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WO2001052877A1 - Compositions et techniques pour traiter des troubles neurodegeneratifs - Google Patents

Compositions et techniques pour traiter des troubles neurodegeneratifs Download PDF

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Publication number
WO2001052877A1
WO2001052877A1 PCT/US2001/001671 US0101671W WO0152877A1 WO 2001052877 A1 WO2001052877 A1 WO 2001052877A1 US 0101671 W US0101671 W US 0101671W WO 0152877 A1 WO0152877 A1 WO 0152877A1
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complexes
heat shock
disease
composition
cells
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PCT/US2001/001671
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Pramod K. Srivastava
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University Of Connecticut Health Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • 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
    • 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
    • A61K38/1716—Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K38/1738—Calcium binding proteins, e.g. calmodulin
    • 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
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/414—Nervous system antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4262—Heat shock proteins
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6043—Heat shock proteins
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/622—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier non-covalent binding

Definitions

  • the present invention relates to compositions comprising complexes of heat shock proteins non-covalently or covalently bound to peptides associated with a neurodegenerative disorder such as Alzheimer's Disease.
  • the invention further relates to methods of using the neurodegenerative disorder-associated heat shock protein-peptide complexes, including Alzheimer's Disease-associated heat shock protein-peptide complexes, in the prevention and treatment of neurodegenerative disorders such as Alzheimer's Disease.
  • Neuropsychiatric and neurodegenerative disorders beginning to be understood at the molecular level. Strikingly similar pathologies commonly associated with the neurodegenerative disorders can be arrived at by a large number of different genetic mechanisms. For example, a pathogenic mutation in the prion gene results in both tangle and Lewy body pathologies of prion disease (Feany and Kickson, 1995, Am. J. Pathol. 146: 1388).
  • AD Alzheimer's Disease
  • PHFs paired helical filaments
  • Other physical changes may include microvascular amyloidosis and dystrophic cortical neurites (for a review on the pathological hallmarks of AD, see Sobow, 1996, Folia Neuropathol. 34:55-62).
  • Neurofibrillary tangles consist of the intermediate filament protein Tau.
  • Tau is an unphosphorylated protein but is found to be phosphorylated in PHFs.
  • Amyloid plaques also called senile plaques, consist of amyloid- ⁇ -peptide (A ⁇ ), a product of the cleavage of an amyloid precursor protein (APP).
  • a ⁇ amyloid- ⁇ -peptide
  • APP amyloid precursor protein
  • AD Alzheimer's disease
  • the presenilins are membrane proteins having eight transmembrane domain and have been recently shown to be required for the processing of Notch protein (De Strooper et al, 1999, Nature 398:518-522; Struhl and Greenwald, ibid. :522-525; Ye et al, ibid. :525-529).
  • mutations in PS2 are not completely penetrant and the age of onset of AD in carriers of PSl mutations varies from 40 to 88 years (Renbaum and Levy-Lahad, 1998, Cell Mol. Life Sci. 54:910- 919).
  • AD late onset AD
  • no specific gene mutations are associated with the inheritance of AD; however, specific alleles of apolipoprotein e4 and 2 macro globulin are associated with increased risk for AD.
  • AD pathology a common theme in AD pathology is the production of aberrant structures that are not normally found in healthy brains. This observation is supported by the finding that PHFs are associated with ubiquitin, which would normally lead to the degradation of the associated molecule but in AD does not do so.
  • AD Alzheimer's Association
  • diagnostic tests based on the presence of Tau peptides in a patient or subject's blood (U.S. Patent No. 5,492,812) or the presence of p33 (Annexin V) proteins in a patient or subject's cerebrospinal fluid (U.S. Patent No. 5,849,600) have been proposed.
  • genetic testing for example to determine which allele(s) of APP, PSl or PS2 an individual carries, can be carried out to determine the individual's risk of developing AD.
  • AD Alzheimer's disease
  • AD cognitive impairment associated with AD
  • AchEIs acetylcholinesterase inhibitors
  • tacrine hydrochloride CognexTM; Warner Lambert
  • Other putative treatments for AD are currently being tested in clinical trials, for example anti- inflammatory drugs and ⁇ -tocopherol (Shadlen and Larson, 1999, Postgrad. Med. 105:109- 118).
  • anti- inflammatory drugs and ⁇ -tocopherol Shadlen and Larson, 1999, Postgrad. Med. 105:109- 118.
  • Heat shock protems also referred to interchangeably as stress protems, were first identified as protems synthesized by a cell in response to heat shock. To date, five major classes of hsps have been identified, based on the molecular weight of the family members. These classes are called shsps (small heat shock proteins), Hsp60, Hsp70, Hsp90, and HsplOO, where the numbers reflect the approximate molecular weight of the hsps in kilodaltons.
  • shsps small heat shock proteins
  • Heat shock proteins are highly conserved proteins. For example, DnaK, the Hsp70 from E.
  • Hsp70 proteins from excoriates (Bardwell et al, 1984, Proc. Natl. Acad. Sci. U.S.A. 81: 848-852).
  • the Hsp60 and Hsp90 families also show similarly high levels of intra-family conservation (Hickey et al, 1989, Mol. Cell. Biol. 9: 2615-2626; Jindal, 1989, Mol. Cell. Biol. 9: 2279-2283).
  • Hsps are involved not only in cellular protection against adverse conditions, but are also involved in essential biochemical and imrnunological processes in unstressed cells.
  • hsps are involved in various kinds of chaperoning functions.
  • Members of the Hsp70 family, located in the cell cytoplasm, nucleus, mitochondria, or endoplasmic reticulum are involved in the presentation of antigens to the cells of the immune system, and are also involved in the transfer, folding and assembly of proteins in normal cells (Lindquist et al, 1988, Ann. Rev. Genetics 22: 631-677).
  • a number of protems thought to be involved in chaperoning are residents of the endoplasmic reticulum (ER) lumen, for example, protein disulfide isomerase (PDI; Gething et al, 1992, Nature 355: 33-45), Grp94 or ERp99 (Sorger & Pelham, 1987, J. Mol. Biol. 194: 991-94) which is related to Hsp90, and Grp78 or BiP, which is related to Hsp70 (Munro et al, 1986, Cell 46: 291-300; Haas & Webl, 1983, Nature 306: 387-389).
  • PDI protein disulfide isomerase
  • Grp94 or ERp99 Sorger & Pelham, 1987, J. Mol. Biol. 194: 991-94
  • Grp78 or BiP which is related to Hsp70 (Munro et al, 1986, Cell 46: 291-300; Haas & Webl, 1983,
  • hsps as protein chaperones extends to forming complexes with antigenic molecules and presenting them to the immune system, for example in cancer cells (Srivastava et al, 1996, Proc. Natl. Acad. Sci. USA 83:3407-3411; Ullrich et al., 1996, Proc. Natl. Acad. Sci. USA 83:3121-3125). Immunization of mice with hsp complexes derived from a particular tumor rendered the mice immune to that particular tumor, but not to antigenically distinct tumors (Udono and Srivastava, 1993, J. Exp. Med. 178:1391-1396).
