NS-specific antigen. Derivatives or analogs of a NS-specific antigen 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 stringent, moderately stringent, or nonstringent conditions. The NS-specific antigen derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, 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 vitro . Additionally, the coding nucleic acid sequence can be mutated in vitro or in vivo , to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new 5 restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C. , et al., 1978, J. Biol. Chem
10 253:6551) , etc.
Manipulations may also be made at the protein level. Included within the scope of the invention are fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by glycosylation,
15 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
20 specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
In addition, analogs and derivatives of a NS-specific
25 antigen can be chemically synthesized. For example, a peptide corresponding to a portion of an antigen which comprises the desired domain or which mediates the desired activity can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical
30 amino acid 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, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, e-Ahx, 6-amino
35 hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, /3-alanine, fluoro-amino acids, designer amino acids such as /3-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary) .
The functional activity of NS-specific antigens and derivatives thereof can be assayed by various methods known in the art, including, but not limited to T-cell proliferation assays (Mor and Cohen, 1995, J. Immunol. 155:3693-3699) .
A NS-specific antigen 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.
5.2.2 USES
The compositions described in Section 5.2 may be used to prevent or inhibit secondary degeneration which may otherwise follow primary CNS injury, e.g. a cut or a crush in a CNS tissue. In addition, such compositions may be used to ameliorate the effects of disease that results in degenerative processes, e.g. degeneration occurring in either grey or white matter (or both) as a result of various diseases or disorders including, without limitation: senile dementias, Alzheimer's disease, Parkinson's Disease, glaucoma, multiple sclerosis, Huntington's disease, ALS, prion diseases such as Creutzfeldt-Jakob disease, etc.
5.3 FORMULATIONS AND ADMINISTRATION
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 term "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, gelatine, starch, lactose or lactose monohydrate; 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.
Methods of administration include, but are not limited to, parenteral e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, and mucosal e.g., oral, nasal, buccal, vaginal, rectal, intraocular) routes. Administration can be systemic or local.
For oral administration, 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) . 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). The tablets may be coated by methods well-known in the art.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
The compounds 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 multi- dose 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. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds 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.
For administration by inhalation, the 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. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. In a preferred embodiment, compositions comprising substance-delivering activated T-cells or NS-specific antiself T-cells are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous or intraperitoneal administration to human beings.
Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.
In an embodiment, pharmaceutical compositions comprising NS-specific antigen are administered with 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.
In a preferred embodiment, the pharmaceutical compositions of the invention are administered to a mammal shortly after injury or detection of a degenerative lesion in the CNS. The therapeutic methods of the invention may comprise administration of activated T-cells or NS-specific ATCs or a NS-specific antigen or any combination thereof.
In an embodiment, the NS-specific ATCs or a NS-specific antigen of the invention are administered in combination with a therapeutic composition which promotes regeneration of axons within the CNS; the latter therapeutic composition may comprise for example, the activated, substance-delivering T- cells of the present invention wherein the said substance promotes nerve regeneration. Alternatively, the therapeutic composition may comprise mononuclear phagocytes as described in PCT Publication No. WO 97/09985, which is incorporated herein by reference in its entirety. Briefly, mononuclear phagocytes which are cultured ex vivo together with a stimulatory tissue, such as der is or a nerve segment, are administered into the central nervous system of a mammal at or near the site of injury or disease-inflicted lesion. In an embodiment, the mononuclear phagocytes are allogeneic. In a preferred embodiment, the mononuclear phagocytes are autologous .
In an embodiment, mononuclear phagocyte cells according 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 ATCs or NS-specific antigen. In an embodiment, administration of substance-delivering activated T-cells, or NS-specific ATCs, a NS-specific antigen, may be administered as a single dose or may be repeated, preferably at 2 week intervals and then 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. In the case of a CNS injury, 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. In chronic human diseases or conditions such as Alzheimer's disease or Parkinson's disease, the therapeutic treatment in accordance with the invention may be for life.
As will be evident to those of skill in the art, 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 activated T-cells or NS-specific antiself T-cells of the invention is proportional to the number of nerve fibers affected by CNS injury or disease at the site being treated. In a preferred embodiment, the dose ranges from about 5 x 105 to about 107 for treating a lesion affecting about 105 nerve fibers, such as a complete transection of a rat optic nerve, and ranges from about 107 to about 108 for treating a lesion affecting about 105 nerve fibers, such as a complete transection of a human optic nerve. As will be evident to those of skill in the art, the dose of T-cells can be scaled up or down in proportion to the number of nerve fibers affected at the lesion or site of injury being treated.