  • hsp-antigen complexes may be used in combination with adoptive immunotherapy, i.e. in combination with antigen presenting cells that contain the hsp-antigen complexes (see e.g. U.S. Patent Nos. 5,830,464 and 5,985,270).
  • the present invention provides a composition comprising a purified population of complexes of heat shock proteins bound to endogenous antigenic molecules, said complexes being purified fiOm mammalian tissues or cells, which tissues or cells display a pathology associated with a ND or which population comprises a complex in which the antigenic molecule displays the antigenicity of an antigen associated with a ND.
  • the ND includes but is not limited to Alzheimer's Disease, age-related loss of cognitive function, senile dementia, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's Disease, cerebral palsy, progressive supranuclear palsy, Guam disease, Lewy body dementia, prion diseases, spongiform encephalopathies, Creutzfeldt- Jakob disease, polyglutamine diseases, Huntington's disease, myotonic dystrophy, Freidrich's ataxia, ataxia, Gilles de la Tourette's syndrome, seizure disorders, epilepsy, chronic seizure disorder, stroke, brain trauma, spinal cord trauma, AIDS dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic function disorder, hypertension, neuropsychiatric disorder, schizophrenia, or schizoaffective disorder.
  • the invention provides compositions comprising a purified population of complexes bound to endogenous antigenic molecules, which population is purified from mammalian tissues or cells, which tissues or cells display a pathology associated with AD or which population comprises a complex in which the antigenic molecule displays the antigenicity of an antigen associated with AD.
  • the purified population of complexes bound to endogenous antigenic molecules is from an individual with AD, for example from brain tissue or other AD-associated tissue such as pancreatic tissue.
  • the purified population of complexes bound to endogenous antigenic molecules is isolated from brain or pancreatic tissue of an individual with Trisomy 21.
  • the tissue is isolated post-mortem from a cadaver of an individual with AD or Trisomy 21.
  • the purified population of complexes bound to endogenous antigenic molecules is isolated from a culture of olfactory neurons from an individual with AD or Trisomy 21.
  • the purified population of complexes bound to endogenous antigenic molecules is from an AD model organism.
  • the organism is a rat which has received infusions of A ⁇ and TGF- ⁇ .
  • the AD model organism is a transgenic mouse.
  • the mouse expresses human APP V717F and optionally another AD-associated protein.
  • the AD model organism is a non-human primate.
  • the purified population of complexes bound to endogenous antigenic molecules is from a tissue or cell culture model system for AD.
  • the tissue or cell culture model system is selected from the group consisting of immortalized murine Trisomy 16 neuronal cells, mouse neuroblastoma cell line S20Y, and mouse embryonal carcinoma cell line PI 9.
  • the purified population of complexes bound to endogenous antigenic molecules is from cells associated with amyloid plaques or cells containing neurofibrillary tangles.
  • compositions of the invention comprising a purified population of complexes of heat shock proteins bound to endogenous antigenic molecules, said complexes being purified from mammalian tissues or cells, which tissues or cells display a pathology associated with a ND or which population comprises a complex in which the antigenic molecule displays the antigenicity of an antigen associated with a ND comprise hsp70, hsp90, gp96, calreticulin (for the ability of calreticulin-anitgenic molecule complexes to elicit an immune response, see Basu and Srivastava, 1999, J. Exp. Med. 189:797-202) or a combination thereof.
  • the invention further provides pharmaceutical composition comprising a purified population of complexes of heat shock proteins bound to endogenous antigenic molecules, said complexes being purified from mammalian tissues or cells, which tissues or cells display a pathology associated with a neurodegenerative disorder or which population comprises a complex in which the antigenic molecule displays the antigenicity of an antigen associated with a neurodegenerative disorder.
  • the present invention provides methods of treating or preventing a neurodegenerative disorder in a mammal comprising administering to the mammal a composition comprising a purified population of complexes of heat shock proteins bound to endogenous antigenic molecules, said complexes being purified from mammalian tissues or cells, which tissues or cells display a pathology associated with a ND or which population comprises a complex in which the antigenic molecule displays the antigenicity of an antigen associated with a ND.
  • the neurodegenerative disorder includes but is not limited to Alzheimer's Disease, age-related loss of cognitive function, senile dementia, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's Disease, cerebral palsy, progressive supranuclear palsy, Guam disease, Lewy body dementia, prion diseases, spongiform encephalopathies, Creutzfeldt- Jakob disease, polyglutamine diseases, Huntington's disease, myotonic dystrophy, Freidrich's ataxia, ataxia, Gilles de la Tourette's syndrome, seizure disorders, epilepsy, chronic seizure disorder, stroke, brain trauma, spinal cord trauma, ADDS dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic function disorder, hypertension, neuropsychiatric disorder, schizophrenia, or schizoaffective disorder.
  • the composition comprises hsp70, hsp90, gp96, calreticulin, or a combination thereof
  • the present invention provides methods of preventing or treating AD in a human subject comprising administering to the subject a composition comprising a purified population of complexes of heat shock proteins bound to endogenous antigenic molecules, said complexes being purified from mammalian tissues or cells, which tissues or cells display a pathology associated with AD or which population comprises a complex in which the antigenic molecule displays the antigenicity of an antigen associated with AD.
  • the AD-associated heat shock protein- peptide complex is autologous.
  • the composition is administered intrathecally or mtradermally.
  • the present invention further provides a kit comprising in one or more separate containers a composition comprising a purified population of complexes of heat shock proteins bound to endogenous antigenic molecules, said complexes being purified from mammalian tissues or cells, which tissues or cells display a pathology associated with a ND or which population comprises a complex in which the antigenic molecule displays the antigenicity of an antigen associated with a ND.
  • the invention further provides a method of treating or preventing a neurodegenerative disorder in a subject having a neurodegenerative disorder or in whom treatment or prevention of a neurodegenerative disorder is desired comprising isolating or culturing a mammalian tissue or cell that displays a pathology associated with a neurodegenerative disorder or expresses an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder; recovering a population of complexes of the heat shock proteins noncovalently associated with the antigenic molecules; and administering to the subject an amount of the recovered complexes effective to treat or protect against the neurodegenerative disorder.
  • the complexes are treated with a cross-linking reagent prior to administeration.