6. EXAMPLE: ACCUMULATION OF ACTIVATED T-CELLS IN INJURED CNS
6.1 MATERIALS AND METHODS
6.1.1 ANIMALS
Female Lewis rats were obtained from Harlan Olac
(Bicester, UK) , matched for age (8-12 weeks) and housed four to a cage in a light and temperature-controlled room.
6.1.2 PROTEINS USED FOR T-CELL ACTIVATION
Myelin basic protein (MBP) was prepared from guinea pig spinal cord as previously described (Ben-Nun et al . , supra (1982)). Chick ovalbumin (OVA) was purchased from Sigma (Israel) . Heat-inactivated Mycobacterium tuberculosis H37RA (M . tuberculosis) and Incomplete Freund's adjuvant (IFA) were purchased from Difco Laboratories (Detroit, MI, USA) .
6.1.3 MEDIA
The proliferation medium of the T-cells contained the following: Dulbecco's modified Eagle's medium (DMEM,
Biological Industries, Israel) supplemented with 2mM L- glutamine (L-Glu , Sigma , USA) , 5xlO~5M 2-mercaptoethanol ( 2- ME, Sigma) , penicillin (100 IU/ml; Biological Industries) , streptomycin (100 μg/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)). Propagation medium contained: DMEM, 2-ME, L-Glu, sodium pyruvate, non-essential amino acids and antibiotics in the same concentration as above and also 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) .
6.1.4 ANTIGENS
MBP from the spinal cords of guinea pigs was prepared as described (Hirshfeld, et al . , 1970, FEBS Lett . 7:317). OVA was purchased from Sigma (St. Louis, Missouri) . The p51-70 of the rat 18.5kDa isoform of MBP (sequence: APKRGSGKDSHTRTTHYG) SEQ ID NO: 2 and the p277 of the human hsp60 (sequence: VLGGGCALLRCPALDSLTPANED) SEQ ID NO: 3 (Elias, et al . , 1991, Proc . Natl . Acad . Sci . USA 88 , 3088-91) were synthesized using the 9-f Luorenylmethoxycarbonyl technique with an automatic multiple peptide synthesizer (AMS 422, ABIMED, Langenfeld, Germany). The purity of the peptides was analyzed by HPLC and amino acid composition.
6.1.5 T CELL LINES
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/ l) and emulsified with an equal volume of incomplete Freund's adjuvant (Difco
Laboratories, Detroit, Michigan) supplemented with 4 mg/ml Mycobacterium tuberculosis (Difco). The emulsion (0.1 ml) was injected into hind foot pads of the rats. Ten days after the antigen was injected, the rats were killed and draining lymph nodes were surgically removed and dissociated. The cells were washed and activated with the antigen (lOμg/ l) in proliferation medium containing Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine (2mM) , 2- mercaptoethanol (5xl0~5M) , sodium pyruvate (ImM) , penicillin (100 IU/ml) , streptomycin (100 μg/ml) , nonessential amino acids (1 ml/100 ml) and autologous rat serum 1% (volume/volume) . After incubation for 72 h at 37 °C, 90% relative humidity and 7% C02, the cells were transferred to propagation medium which additionally contained 10% fetal calf serum (FCS) (volume/volume) and 10% T cell growth factor derived from the supernatant of concanavalin A-stimulated spleen cells. Cells were grown in propagation medium for 4- 10 days before being re-exposed to antigen (lOμg/ml) in the presence of irradiated (2000 rad) thymus cells (107 cells/ml) in proliferation medium. The T-cell lines were expanded by repeated re-exposure and propagation.
6.1.6 LABELING OF T-CELLS
T-cells were washed and suspended in 10.7μm Hoechst 33342 Stain (Molecular Probes, USA) for 10 minutes at 37°C. The cells were washed twice with 50 ml volumes of PBS and then resuspended at 5 x 10ε cells/ml on ice until injection.
6.1.7 CRUSH INJURY OF RAT OPTIC NERVE
Crush injuries were performed as previously described (Hirschberg et al . , 1994, J. Neuroimmunol. 50:9-16). Briefly, rats were deeply anesthetized by i.p. injection of xylazine (10 mg/kg; Rompun) and ketamine (50 mg/kg; Velalar) . Under 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. A moderate crush injury was inflicted on the optic nerve, 2 mm from the eye, using a calibrated cross-action forceps (Duvdevani et al . , Instructure Neurology and Neuroscience , 2 : 31, 1990). The contralateral nerve was left undisturbed and was used as a control. 6.1.8 SECTIONING OF NERVES
At specified time points, rats were euthanized by over- anesthesia with ether and their optic nerves were surgically removed, immersed in Tissue-Tek (Miles Inc., USA), and frozen in liquid nitrogen cooled in iso-pentane (BDH, UK) . The nerves were then transferred to dry ice and stored at -70 °C until sectioning. Longitudinal cryostat nerve sections (20 μm thick) were picked up onto gelatin-coated glass slides (four sections per slide) and frozen at -20°C until viewed or prepared for fluorescence staining.
6.1.9 DATA ANALYSIS OF T-CELLS IN NERVE SECTIONS
Nerves excised at various time periods after injury were prepared and sectioned. Hoechst-labeled nuclei or immunostained cells in each section were counted using the fluorescence microscope. For each time point five sections were counted, and the numbers were averaged.
6.1.10 IMMUNOLABELING OF NERVE SECTIONS Longitudinal cryostat nerve sections (20 μm thick) were picked up onto gelatin glass slides and frozen until preparation for fluorescent staining. Sections were thawed and fixed in ethanol for 10 minutes at room temperature, washed twice with double-distilled water (ddH20) , and incubated for 3 minutes in PBS containing 0.05% polyoxyethylene-sorbitan monolaurate (Tween-20; Sigma, USA) . They were then incubated overnight at 4°C with a mouse monoclonal antibody directed against rat macrophages (EDI; 1:400; Serotec, UK) and antibody against rat glial fibrillary acidic protein (GFAP; 1:100; BioMakor) , all diluted in PBS containing 3% FCS. Staining of T-cells was accomplished by incubating nerve sections for 1 hr at room temperature with a mouse monoclonal antibody directed against rat T-cell receptor (TCR) (1:100, Hunig et al . , J . Exp . Med . , 169:73. 1989) , in PBS containing 3% FCS and 2% BSA. After three washes with PBS containing 0.05% Tween-20, the sections were incubated with goat anti-mouse F(ab')2 conjugated to either fluorescein isothiocyanate (FITC; BioMakor) or tetramethyl rhodamine isothiocyanate (TRITC; BioMakor) at a dilution of 1:100 and 1:50 respectively, for 1 hr at room temperature. They were then washed with PBS containing Tween-20 and treated with glycerol containing 1, 4-diazobicyclo- (2 ,2 ,2) octane (Sigma) , to inhibit quenching of fluorescence. The sections were viewed with a Zeiss Universal fluorescence microscope using filters that detect TRITC, FITC and Hoechst stains (Blaugrund et al . , Exp. Neurol . , 118: 105, 1992, Blaugrund et al . , Brain Res . , 574:244, 1992).
6.2 RESULTS
6.2.1 ACCUMULATION OF ACTIVATED T-CELLS
T-cell clones primed to MBP
Figure imgf000035_0001
were activated with MBP for 2 days before being labeled with Hoechst stain and injected into animals i.p. at the time of injury. At 3, 7, 14 and 21 days after injury, the nerves were excised, cryosectioned and analyzed microscopically for the presence of labeled T-cells. Tmp cells were detected in the injured optic nerves at day 3 and accumulated until a peak at day 14 (Fig. 1) . Large clusters of
Figure imgf000035_0002
cells were observed at the injury site and fewer individual cells were seen proximal and distal (Fig. 2) . Four weeks after injury, labeled T-cells were still detectable in the degenerating optic nerves. No T-cells were found in the non-injured optic nerves (Fig. 3) , non-injured sciatic nerve or injured sciatic nerve at any time after injury. Labeled T-cells were occasionally found in capillaries and in connective tissue but were not concentrated or localized into any specific areas. T-cells that were not prestimulated with antigen did not accumulate in any of the nerves, including damaged nerves.
The accumulation of Tmp cells in injured CNS, but not in injured PNS, suggests that there might be some specific interaction between the primed T-cells and the CNS tissue from which the MBP antigen was originally derived. To determine whether the injured CNS interacted with T-cells in general, or specifically with T-cells primed with a CNS antigen, the previous experiments were repeated using a clone that responds to chick ovalbumin (T0VA) . Rats were injected with a labeled T0VA clone prestimulated with ovalbumin (OVA) using the same protocol as with the
Figure imgf000036_0001
cells. The labeled T0VA cells accumulated in injured optic nerve, and the pattern of accumulation was similar to that of the
Figure imgf000036_0002
cells. Labeled T0VA and
Figure imgf000036_0003
cells were counted in longitudinal sections of optic nerve prepared, 3, 7, 14 and 21 days after injury. No significant different was observed in numbers of Trøp and T0VA cells in injured optic nerve (Fig. 4) , indicating that antigen specificity has little to do with the accumulation of T-cells in CNS injury sites.
Figure imgf000036_0004
cells were detectable slightly earlier than T0VA cells in the optic nerve injury site, and antigen specificity may play a role in this but is not sufficient to explain the large accumulation of T0VA cells in the site of injury.
Fig. 5 shows accumulation of T-cells measured immunocytochemically using antibodies to T-cell receptors. This detection technique rules out the possibility that the observed labeling is due to phagocytic cells which had phagocytized the pre-labeled T-cells shown in Fig. 1. The graph shows a striking elevation in T-cell accumulation following injury, regardless of whether the systematically injected T-cells are specific to a self-epitope (MBP) or to a non-self epitope (OVA) .
Fig. 6 shows that accumulation of T-cells is dependent on the lesion and not the breakdown of the blood-brain barrier. T-cells specific to either MBP or OVA were injected 2 weeks after injury and their accumulation analyzed a week later, namely 21 days following the primary lesion. Their accumulation was compared to that of T-cells injected immediately after injury and detected either 7 or 21 days later. It appears that the time elapsed between the injury and the injection of T-cells, which is a factor in the sealing of the blood-brain barrier, is not a factor in the T- cell accumulation. 7. EXAMPLE: USES OF ACTIVATED T-CELLS AND NS-SPECIFIC ATCs 7.1 MATERIALS AND METHODS
Animals, proteins used for T-cell stimulation, media, crush injury of rat optic nerve, sectioning of nerves, immunolabeling of nerve sections, and data analysis of
T-cells in nerve sections are described in Section 6, supra.
7.1.1 ESTABLISHMENT OF T-CELL LINES
WITH ACTIVE EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE)
MBP and OVA were dissolved in PBS (1 mg/ml) and emulsified with an equal volume of Incomplete Freund's Adjuvant (IFA) supplemented with 4 mg/ml M . tuberculosis . Rats were immunized subcutaneously in the hind footpads with 0.1 ml of the emulsion. At day 9 (1-3 days before clinical onset of disease) , animals were euthanized and draining lymph nodes were surgically removed and dissociated under sterile conditions. The cells were washed and placed in proliferation medium with irradiated thy ocytes (2000 rads) and either 10 μg/ml of MBP, OVA or M . tuberculosis for 3 days. Cells were then washed and placed in propagation medium for 5 to 10 days at which time they were re-exposed to irradiated thymocytes and peptides in proliferation medium. T-cell lines were expanded by re-exposure and propagation and tested for specificity in an antigen specific T cell proliferation assay. Lines were expanded and stocks were frozen in liquid nitrogen. The cells were thawed and stimulated once before being used in experiments.
7.1.2 PASSIVE TRANSFER OF T-CELL LINES
T-cell lines were activated by restimulation in vitro with their own antigen (10 μg/ml) in proliferation medium. After incubation for 48-72 hrs at 37 °C 90% relative humidity and 7.5% C02, the cells were washed. Viable cells were isolated on Percoll and suspended in PBS. Animals were injected with 10 x 106 cells/ml i.p. Control animals were injected with 1 ml PBS i.p. 7.1.3 CRUSH INJURY OF RAT SCIATIC NERVE
Under deep anesthesia as described in Section 6.1.5, the sciatic nerve was exposed and a similar crush injury was inflicted. At the end of the operation the skin was sutured.
7.1.4 RETROGRADE LABELING OF RGCs
The optic nerve was exposed, without damaging the retinal blood supply. Solid crystals of the dye, 4- (4- (didecylamino) styryl) -n-methyl-pyridinium iodide (4-Di-10- Asp) (Molecular Probes, Europe BV) , were deposited 1-2 mm from the distal border of the injury site. Non-injured optic nerves were similarly labeled at approximately the same distance from the globe. Five days after dye application, the retinas were excised under deep anesthesia, flat mounted in 4% paraformaldehyde solution, and labeled retinal ganglion cells (RGCs) were counted by fluorescence microscopy.
7.1.5 ASSESSMENT OF EFFECTS OF INJECTED T-CELLS
The effect of injected T-cells on the numbers of surviving optic nerve fibers was monitored by retrograde labeling of RGCs (see above) immediately after injury in order to assess primary degeneration and two weeks later in order to assess secondary degeneration. Five days after dye (4-Di-10-Asp) application, the retinas were excised, whole mounted and their RGCs were counted. The counting was done in five randomly selected fields in each retina (all located at approximately the same distance from the optic disc) . In all cases the dye was applied 2 ml distally to the site of the prior insert. Using this lengthening approach, only those RGCs whose axons were still viable could be labeled. The numbers of RGCs in each group of injured nerves treated with PBS only were injected with