  • the invention further provides a method for eliciting in an individual an immune response against a tissue or cell which displays a pathology associated with a neurodegenerative disorder comprising administering to the individual a composition comprising a purified population of complexes of heat shock proteins bound to endogenous antigenic molecules, said complexes being purified from mammalian tissues or cells, which tissues or cells display a pathology associated with a neurodegenerative disorder.
  • the invention further provides a method for eliciting in an individual an immune response against an antigen associated with a neurodegenerative disorder comprising administering to the individual a composition comprising a purified population of complexes of heat shock proteins bound to endogenous antigenic molecules, said population being purified from mammalian tissues or cells and comprising a complex in which the antigenic molecule displays the antigenicity of an antigen associated with a neurodegenerative disorder.
  • AD Alzheimer's Disease
  • a ⁇ 40 The 40 kilodalton cleavage product of APP; the prevalent cleavage product of APP in healthy individuals.
  • a ⁇ 42 The 42 kilodalton cleavage product of APP; the prevalent cleavage product of APP in individuals with AD.
  • CSF Cerebrospinal fluid
  • FAD Familial AD hsp: heat shock protein
  • ND Neurodegenerative disorder or disease
  • ND-associated antigen a proteinaceous molecule whose expression or overexpression (by increased transcription of the encoding gene, increased translation of the encoding RNA, increased or aberrant processing of a precursor protein, or increased or aberrant posttranslational modification of the molecule or a precursor thereof) is associated with a neurodegenerative disorder or disease.
  • An ND-associated antigen is expressed in mammalian tissues or cells which display a pathology associated with a neurodegenerative disorder and/or displays the antigenicity of an antigen associated with a neurodegenerative disorder.
  • NFT Neurofibrillary tangle
  • PHF Paired helical filament, a type of neuropathological structure associated with AD.
  • NDs neurodegenerative diseases or disorders
  • AD Alzheimer's Disease
  • ND-associated tissues or cells refers to tissues or cells which display the antigenicity of an antigen associated with a neurodegerative disorder.
  • AD-associated tissues or cells include but are not limited to the brain lesions or pathologic structures (such as amyloid plaques and neurofibrillary tangles that are associated with Alzheimer's Disease), brain tissue of rodent or non-human primate models of Alzheimer's Disease, cell culture cells that overexpress AD antigens, or non-brain tissue from individuals with AD or from individuals with a genetic predisposition to the disease.
  • pathologic structures such as amyloid plaques and neurofibrillary tangles that are associated with Alzheimer's Disease
  • cell culture cells that overexpress AD antigens
  • non-brain tissue from individuals with AD or from individuals with a genetic predisposition to the disease are described in Section 4.1, infra.
  • the ND-associated hsp-peptide complexes of the invention include any complex derived from a neurodegenerative disease or disorder tissue or a model cell or tissue thereof containing an hsp and a peptide that is capable of stimulating an immuno- protective or -therapeutic response against the neurodegenerative disease.
  • the peptides of the complexes can be noncovalently or covalently associated with the hsp.
  • the complexes are purified in a non-covalent form and, optionally, their components covalently coupled after they are purified (see Section 4.5, infra).
  • the ND-associated hsp-peptide complexes used in accordance with the invention comprise a human hsp.
  • the hsps that can be used according to the present invention include but are not limited to, gp96, hsp90, hsp 70, and calreticulin, either alone or in combination with each other.
  • Preferred complexes include, but are not limited to, ND-associated hsp90- peptide complexes, ND-associated hsp70-peptide complexes, ND-associated hsp60-peptide complexes and combinations thereof.
  • an hsp called gp96 which is present in the endoplasmic reticulum of eukaryotic cells and is related to the cytoplasmic hsp90s (i.e., is a member of the hsp90 family) can be used to generate an effective therapeutic composition containing a gp96-peptide complex.
  • Heat shock proteins which are also referred to interchangeably herein as stress proteins, useful in the practice of the instant invention can be selected from among any cellular protein that satisfies any one of the following criteria.
  • a heat shock protein is characterized by having its intracelmlar concentration increase when a cell is exposed to a stressful stimuli, by being capable of binding other proteins or peptides, by being capable of releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or low pH, or by having at least 35% homology with any cellular protein having any of the above properties.
  • ATP adenosine triphosphate
  • hsps The heat shock proteins
  • Hsps heat shock proteins
  • Hsp60 the heat shock proteins
  • Hsp70 the heat shock proteins
  • Hsp90 the heat shock proteins
  • Hsp 100 the number of the hsps in kilodaltons.
  • Mammalian hsp90 and gp96 each are members of the hsp90 family.
  • hsps can accumulate to very high levels in stressed cells, but they occur at low to moderate levels in cells that have not been stressed.
  • the highly inducible mammalian hsp70 is hardly detectable at normal temperatures but becomes one of the most actively synthesized proteins in the cell upon heat shock (Welch, et al., 1985, J. Cell. Biol. 101 :1198-1211).
  • hsp90 and hsp60 proteins are abundant at normal temperatures in most, but not all, mammalian cells and are further induced by heat (Lai, et al., 1984, Mol. Cell. Biol. 4:2802-10; van Bergen en Henegouwen, et al., 1987, Genes Dev. 1:525-31).
  • Heat shock protems are among the most highly conserved proteins in existence.
  • DnaK the hsp70 from E. coli has about 50% amino acid sequence identity with hsp70 proteins from excoriates (Bardwell, et al., 1984, Proc. Natl. Acad. Sci. 81:848-852).
  • the hsp60 and hsp90 families also show similarly high levels of infra families conservation (Hickey, et al, 1989, Mol. Cell. Biol. 9:2615-2626; Jindal, 1989, Mol. Cell. Biol. 9:2279-2283).
  • the hsp60, hs ⁇ 70 and hsp90 families are composed of proteins that are related to the stress proteins in sequence, for example, having greater than 35% amino acid identity, but whose expression levels are not altered by stress. Therefore it is contemplated that the definition of stress protein, as used herein, embraces other proteins, muteins, analogs, and variants thereof having at least 35% to 55%, preferably 55% to 75%, and most preferably 75% to 85%> amino acid identity with members of the three families whose expression levels in a cell are enhanced in response to a stressful stimulus. The purification of stress proteins belonging to these three families is described below.
  • Sources from which the AD-associated hsp-peptide complexes of the present invention include, but are not limited to, human tissues, tissues from non-human AD model organisms, and in vitro cell cultures.
  • Sources from which the AD-associated hsp-peptide complexes of the present invention include, but are not limited to, human tissues, tissues from non-human AD model organisms, and in vitro cell cultures.