Figure imgf000038_0001
or T0VA cells. Results were expressed as percentage of axons, out of those which survived the primary insult (42% the axons remained after the primary insult) . 7.1.6 CLINICAL EVALUATION OF EAE
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, moribound state.
7.2 RESULTS
7.2.1 ACCUMULATION OF NS-SPECIFIC ATCS
The injured optic nerve was analyzed for T-cell accumulation. As shown in Fig. 7, in the uninjured optic nerves of control rats injected with phosphate-buffered saline (PBS) no T-cells could be detected. Small but significant numbers of T-cells were observed in the uninjured optic nerves of rats injected with anti-MBP T-cells (primed against a peptide comprising amino acids 51-70 of MBP \P52- 70 " known to be capable of inducing experimental autoimmune encephalomyelitis (EAE) under these experimental conditions) , but not of rats injected with anti-OVA T-cells. Crush injury of the optic nerve was accompanied by a small but significant accumulation of endogenous T-cells, possibly reflecting a response to self antigens triggered by the injury. In the injured optic nerves, T-cell accumulation was significantly increased by 5- to 6-fold) in rats injected with anti-OVA, anti-hsp60, or anti-MBP T-cells. These observations confirmed our previous finding that axonal injury in the CNS is accompanied by the accumulation of endogenous T-cells and that this accumulation is augmented by systemic injection of activated T-cells irrespective of their antigenic specificity.
7.2.2 PROTECTION OF SECONDARY NERVE
DEGENERATION BY MBP SPECIFIC T-CELLS
The course of secondary degeneration as a result of the injected T-cells was then examined. Previous studies have shown that a time lapse of 2 weeks between a crush injury (of similar severity to the present one) and dye application is optimal for demonstrating differences (in terms of the numbers of still-viable, i.e. labeled, neurons) in degeneration with and without neuroprotection. As shown in Fig. 8, in retinas of injured nerves that were subjected to dye application 2 weeks after injury and excised a week later, the number of labeled ganglion cells (reflecting still-viable axons) was about 2.5-fold greater in animals injected at the time of injury, with T-cells specific to MBP (primed against P51-70) than with PBS. In contrast, labeled ganglion cells in the retinas of rats injected with anti-OVA or anti-hsp60 T-cells were not significantly more numerous than in the retinas of rats injected with PBS. Fig. 9 represents micrographs of retrogradeably labeled retinas of injured optic nerves of rats injected with PBS, anti-hsp60 T- cells, or anti-MBP T-cells. Since only the anti-MBP T-cells showed a neuroprotective effect, and as hsp60, like MBP, is a self antigen which is expressed in injured tissues including EAE lesions, it was interesting to find out whether the protective effect of the anti-MBP T-cells is a function of their aggressiveness in causing an autoimmune disease. If so, this would explain the lack of a protective effect by hsp60, which is also a self antigen, but, unlike MBP, is not restricted to the CNS and the T-cells specific to it do not cause a disease. To explore the possibility of a connection between the observed neuroprotective effect and autoimmune disease, the effect of T-cells generated against an epitope in MBP (P51-70) that does not cause an autoimmune disease was examined. As shown in Fig. 10, the neuroprotective effect of these non- aggressive anti-MBP T-cells was similar to that of the anti- MBP T-cells that cause autoimmune diseases. It thus seems that the observed beneficial effect of the T-cells on secondary degeneration is not common to all self antigens, but in this study is restricted to NS-specific antigens. Furthermore, T-cells activated with a fragment of a NS- specific antigen that does not cause autoimmune disease were substantially as effective in inhibiting secondary degeneration as T-cells activated with full-length NS- specific antigen that does cause autoimmune disease.
7.2.3 CLINICAL SEVERITY OF EAE Animals were injected i.p. with 107
Figure imgf000041_0001
cells with or without concurrent optic nerve crash injury. The clinical course of the rats injected with the Tmp cells was evaluated according to the neurological paralysis scale. Each group contained 5-9 rats. As can be seen in Fig. 11, the course and degree of the EAE was not affected by whether or not the rats had been subjected to an optic nerve crash.
7.2.4 SURVIVAL OF RGCS IN NON-INJURED NERVES
Animals were injected i.p. with 107 Tmp cells or PBS. Two weeks later, 4-Di-10-Asp was applied to the optic nerves. After five days the retinas were excised and flat mounted. Labeled RGCs from five fields, in each retina were counted and their average number per mm2 was calculated.
As can be seen in Fig. 12, there is no difference in the number of surviving RGCs in non-injured optic nerves of rats injected with anti-MBP T-cells compared to rats injected with PBS.
8. EXAMPLE: NEUROPROTECTIVE EFFECTS OF NS-SPECIFIC ANTIGEN 8.1 MATERIALS AND METHODS
Animals, crush injury of rat optic nerve, and retrograde labeling are described above in Sections 6 and 7. A peptide based on amino acids 35-55 of myelin/oligodendrocyte glycoprotein (MOG p35-55) was chemically synthesized at the Weizmann Institute, Israel.
8.1.1 INHIBITION OF SECONDARY DEGENERATION
Rats were injected intradermally in the footpads with MOG p35-55 (50 μg/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. 5
8.2 RESULTS
As shown in Fig. 13, the number of labeled ganglion cells (indicating viable axons) was about 12.5 fold greater in animals injected with MOG p35-55 compared to animals 0 receiving PBS.
9. EXAMPLE: NEUROPROTECTIVE EFFECTS OF MBP ADMINISTERED ORALLY
9.1 MATERIALS AND METHODS 5 Animals, crush injury of rat optic nerve, and retrograde labeling of RGCs are described above in Sections 6 and 7.
9.1.1 INHIBITION OF SECONDARY DEGENERATION
Bovine MBP (Sigma, Israel) (1 mg/dose) was administered to rats by gavage using a blunt needle. MBP was administered 0 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. 5
9.2 RESULTS
As shown in Fig. 14, the number of labeled RGCs was about 1.3 fold greater in animals treated with MBP compared to untreated animals.
30
10. DISCUSSION
The results of the experiments described in Sections 6 and 7 show that activated T-cells accumulate at a site of __ injury in the CNS. Furthermore, the results also demonstrate