  • AD-associated hsp-peptide complexes are produced from human tissues, the preferred type of tissue is brain tissue, including cells and pathological structures, for example from cadavers of AD patients. Other tissue type that express AD-associated antigens, such as the pancreas, can also be used. In another embodiment, brain tissue as well as other tissue types from rodents or other mammals with AD-like pathologies can be used. In yet another embodiment, cultured cells that may contain hsp-peptide complexes comprising AD- associated antigens complexed to endogenous or recombinantiy expressed hsps, similar to those hsp-peptide complexes found in AD brains, are used. In a specific embodiment, the cultured cells overexpress hsps by recombinant methods (e.g. as described in WO 97/10000) to improve the recovery of AD-associated antigens.
  • recombinant methods e.g. as described in WO 97/10000
  • the AD-associated hsp-peptide complexes of the invention can be autologous to the patient, i.e. the complexes are derived originally from the patient to whom they are administered. In an alternative embodiment, the complexes are non-autologous.
  • the AD-associated hsp- complexes of the invention are obtained from human brain tissue.
  • the brain tissue can originate from a symptomatic AD patient, e.g. a senile adult diagnosed with AD; an asymptomatic AD patient, for example an individual who is genetically predisposed to AD who has developed the pathological structures but not the clinical manifestations of AD; or an individual with trisomy 21.
  • the brain tissue may be obtained ante mortem (for example from a biopsy) or post mortem.
  • the AD-associated hsp-complexes may be prepared from whole brain tissue or, in a certain embodiment, from specific anatomical structures containing cells with neurofibrillary tangles or cells that secrete amyloid plaques.
  • the complexes can be isolated from other organs such as but not limited to the pancreas, the kidney, or skeletal muscle. These organs in particular have been noted to contain pathological structures similar to those observed in AD brains. For example, overexpression of a C-terminal APP fragment in transgenic mice results in A ⁇ deposits and amyloid fibrils in the brains, kidneys and pancreases of the mice (Kawarabayashi et al., 1996, Neurolbiol.
  • tissue can serve as good sources of AD-associated hsp-peptides complexes in subjects with APP abnormalities, such as AD patients, asymptomatic individuals having APP-related FAD, or individuals with trisomy 21.
  • the tissue is obtained post mortem, although the tissue can also be obtained from tissue biopsies ante mortem or from tissue explants.
  • transgenic mice created as models for AD are used as sources of tissues (e.g. brain, pancreas, kidneys, muscle) for the isolation of AD-associated hsp-peptide complexes.
  • tissues e.g. brain, pancreas, kidneys, muscle
  • transgenic mouse models of AD have been described in the scientific literature, most of which express various forms of AD- associated antigens.
  • the first successful transgenic mouse models of AD harbored the APP mutant V717F, a form of APP that causes FAD.
  • mice displayed AD-like neuropathologies such as A ⁇ deposits and neuritic plaques (Games et al., 1995, Nature 373:523-527) although they fail to acquire neurofibrillary tangles (Chen et al., 1998, Prog. Brain Res. 117:327-334).
  • transgenic mice that express the longest human brain tau protein isoform accumulate paired helical filaments in their brain tissues but not neurofibrillary tangles (Gotz et al, 1995, EMBO J. 14:1304-1313).
  • transgenic mouse models for AD include but are not limited to mice expressing or overexpressing the APP isoforms APP695, APP751, APP770, and mice which overexpress APP or presenilin protems having mutations corresponding to those seen in patients with FAD (for example PSl I213T; Nakano et al., 1999, Eur. J. Neurosci:2577-2581).
  • the transgenic mice from which the AD-associated hsp- peptide complexes of the invention are obtained are mice that have been cross-bred to overexpress more than one of the AD-associated protems described above.
  • mice from which AD-associated hsp- peptide complexes are isolated are trisomy 16 mutant mice.
  • Mouse chromosome 16 contains the mouse counterpart of APP and trisomy 16 mice serve as a model system for Down's syndrome (Epstein et al., 1985, Ann. NY Acad. Sci. 450:157-68). However, the mice die during the third week of gestation. Accordingly, the AD-associated hsp-peptide complexes of the invention are isolated from fetal tissue.
  • the AD-associated hsp-peptide complexes of the invention are isolated from non-human primates.
  • the non-human primate brain has been shown to develop the same classes of pathological structures seen in the human brain as a result of aging or AD (Voytko, 1998, Lab. Animal Sci. 48:611-617), making them excellent sources of AD-associated hsp-peptide complexes.
  • the AD-associated hsp-peptide complexes of the invention are isolated from non-human primates whose brains have been infused with A ⁇ and TGF ⁇ , as described supra.
  • the AD-associated hsp-peptide complexes of the invention are isolated from in vitro cultured cells that express antigens associated with AD.
  • the complexes can be isolated from primary culture cells or from cells of immortalized cell lines. Any cell line that has been characterized to express at least one AD-associated antigen can be used to isolate the hsp-peptide complexes of the present invention.
  • the cells lines can be but are not limited to being of central nervous system, kidney, pancreas or muscle origin.
  • the cells can be manipulated to overexpress an hsp molecule.
  • the cells may be transfected with an expression vector comprising an hsp70, hsp90 or gp96 open reading frame and grown under conditions that allow for the expression of corresponding protein prior to hsp-peptide complex isolation.
  • Human olfactory neurons isolated from the central nervous system can be made to divide in cell culture (U.S. Patent No. 5,869,266). Thus, they provide replicable cells that, when isolated from an AD individual, are likely to express the full repertoire of AD-associated antigens, making them an ideal renewable source for the isolation of AD- associated hsp-peptide complexes, particularly if they are made to overexpress hsps, for example by transfecting into the neurons an expression vector harboring an open reading frame encoding an hsp molecule.
  • the neurons are isolated from a cadaver of an AD or trisomy 21 individual.
  • the neurons originate from the patient or subject to whom the compositions of the invention are administered, for example by way of a tissue biopsy, such that the complexes are autologous to said patient or subject.
  • Several neuronal cell lines have been immortalized from trisomy 16 mice (Frederiksen et al., 1996, Arm. NY Acad. Sci. 777:415-420). These can be used as sources of AD-associated hsp-complexes.
  • S20Y a murine cholinergic neuroblastoma cell line that has been shown to become immunoreactive to PHF antibodies when grown under certain conditions (Ko et al., 1990, Acta Neuropathol. (Berl.) 81:30-40), can be used to isolate the complexes.
  • the cell line used to isolate AD- associated hsp-peptide complexes is PI 9.
  • P19 is a murine embryonal carcinoma cell line that differentiates into cholinergic neurons upon retinoic acid treatment, resulting in high levels of APP and Tau protein expression (Fukuchi et al., 1992, J. Neurochem., 58:1863- 1873).