•39 that the accumulation of T-cells at the site of injury is a non-specific process, i.e., 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 a 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 causes autoimmune disease (1mτ,) , 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 a 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 of T-cells which recognize a NS-specific self antigen which is present at a site of injury.
In addition, the studies described in Sections 8 and 9 show that activation of T-cells by administering an immunogenic antigen (e.g. MBP) or immunogenic epitope of an antigen ( e .g. MOG p35-55) , may be used for preventing or inhibiting secondary CNS degeneration following injury.
The present application claims priority benefits of
Israeli patent application IL 124550, filed May 19, 1998, the disclosure of which is incorporated herein by reference in its entirety.
The present invention is not to be limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims . All publications cited herein are incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS:
1. A method for delivering a therapeutic or detectable substance to a site of injury or disease of the central nervous system (CNS) , comprising administering activated T-cells that do not recognize a nervous system specific (NS-specific) antigen in which the activated T-cells contain or express a therapeutic or detectable substance to a mammal.
2. The method according to claim 1 in which said activated T-cells are generated by exposing T-cells to a cognate non-self antigen or a mitogen.
3. The method according to claim 1 in which said activated T-cells endogenously produce said substance.
4. The method according to claim 1 in which said activated T-cells are T-cells that have been genetically- engineered to express said therapeutic substance or to express an enzyme which catalyzes production of said therapeutic substance or to express a regulatory product, that induces production of said therapeutic substance.
5. The method according to claim 1 in which said injury comprises blunt trauma, penetrating trauma, hemorrhagic stroke, or ischemic stroke.
6. The method according to claim 1 in which said disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, glaucoma, Huntington's disease, amyotrophic lateral sclerosis, and Creutzfeldt-Jakob disease.
7. A recombinant T-cell comprising a promoter operably linked to a nucleotide sequence that encodes a protein that ameliorates the effects of an injury or disease of the central nervous system (CNS) wherein the recombinant T-cells do not recognize a nervous system specific (NS- specific) antigen.
8. A method for delivering a substance to a site of injury or disease of the CNS, comprising administering to a mammal the recombinant T-cell of claim 7.
9. The method according to claim 1 or 8 in which said mammal is a human.
10. The method according to claim 8 in which said recombinant T-cell is produced using an autologous T-cell.
11. A method for detecting a site of injury or disease of the central nervous system (CNS) in a mammal, comprising: a) administering to a mammal an effective amount of labeled activated T-cells that do not recognize a nervous system specific (NS- specific) antigen; and b) detecting in the mammal the labeled activated T-cells that have accumulated at said site of injury or disease, in which step (b) is performed after an interval sufficient to permit said labeled activated T-cells administered in step (a) to accumulate at said site of injury or disease.
12. The method according to claim 11 in which the mammal is a human.
13. The method according to claim 1, 8 or 11 in which the activated T-cells recognize a non-self antigen.
14. The method according to claim 11 in which the detectable substance is detected in vivo.
15. The method of claim 11 in which the labeled activated T-cells are labeled with a radioisotope or a contrast agent.
16. The method of claim 1, 8 or 11 in which the labeled activated T-cells are administered intravenously or intraperitoneally .
17. A method for preventing or inhibiting degeneration in the central nervous system (CNS) of a mammal comprising administering non-recombinant, NS-specific antiself activated T-cells in which said activated T-cells ameliorate the effects of injury or disease.
18. A method for preventing or inhibiting degeneration in the central nervous system (CNS) of a mammal comprising administering an effective amount of a NS-specific antigen.
19. The method according to claim 18 in which said
NS-specific antigen activates T-cells in vivo resulting in a population of T-cells that accumulate at a site of injury or disease of the CNS.
20. The method according to claim 18 in which said
NS-specific antigen is administered intravenously, intraperitoneally, orally, nasally or buccally.
21. The method according to claim 2 in which said T-cells are autologous.
22. A pharmaceutical composition comprising isolated, activated T-cells that do not recognize a nervous system specific (NS-specific) antigen in which the activated T-cells contain an exogenously added detectable substance or contain a therapeutic substance or express a recombinant substance having a therapeutic effect when administered in vivo to a mammal; and a pharmaceutically acceptable carrier.
23. A pharmaceutical composition comprising a therapeutically effective amount of an isolated, NS-specific antigen; and a pharmaceutically acceptable carrier.
24. Use of an active ingredient comprising non- recombinant NS-specific antiself T-cells or a NS-specific antigen, for the preparation of a pharmaceutical composition for the treatment of a human condition or disease of the CNS.
25. Use according to claim 24 in which the untreated human condition or disease results in central nervous system degeneration.
26. Use of an active ingredient comprising recombinant, activated T-cells that do not recognize a nervous system specific (NS-specific) antigen, for the preparation of a pharmaceutical composition for the treatment of a human condition or disease of the CNS.
AMENDED CLAIMS
[received by the International Bureau on 21 December 1998 (21.12.98); original claims 1-26 replaced by new claims 1-32 (5 pages)]
1. A method for delivering a therapeutic or detectable substance to a site of injury or disease of the central nervous system (CNS), comprising administering to a
5 mammal activated T-cells that do not recognize a nervous system specific (NS-specific) antigen in which the activated T-cells contain or express a therapeutic or detectable substance.
2. The method according to claim 1 in which said activated T-cells are 0 generated by exposing T-cells to a non-self antigen or a mitogen.