  • ND-associated tissue from humans or animals can be isolated by standard methods in the art, e.g. dissection. For example, simply using a scalpel, it is possible to dissect out specific anatomical structures of the brain, for example to separate grey matter from white matter or the cortex from subcortical nuclei. Any other technique known in the art for isolation of the tissue may be used.
  • the methods described in the sections that follow can be used to isolate the ND-associated hsp-peptide complexes of the invention from cells that express hsps endogenously as well as from cells that express higher levels of hsps through recombinant means. Recombinant expression of hsps is described in Section 4.4, infra.
  • the ND-associated hsp-peptide complexes of the invention are used in purified form, preferably to apparent homogeneity as viewed on an SDS-PAGE gel, or to at least 60%, 70%, 80%, or 90% of total protein.
  • a pellet of eukaryotic cells (e.g., from the Alzheimer diseased brain of a cadaver, or more specifically the regions of the brain containing amyloid plaques and/or neurofibrillary tangles) is resuspended in 3 volumes of buffer consisting of 30mM sodium bicarbonate buffer (pH 7.5) and lmM PMSF and the cells allowed to swell on ice 20 minutes.
  • the cell pellet then is homogenized in a Dounce homogenizer (the appropriate clearance of the homogenizer will vary according to each cell type) on ice until >95% cells are lysed.
  • the lysate is centrifuged at l,000Xg for 10 minutes to remove unbroken cells, nuclei and other debris.
  • the supernatant from this centrifugation step then is recentrifuged at 100,000Xg for 90 minutes.
  • the gp96-peptide complex can be purified either from the 100,000Xg pellet or from the supernatant.
  • the supernatant When purified from the supernatant, the supernatant is diluted with equal volume of 2X lysis buffer and the supernatant mixed for 2-3 hours at 4°C with Con A- Sepharose® (Pharmacia, Inc., Sweden) equilibrated with PBS containing 2mM Ca 2+ and 2mM Mg 2+ . Then, the slurry is packed into a column and washed with IX lysis buffer until the OD 280 drops to baseline.
  • Con A- Sepharose® Pharmacia, Inc., Sweden
  • the column is washed with 1/3 column bed volume of 10% ⁇ -methyl mannoside ( -MM) dissolved in PBS containing 2mM Ca 2+ and 2mM Mg 2+ , the column sealed with a piece of parafilm, and incubated at 37 °C for 15 minutes. Then the column is cooled to room temperature and the parafilm removed from the bottom of the column. Five column volumes of the oc-MM buffer are applied to the column and the eluate analyzed by SDS-PAGE. Typically the resulting material is about 60-95% pure, however this depends upon the cell type and the tissue-to-lysis buffer ratio used.
  • the sample is applied to a Mono Q® FPLC ion-exchange chromatographic column (Pharmacia, Inc., Piscataway, NJ) equilibrated with a buffer containing 5mM sodium phosphate, pH 7.
  • the proteins are then eluted from the column with a 0-1M NaCl gradient.
  • the gp96 fraction elutes between 400mM and 550mM NaCl.
  • the procedure can be modified by two additional steps, used either alone or in combination, to consistently produce apparently homogeneous gp96- peptide complexes.
  • One optional step involves an ammonium sulfate precipitation prior to the Con A purification step and the other optional step involves DEAE-Sepharose® purification after the Con A purification step but before the Mono Q® FPLC step.
  • the supernatant resulting from the 100,000Xg centrifugation step is brought to a final concentration of 50% ammonium sulfate by the addition of ammonium sulfate.
  • the ammonium sulfate is added slowly while gently stirring the solution in a beaker placed in a tray of ice water.
  • the solution is stirred from about 1/2 to 12 hours at 4°C and the resulting solution centrifuged at 6,000 rpm (Sorvall SS34 rotor).
  • the supernatant resulting from this step is removed, brought to 70% ammonium sulfate saturation by the addition of ammonium sulfate solution, and centrifuged at 6,000 rpm (Sorvall SS34 rotor).
  • the resulting pellet from this step is harvested and suspended in PBS containing 70% ammonium sulfate in order to rinse the pellet.
  • This mixture is centrifuged at 6,000 rpm (Sorvall SS34 rotor) and the pellet dissolved in PBS containing 2mM Ca + and Mg 2+ . Undissolved material is removed by a brief centrifugation at 15,000 rpm (Sorvall SS34 rotor). Then, the solution is mixed with Con A Sepharose® and the procedure followed as before.
  • the gp96 containing fractions eluted from the Con A column are pooled and the buffer exchanged for 5mM sodium phosphate buffer, pH 7, 300mM NaCl by dialysis, or preferably by buffer exchange on a Sephadex® G25 column (Pharmacia, Inc., Sweden).
  • the solution is mixed with DEAE- Sepharose® previously equilibrated with 5mM sodium phosphate buffer, pH 7, 300mM NaCl.
  • the protein solution and the beads are mixed gently for 1 hour and poured into a column. Then, the column is washed with 5mM sodium phosphate buffer, pH 7, 300mM NaCl, until the absorbance at 280nM drops to baseline.
  • the bound protein is eluted from the column with five volumes of 5mM sodium phosphate buffer, pH 7, 700mM NaCl. Protein containing fractions are pooled and diluted with 5mM sodium phosphate buffer, pH 7 in order to lower the salt concentration to 175mM. The resulting material then is applied to the Mono Q® FPLC column (Pharmacia) equilibrated with 5mM sodium phosphate buffer, pH 7 and the protein that binds to the Mono Q® FPLC column (Pharmacia) is eluted as described before.
  • the pellet When the gp96 fraction is isolated from the 100,000Xg pellet, the pellet is suspended in 5 volumes of PBS containing either 1% sodium deoxycholate or 1% octyl glucopyranoside (but without the Mg 2+ and Ca 2+ ) and incubated on ice for 1 hour. The suspension is centrifuged at 20,000Xg for 30 minutes and the resulting supernatant dialyzed against several changes of PBS (also without the Mg 2+ and Ca 2+ ) to remove the detergent. The dialysate is centrifuged at 100,000Xg for 90 minutes, the supernatant harvested, and calcium and magnesium are added to the supernatant to give final concentrations of 2mM, respectively. Then the sample is purified by either the unmodified or the modified method for isolating gp96-peptide complex from the 100,000Xg supernatant, see above.
  • the AD-associated gp96-peptide complexes can be purified to apparent homogeneity using this procedure. About 10-20 ⁇ g of AD-associated gp96-peptide complex can be isolated from lg cells or tissue.
  • cells e.g., from the Alzheimer-diseased brain of a cadaver, or more specifically the regions of the brain containing amyloid plaques and/or neurofibrillary tangles
  • IX lysis buffer consisting of 5mM sodium phosphate buffer, pH 7, 150mM NaCl, 2mM CaCl 2 , 2mM MgCl 2 and lmM phenyl methyl sulfonyl fluoride (PMSF).