3. The method according to claim 1 in which said activated T-cells endogenously produce said therapeutic substance.
" 4. The method according to claim 1 in which said activated T-cells are
T-cells that have been genetically-engineered to express said therapeutic substance or to express an enzyme which catalyzes production of said therapeutic substance or to express a regulatory product, that induces production of said therapeutic substance.
20 5. The method according to claim 4 in which the genetically engineered T-cells express said therapeutic substance or enzyme or regulatory product under the control of necessary elements for transcription or translation continuously or after induction.
6. The method according to claim 1 in which said injury comprises
25 blunt trauma, penetrating trauma, trauma sustained duπng a neurosurgical operation, hemorrhagic stroke, or ischemic stroke.
7. The method according to claim 1 in which said disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, glaucoma, optic nerve injury accompanying optic neuropathy, Huntington's disease, amyotrophic lateral sclerosis, and Creutzfeldt-Jakob disease.
AMENDED~SHEET (ARTICLE 19)
8. A recombinant T-cell comprising a promoter operably linked to a nucleotide sequence that encodes a protein that ameliorates the effects of an injury or disease of the central nervous system (CNS) wherein the recombinant T-cells do not recognize a nervous system specific (NS-specific) antigen.
9. The recombinant T-cell according to claim 8 in which the protein is a therapeutic substance or an enzyme which catalyzes production of said therapeutic substance or a regulatory product, that induces production of a therapeutic substance.
10. A method for delivering a substance to a site of injury or disease of the CNS, comprising administering to a mammal the recombinant T-cell of claim 8.
11. The method according to claim 1 or 10 in which said mammal is a human.
12. The method according to claim 10 in which said recombinant T-cell is produced using an autologous T-cell.
13. A method for detecting a site of injury or disease of the central nervous system (CNS) in a mammal, comprising: a) administering to a mammal an effective amount of labeled activated T-cells that do not recognize a nervous system specific (NS-specific) antigen; and b) detecting in the mammal the labeled activated T-cells that have accumulated at said site of injury or disease, in which step (b) is performed after an interval sufficient to permit said labeled activated T-cells administered in step (a) to accumulate at said site of injury or disease.
14. The method according to claim 13 in which the mammal is a human.
15. The method according to claim 1, 10 or 13 in which the activated T- cells recognize a non-self antigen.
16. The method of claim 13 in which the labeled activated T-cells are 5 labeled with a radioisotope, a contrast agent or fluorescence-emitting metal.
17. The method of claim 1, 10 or 13 in which the activated T-cells are administered intravenously, intraperitoneally, intramuscularly or subcutaneously.
0 18. The method according to claim 2 in which said T-cells are autologous.
19. A pharmaceutical composition comprising isolated, activated T-cells that do not recognize a nervous system specific (NS-specific) antigen for use to deliver a
" therapeutic or detectable substance in which the activated T-cells contain a therapeutic substance or express a recombinant substance having a therapeutic effect or contain an exogenously added detectable substance when administered in vivo to a mammal; and a pharmaceutically acceptable carrier.
20 20. The pharmaceutical composition according to claim 19 in which said activated T-cells are generated by exposing T-cells to a non-self antigen or a mitogen.
21. The pharmaceutical composition according to claim 19 in which said activated T-cells endogenously produce said therapeutic substance. 25
22. The pharmaceutical composition according to claim 19 in which said activated T-cells are T-cells that have been genetically-engineered to express said therapeutic substance or to express an enzyme which catalyzes production of said therapeutic substance or a regulatory product, that induces production of said therapeutic substance.
23. The pharmaceutical composition according to claim 19 in which said activated T-cells express said therapeutic substance or enzyme or regulatory product under the control of necessary elements for transcription or translation continuously or after induction.
24. The pharmaceutical composition according to claim 19 in which said activated T-cells are used to treat injury comprising blunt trauma, penetrating trauma, penetrating trauma, trauma sustained during a neurosurgical operation, hemorrhagic stroke, or ischemic stroke.
25. The pharmaceutical composition according to claim 19 in which said activated T-Cells are used to treat disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, glaucoma, optic nerve injury accompanying optic neuropathy, Huntington's disease, amyotrophic lateral sclerosis, and Creutzfeldt- Jakob disease.
26. Use of an active ingredient comprising activated T-cells that do not recognize a nervous system specific (NS-specific) antigen, for the preparation of a pharmaceutical composition for the treatment or detection of a human condition or disease of the CNS.
27. The use according to claim 26 in which said activated T-cells are generated by exposing T-cells to a non-self antigen or a mitogen.
28. The use according to claim 26 in which said activated T-cells endogenously product said therapeutic substance.
29. The use according to claim 26 in which said activated T-cells are T- cells that have been genetically-engineered to express said therapeutic substance or to express an enzyme which catalyzes production of said therapeutic substance or to express a regulatory product, that induces production of said therapeutic substance.
30. The use according to claims 26 in which said activated T-cells express said therapeutic substance or enzyme or regulatory product under the control of necessary elements for transcription or translation continuously or after induction.
31. The use according to claim 26 in which said activated T-cells are used to treat injury comprising blunt trauma, penetrating trauma, trauma sustained during a neurosurgical operation, hemorrhagic stroke, or ischemic stroke.
32. The use according to claim 26 in which said activated T-cells are used to treat disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, glaucoma, optic nerve injury accompanying optic neuropathy, Huntington's disease, amyotrophic lateral sclerosis, and Creutzfeldt-Jakob disease.
PCT/US1998/014715 1997-07-21 1998-07-21 Activated t-cells and their uses WO1999034827A1 (en)

Priority Applications (16)

Application Number Priority Date Filing Date Title
JP2000527274A JP2002500199A (en) 1998-07-21 1998-07-21 Activated T cells and their uses
IL13415998A IL134159A0 (en) 1997-07-21 1998-07-21 Activated t-cells and their uses
PCT/US1998/014715 WO1999034827A1 (en) 1998-07-21 1998-07-21 Activated t-cells and their uses
CA002296999A CA2296999A1 (en) 1997-07-21 1998-07-21 Activated t-cells and their uses
EP98936862A EP1039929A1 (en) 1998-07-21 1998-07-21 Activated t-cells and their uses
AU85717/98A AU755872B2 (en) 1997-07-21 1998-07-21 Activated T-cells and their uses
US09/218,277 US20030108528A1 (en) 1998-05-19 1998-12-22 Activated t-cells, nervous system-specific antigens and their uses
PCT/US1999/010953 WO1999060021A2 (en) 1998-05-19 1999-05-19 Use of activated t cells, nervous system-specific antigens for treating disorders of the nevrous system
AU40017/99A AU768763B2 (en) 1998-05-19 1999-05-19 Activated T cells, nervous system-specific antigens and their uses
IL13952599A IL139525A0 (en) 1998-05-19 1999-05-19 Activated t cells nervous system-specific antigens and their uses
CA002328612A CA2328612A1 (en) 1998-05-19 1999-05-19 Activated t cells, nervous system-specific antigens and their uses
EP99923190A EP1080110A2 (en) 1998-05-19 1999-05-19 Use of activated t cells, nervous system-specific antigens for treating disorders of the nevrous system
JP2000549639A JP2003532618A (en) 1998-05-19 1999-05-19 Cells, nervous system-specific antigens and their uses
US09/893,348 US20020072493A1 (en) 1998-05-19 2001-06-28 Activated T cells, nervous system-specific antigens and their uses
US10/810,653 US7560102B2 (en) 1998-05-19 2004-03-29 Method for reducing neuronal degeneration so as to ameliorate the effects of injury or disease
US11/563,630 US20080279869A1 (en) 1998-05-14 2006-11-27 Method for reducing neuronal degeneration by administering cns-derived peptides or activated t cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1998/014715 WO1999034827A1 (en) 1998-07-21 1998-07-21 Activated t-cells and their uses