  • PMSF phenyl methyl sulfonyl fluoride
  • the cells can be lysed by mechanical shearing and in this approach the cells typically are resusp ended in 30mM sodium bicarbonate pH 7.5, lmM PMSF, incubated on ice for 20 minutes and then homogenized in a dounce homogenizer until >95% cells are lysed.
  • the lysate is centrifuged at l,000Xg for 10 minutes to remove unbroken cells, nuclei and other cellular debris.
  • the resulting supernatant is recentrifuged at 100,000Xg for 90 minutes, the supernatant harvested and then mixed with Con A Sepharose® equilibrated with phosphate buffered saline (PBS) containing 2mM Ca 2+ and 2mM Mg 2+ .
  • PBS phosphate buffered saline
  • the supernatant is diluted with an equal volume of 2X lysis buffer prior to mixing with Con A Sepharose®.
  • the supernatant is then allowed to bind to the Con A Sepharose® for 2-3 hours at 4°C.
  • the material that fails to bind is harvested and dialyzed for 36 hours (three times, 100 volumes each time) against lOmM Tris-Acetate pH 7.5, O.lmM EDTA, lOmM NaCl, lmM PMSF. Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Then the resulting supernatant is harvested and applied to a Mono Q® FPLC column equilibrated in 20mM Tris-Acetate pH 7.5, 20mM NaCl, O.lmM EDTA and 15mM 2-mercaptoethanol.
  • the column is then developed with a 20mM to 500mM NaCl gradient and eluted fractions fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized by immunoblotting using an appropriate anti-hsp70 antibody (such as from clone N27F3-4, from StressGen, Victoria, British Columbia, Canada).
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • Fractions strongly immunoreactive with the anti-hsp70 antibody are pooled and the hsp70-peptide complexes precipitated with ammonium sulfate; specifically with a 50%-70% ammonium sulfate cut.
  • the resulting precipitate is then harvested by centrifugation at 17,000 rpm (SS34 Sorvall rotor) and washed with 70% ammonium sulfate.
  • the washed precipitate is then solubilized and any residual ammonium sulfate removed by gel filtration on a Sephadex® G25 column (Pharmacia). If necessary the hsp70 preparation thus obtained can be repurified through the Mono Q® FPLC column as described above.
  • the hsp70-peptide complex can be purified to apparent homogeneity using this method. Typically lmg of hsp70-peptide complex can be purified from lg of cells/tissue.
  • the present invention further describes a rapid method for purification of hsp70-peptide complexes.
  • This improved method comprises contacting cellular proteins with ADP or a nonhydrolyzable analog of ATP affixed to a solid substrate, such that hsp70 in the lysate can bind to the ADP or nonliydrolyzable ATP analog, and eluting the bound hsp70.
  • a preferred method uses column chromatography with ADP affixed to a solid substratum (e.g., ADP-agarose).
  • the resulting hsp70 preparations are higher in purity and devoid of contaminating peptides.
  • the hsp70 yields are also increased significantly by about more than 10 fold.
  • chromatography with nonhydrolyzable analogs of ATP instead of ADP, can be used for purification of hsp70-peptide complexes.
  • 500 million cells are homogenized in hypotonic buffer and the lysate is centrifuged at 100,000Xg for 90 minutes at 4°C. The supernatant is applied to an ADP-agarose column. The column is washed in buffer and is eluted with 5 column volumes of 3 mM ADP. The hsp70-peptide complexes elute in fractions 2 through 10 of the total 15 fractions which elute. The eluted fractions are analyzed by SDS-PAGE. The AD-associated hsp70-peptide complexes can be purified to apparent homogeneity using this procedure.
  • cells e.g., from the Alzheimer diseased brain of a cadaver, or more specifically the regions of the brain containing amyloid plaques and/or neurofibrillary tangles
  • IX Lysis buffer consisting of 5mM sodium phosphate buffer (pH7), 150mM NaCl, 2mM CaCl 2 , 2mM MgCl 2 and lmM phenyl methyl sulfonyl fluoride (PMSF).
  • PMSF phenyl methyl sulfonyl fluoride
  • the cells can be lysed by mechanical shearing and in this approach the cells typically are resuspended in 30mM sodium bicarbonate pH 7.5, lmM PMSF, incubated on ice for 20 minutes and then homogenized in a dounce homogenizer until >95% cells are lysed.
  • the lysate is centrifuged at l,000Xg for 10 minutes to remove unbroken cells, nuclei and other cellular debris.
  • the resulting supernatant is recentrifuged at 100,000Xg for 90 minutes, the supernatant harvested and then mixed with Con A Sepharose® equilibrated with PBS containing 2mM Ca 2+ and 2mM Mg 2+ .
  • Con A Sepharose® equilibrated with PBS containing 2mM Ca 2+ and 2mM Mg 2+ .
  • the supernatant is diluted with an equal volume of 2X Lysis buffer prior to mixing with Con A Sepharose®.
  • the supernatant is then allowed to bind to the Con A Sepharose® for 2-3 hours at 4°C.
  • the material that fails to bind is harvested and dialyzed for 36 hours (three times, 100 volumes each time) against 20 mM sodium phosphate, pH 7.4, 1 mM EDTA, 250 mM NaCl, 1 mM PMSF. Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Then the resulting supernatant is harvested and applied to a Mono Q® FPLC column equilibrated with a buffer containing 20 mM sodium phosphate, pH 7.4, 1 mM EDTA, 250 mM NaCl, 1 mM PMSF. The protems are then eluted with a salt gradient of 200mM to 600mM NaCl.
  • AD-associated hsp90-peptide complexes can be purified to apparent homogeneity using this procedure.
  • 150-200 ⁇ g of AD- associated hsp90-peptide complex can be purified from lg of cells or tissue.
  • ND-associated hsp-peptide complexes are prepared from cells that express higher levels of hsps through recombinant means.
  • Amino acid sequences and nucleotide sequences of many hsps are generally available in sequence databases, such as GenBank.
  • Computer programs, such as Entrez can be used to browse the database, and retrieve any amino acid sequence and genetic sequence data of interest by accession number. These databases can also be searched to identify sequences with various degrees of similarities to a query sequence using programs, such as FASTA and BLAST, which rank the similar sequences by alignment scores and statistics.
  • nucleotide sequences of non-limiting examples of hsps that can be used for the compositions, methods, and for preparation of the hsp peptide-complexs of the invention are as follows: human hsp70, Genbank Accession No. M24743, Hunt et al, 1995, Proc. Natl. Acad. Sci. U.S.A., 82: 6455-6489; human Hsp90, Genbank Accession No. XI 5183, Yamazaki et al, Nucl. Acids Res. 17: 7108; human gp96: Genbank Accession No. XI 5187, Maki et al, 1990, Proc. Natl. Acad. Sci. U.S.A.
  • hsp gene refers not only to the naturally occurring nucleotide sequence but also encompasses all the other degenerate DNA sequences that encode the hsp.
  • the nucleotide sequence encoding the hsp of choice can be obtained and cloned into an expression vector for recombinant expression.
  • the expression vector can then be introduced into a host cell for propogation of the hsp. Methods for recombinant production of hsps are described in detail herein.
  • the DNA may be obtained by DNA amplification or molecular cloning directly from a tissue, cell culture, or cloned DNA (e.g., a DNA "library”) using standard molecular biology techniques (see e.g., Methods in Enzymology, 1987, volume 154, Academic Press; Sambrook et al. 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York; and Current Protocols in Molecular Biology, Ausubel et al. (eds.), Greene Publishing Associates and Wiley Interscience, New York, each of which is incorporated herein by reference in its entirety).
  • standard molecular biology techniques see e.g., Methods in Enzymology, 1987, volume 154, Academic Press; Sambrook et al. 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York; and Current Protocols in Molecular Biology, Ausubel et al. (eds.), Greene Publishing Associates and Wiley
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences. Whatever the source, the hsp gene should be cloned into a suitable vector for propagation of the gene.
  • DNA can be amplified from genomic or cDNA by polymerase chain reaction (PCR) amplification using primers designed from the known sequence of a related or homologous hsp.
  • PCR is used to amplify the desired sequence in DNA clone or a genomic or cDNA library, prior to selection.
  • PCR can be carried out, e.g., by use of a thermal cycler and Taq polymerase (Gene Amp®).
  • the polymerase chain reaction (PCR) is commonly used for obtaining genes or gene fragments of interest.
  • a nucleotide sequence encoding an hsp of any desired length can be generated using PCR primers that flank the nucleotide sequence encoding open reading fram.
  • an hsp gene sequence can be cleaved at appropriate sites with restriction endonuclease(s) if such sites are available, releasing a fragment of DNA encoding the hsp gene. If convenient restriction sites are not available, they may be created in the appropriate positions by site-directed mutagenesis and/or DNA amplification methods known in the art (see, for example, Shankarappa et al, 1992, PCR Method Appl. 1: 277-278). The DNA fragment that encodes the hsp is then isolated, and ligated into an appropriate expression vector, care being taken to ensure that the proper translation reading frame is maintained.
  • DNA fragments are generated to form a genomic library. Since some of the sequences encoding related hsps are available and can be purified and labeled, the cloned DNA fragments in the genomic DNA library may be screened by nucleic acid hybridization to a labeled probe (Benton and Davis, 1977, Science 196: 180; Granstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72: 3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify an appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map.
  • RNA for cDNA cloning of the hsp gene can be isolated from cells which express the hsp.
  • a cDNA library may be generated by methods known in the art and screened by methods, such as those disclosed for screening a genomic DNA library. If an antibody to the hsp is available, the hsp may be identified by binding of a labeled antibody to the hsp-synthesizing clones.
  • nucleotide sequences encoding an hsp can be identified and obtained by hybridization with a probe comprising a nucleotide sequence encoding hsp under conditions of low to medium stringency.
  • procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 6789-6792).
  • Filters containing DNA are pretreated for 6 h at 40 °C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA.
  • Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 6 cpm 3 P -labeled probe is used.
  • Filters are incubated in hybridization mixture for 18-20 h at 40°C, and then washed for 1.5 h at 55 °C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68 °C and reexposed to film. Other conditions of low stringency which may be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • Any technique for mutagenesis known in the art can be used to modify individual nucleotides in a DNA sequence, for purpose of making amino acid substitution(s) in the expressed peptide sequence, or for creating/deleting restriction sites to facilitate further manipulations.
  • Such techniques include but are not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson et al, 1978, J. Biol. Chem. 253: 6551), oligonucleotide-directed mutagenesis (Smith, 1985, Ann. Rev. Genet. 19: 423- 463; Hill et al, 1987, Methods Enzymol.
  • a nucleic acid encoding a secretory form of the hsp of choice is used to practice the methods of the present invention.
  • a nucleic acid can be constructed by deleting the coding sequence for the ER retention signal, KDEL.
  • the KDEL coding sequence is replaced with a molecular tag to facilitate the recognition and purification of the hsp, such as the Fc portion of murine IgGl.
  • 09/253,439 demonstrates that deletion of the ER retention signal of gp96 resulted in the secretion of gp96-Ig peptide-complexes from transfected tumor cells, and the fusion of the KDEL-deleted gp96 with murine IgGl facilitated its detection by ELISA and FACS analysis and its purification by affinity chromatography with the aid of Protein A.
  • Nucleotide sequences encoding an hsp can be inserted into the expression vector for propagation and expression in recombinant cells.
  • An expression construct refers to a nucleotide sequence encoding an hsp operably associated with one or more regulatory regions which allows expression of the hsp in an appropriate host cell. "Operably-associated” refers to an association in which the regulatory regions and the hsp polypeptide sequence to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation of the hsp sequence.
  • expression vectors may be used for the expression of hsps, including, but not limited to, plasmids, cosmids, phage, phagemids, or modified viruses. Examples include bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene). Typically, such expression vectors comprise a functional origin of replication for propagation of the vector in an appropriate host cell, one or more restriction endonuclease sites for insertion of the hsp gene sequence, and one or more selection markers.
  • hsps for expression of hsps in mammalian host cells, a variety of regulatory regions can be used, for example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV- LTR) promoter.
  • Inducible promoters that may be useful in mammalian cells include but are not limited to those associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the ⁇ -interferon gene, and the Hsp70 gene (Williams et al, 1989, Cancer Res. 49: 2735-42 ; Taylor et al, 1990, Mol. Cell. Biol. 10: 165-75).
  • elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38: 639-646; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol.
  • beta-globin gene control region which is active in myeloid cells (Mogram et al, 1985, Nature 315: 338-340; Kollias et al, 1986, Cell 46: 89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48: 703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314: 283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al, 1986, Science 234: 1372-1378).
  • the efficiency of expression of the hsp in a host cell may be enhanced by the inclusion of appropriate transcription enhancer elements in the expression vector, such as those found in SV40 virus, Hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, ⁇ -actin (see Bittner et al, 1987, Methods in Enzymol. 153: 516-544; Gomian, 1990, Curr. Op. in Biotechnol. 1: 36-47).