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09/218,277 Continuation-In-Part US20030108528A1 (en) 1998-05-14 1998-12-22 Activated t-cells, nervous system-specific antigens and their uses

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09/218,277 Continuation-In-Part US20030108528A1 (en) 1998-05-14 1998-12-22 Activated t-cells, nervous system-specific antigens and their uses
US31416199A Continuation-In-Part 1998-05-14 1999-05-19

Publications (1)

Publication Number Publication Date
WO1999034827A1 true WO1999034827A1 (en) 1999-07-15

Family

ID=22267491

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/014715 WO1999034827A1 (en) 1997-07-21 1998-07-21 Activated t-cells and their uses

Country Status (3)

Country Link
EP (1) EP1039929A1 (en)
JP (1) JP2002500199A (en)
WO (1) WO1999034827A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003002140A1 (en) * 2001-06-28 2003-01-09 Yeda Research And Development Co. Ltd Use of poly-glu, tyr and t cells treated therewith for neuroprotection therapy
EP1572064A2 (en) * 2001-01-14 2005-09-14 YEDA RESEARCH AND DEVELOPMENT Co. LTD. Pharmaceutical compositions comprising modified cns-derived peptides for promoting nerve regeneration and prevention of nerve degeneration
US7351686B2 (en) 2001-12-06 2008-04-01 Yeda Research And Development Co. Ltd. Method for neuronal protection in amyotrophic lateral sclerosis by a vaccine comprising Copolymer-1 or Copolymer-1 related peptides
US7399740B2 (en) 2001-06-28 2008-07-15 Yeda Research And Development Co. Ltd. Poly-Glu,Tyr for neuroprotective therapy
US7560102B2 (en) 1998-05-19 2009-07-14 Yeda Research And Development Co., Ltd Method for reducing neuronal degeneration so as to ameliorate the effects of injury or disease
WO2014176604A1 (en) * 2013-04-26 2014-10-30 Cel-Sci Corporation Methods of preparation and composition of peptide constructs useful for treatment of rheumatoid arthritis
US10179174B2 (en) 2011-05-25 2019-01-15 Cel-Sci Corp. Method for inducing an immune response and formulations thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BROD S, ET AL.: "AUTOLOGOUS T-T CELL ACTIVATION MEDIATED BY CELL ADHESION MOLECULES", THE FASEB JOURNAL, FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY, US, vol. 03, no. 03, 1 January 1989 (1989-01-01), US, pages A514, XP002913572, ISSN: 0892-6638 *
LOHSE A W, ET AL.: "CONTROL OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS BY T CELLS RESPONDING TO ACTIVATED T CELLS", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 244, 19 May 1989 (1989-05-19), US, pages 820 - 822, XP002913568, ISSN: 0036-8075, DOI: 10.1126/science.2471264 *
LOHSE A. W., ET AL.: "INDUCTION OF THE ANTI-ERGOTYPIC RESPONSE.", INTERNATIONAL IMMUNOLOGY., OXFORD UNIVERSITY PRESS., GB, vol. 05., no. 05., 1 May 1993 (1993-05-01), GB, pages 533 - 539., XP002913571, ISSN: 0953-8178 *
LOHSE A. W., ET AL.: "INHIBITION OF THE MIXED LYMPHOCYTE REACTION BY T CELL VACCINATION.", EUROPEAN JOURNAL OF IMMUNOLOGY, WILEY - V C H VERLAG GMBH & CO. KGAA, DE, vol. 20., 1 November 1990 (1990-11-01), DE, pages 2521 - 2524., XP002913570, ISSN: 0014-2980, DOI: 10.1002/eji.1830201126 *
YUEN M.-H., ET AL.: "IMMUNOREGULATORY CD8+ CELLS RECOGNIZE ANTIGEN-ACTIVATED CD4+ CELLS IN MYASTHENIA GRAVIS PATIENTS AND IN HEALTHY CONTROLS.", THE JOURNAL OF IMMUNOLOGY, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, US, vol. 154., no. 03., 1 January 1995 (1995-01-01), US, pages 1508 - 1520., XP002913569, ISSN: 0022-1767 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7560102B2 (en) 1998-05-19 2009-07-14 Yeda Research And Development Co., Ltd Method for reducing neuronal degeneration so as to ameliorate the effects of injury or disease
EP1572064A2 (en) * 2001-01-14 2005-09-14 YEDA RESEARCH AND DEVELOPMENT Co. LTD. Pharmaceutical compositions comprising modified cns-derived peptides for promoting nerve regeneration and prevention of nerve degeneration
EP1572064A4 (en) * 2001-01-14 2006-12-06 Yeda Res & Dev Pharmaceutical compositions comprising modified cns-derived peptides for promoting nerve regeneration and prevention of nerve degeneration
WO2003002140A1 (en) * 2001-06-28 2003-01-09 Yeda Research And Development Co. Ltd Use of poly-glu, tyr and t cells treated therewith for neuroprotection therapy
US6835711B2 (en) 2001-06-28 2004-12-28 Yeda Research And Development Co. Ltd. Use of poly-Glu,Tyr for neuroprotective therapy
CN1326560C (en) * 2001-06-28 2007-07-18 耶达研究及发展有限公司 Use of POLY-GLU,TYR and T cells treated therewith for neuroprotective therapy
US7399740B2 (en) 2001-06-28 2008-07-15 Yeda Research And Development Co. Ltd. Poly-Glu,Tyr for neuroprotective therapy
US7351686B2 (en) 2001-12-06 2008-04-01 Yeda Research And Development Co. Ltd. Method for neuronal protection in amyotrophic lateral sclerosis by a vaccine comprising Copolymer-1 or Copolymer-1 related peptides
US10179174B2 (en) 2011-05-25 2019-01-15 Cel-Sci Corp. Method for inducing an immune response and formulations thereof
WO2014176604A1 (en) * 2013-04-26 2014-10-30 Cel-Sci Corporation Methods of preparation and composition of peptide constructs useful for treatment of rheumatoid arthritis

Also Published As

Publication number Publication date
JP2002500199A (en) 2002-01-08
EP1039929A1 (en) 2000-10-04

Similar Documents

Publication Publication Date Title
EP1248643B1 (en) The use of copolymer 1 and related peptides and polypeptides and t cells treated therewith for neuroprotective therapy
US7560102B2 (en) Method for reducing neuronal degeneration so as to ameliorate the effects of injury or disease
EP1294390B1 (en) The use of copolymer 1 and related peptides and polypeptides and t cells treated therewith for neuroprotection from glutamate toxicity
WO1999060021A2 (en) Use of activated t cells, nervous system-specific antigens for treating disorders of the nevrous system
US6835711B2 (en) Use of poly-Glu,Tyr for neuroprotective therapy
WO1999034827A1 (en) Activated t-cells and their uses
AU755872B2 (en) Activated T-cells and their uses
AU768763B2 (en) Activated T cells, nervous system-specific antigens and their uses
JP2005504716A (en) Pharmaceutical composition comprising a modified CNS-derived peptide for promoting nerve regeneration and preventing neurodegeneration
MXPA00000840A (en) Activated t-cells and their uses
EP2234633A1 (en) A method of treating neurodegenerative diseases
MXPA00011385A (en) Activated t cells, nervous system-specific antigens and their uses

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 134159

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: 09218277

Country of ref document: US

AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AU AZ BA BB BG BR BY CA CN CU CZ EE GE HR HU ID IL IS JP KG KP KR KZ LC LK LR LT LV MD MG MK MN MX NO NZ PL RO RU SG SI SK SL TJ TM TR TT UA US UZ VN YU

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2296999

Country of ref document: CA

Ref country code: CA

Ref document number: 2296999

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 09463186

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: PA/a/2000/000840

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 1998936862

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 85717/98

Country of ref document: AU

WWP Wipo information: published in national office

Ref document number: 1998936862

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 85717/98

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 1998936862

Country of ref document: EP