  • appropriate transcription enhancer elements such as those found in SV40 virus, Hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, ⁇ -actin (see Bittner et al, 1987, Methods in Enzymol. 153: 516-544; Gomian, 1990, Curr. Op. in Biotechnol. 1: 36-47).
  • the expression vector may also contain sequences that permit maintenance and replication of the vector in more than one type of host cell, or integration of the vector into the host chromosome.
  • sequences may include but are not limited to replication origins, autonomously replicating sequences (ARS), centromere DNA, and telomere DNA. It may also be advantageous to use shuttle vectors that can be replicated and maintained in at least two types of host cells.
  • the expression vector may contain selectable or screenable marker genes for initially isolating or identifying host cells that contain DNA encoding an hsp. For long term, high yield production of hsps, stable expression in mammalian cells is preferred.
  • selectable or screenable marker genes for initially isolating or identifying host cells that contain DNA encoding an hsp. For long term, high yield production of hsps, stable expression in mammalian cells is preferred.
  • a number of selection systems may be used for mammalian cells, including, but not limited, to the Herpes simplex virus thymidine kinase (Wigler et al, 1977, Cell H: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski and Szybalski, 1962, Proc. Natl. Acad. Sci. U.S.A.
  • genes can be employed in ik ⁇ , hgprf or aprf cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), which confers resistance to methotrexate (Wigler et al, 1980, Natl. Acad. Sci. U.S.A. 77: 3567; O'Hare et al, 1981, Proc. Natl. Acad. Sci. U.S.A.
  • gpt which confers resistance to mycophenolic acid
  • neo neomycin phosphotransferase
  • hyg hygromycin phosphotransferase
  • linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of cDNA or synthetic DNA encoding an hsp, by techniques well known in the art (Wu et al, 1987, Methods Enzymol. 152: 343-349). Cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation. Alternatively, a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA by use of PCR with primers containing the desired restriction enzyme site.
  • the expression construct comprising an hsp coding sequence operably associated with regulatory regions can be directly introduced into appropriate host cells for expression and production of the hsp-peptide complexes of the invention without further cloning (see, for example, U.S. Patent No. 5,580,859).
  • the expression constructs may also contain DNA sequences that facilitate integration of the hsp coding sequence into the genome of the host cell, e.g., via homologous recombination. In this instance, it is not necessary to employ an expression vector comprising a replication origin suitable for appropriate host cells in order to propagate and express the hsp in the host cells.
  • Expression constructs containing cloned hsp coding sequences can be introduced into the mammalian host cell by a variety of techniques known in the art, including but not limited to calcium phosphate mediated transfection (Wigler et al, 1977, Cell 11: 223-232), liposome-mediated transfection (Schaefer-Ridder et al, 1982, Science 215: 166-168), elecfroporation (Wolff et al, 1987, Proc. Natl. Acad. Sci. 84: 3344), and microinjection (Cappechi, 1980, Cell 22: 479-488).
  • Cell lines that stably express hsps to produce the hsp-peptide complexes of the present invention may be engineered by using a vector that contains a selectable marker.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the expression construct confers resistance to the selection and optimally allows cells to stably integrate the expression construct into their chromosomes and to grow in culture and to be expanded into cell lines. Such cells can be cultured for a long period of time while the hsp is expressed continuously.
  • any of the cloning and expression vectors described herein may be synthesized and assembled from known DNA sequences by techniques well known in the art.
  • the regulatory regions and enhancer elements can be of a variety of origins, both natural and synthetic.
  • Some vectors and host cells may be obtained commercially. Non- limiting examples of useful vectors are described in Appendix 5 of Current Protocols in Molecular Biology, 1988, ed. Ausubel et al, Greene Publish. Assoc. & Wiley Interscience, which is incorporated herein by reference; and the catalogs of commercial suppliers such as Clontech Laboratories, Stratagene Inc., and Invitrogen, Inc.
  • viral-based expression systems may also be utilized with mammalian cells for recombinant expression of hsps.
  • Vectors using DNA virus backbones have been derived from simian virus 40 (SV40) (Hamer et al, 1979, Cell 17: 725), adenovirus (Van Doren et al, 1984, Mol. Cell Biol. 4: 1653), adeno-associated virus (McLaughlin et al, 1988, J. Virol. 62: 1963), and bovine papillomas virus (Zinn et al, 1982, Proc. Natl. Acad. Sci. 79: 4897).
  • SV40 simian virus 40
  • adenovirus Van Doren et al, 1984, Mol. Cell Biol. 4: 1653
  • adeno-associated virus McLaughlin et al, 1988, J. Virol. 62: 1963
  • bovine papillomas virus Zainn et al, 1982
  • the donor DNA sequence may be ligated to an adenovirus transcription/translation control region, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing heterologous products in infected hosts (see, e.g., Logan and Shenk, 1984, Proc. Natl. Acad. Sci. U.S.A. 81: 3655-3659).
  • Bovine papillomavirus can infect many higher vertebrates, including man, and its DNA replicates as an episome.
  • a number of shuttle vectors have been developed for recombinant gene expression which exist as stable, multicopy (20-300 copies/cell) extrachromosomal elements in mammalian cells.
  • these vectors typically contain a segment of BPV DNA (the entire genome or a 69% transforming fragment), a promoter with a broad host range, a polyadenylation signal, splice signals, a selectable marker, and "poisonless" plasmid sequences that allow the vector to be propagated in E.
  • the expression gene construct is transfected into cultured mammalian cells, for example, by the techniques of calcium phosphate coprecipitation or electroporation.
  • selection of transformants is achieved by use of a dominant selectable marker, such as histidinol and G418 resistance.
  • BPV vectors such as pBCMGSNeo and pBCMGHis may be used to express hsps (Karasuyama et al, ⁇ ur. J. Immunol. 18: 97-104; Ohe et al, Human Gene Therapy 6: 325-33) which may then be transfected into a diverse range of cell types for hsp expression.
  • the vaccinia 7.5K promoter may be used (see, e.g., Mackett et al, 1982, Proc. Natl. Acad. Sci. U.S.A. 79: 7415-7419; Mackett et al, 1984, J. Virol. 49: 857-864; Panicali et al, 1982, Proc. Natl. Acad. Sci. U.S.A. 79: 4927-4931)
  • vectors based on the ⁇ pstein-Barr virus ( ⁇ BV) origin (OriP) and ⁇ BV nuclear antigen 1 ( ⁇ BNA-1; a trans-acting replication factor) may be used.
  • Such vectors can be used with a broad range of human host cells, e.g., EBO-pCD (Spickofsky et al, 1990, DNA Prot. Eng. Tech. 2: 14-18), pDR2 and ⁇ DR2 (available from Clontech Laboratories).
  • EBO-pCD Spickofsky et al, 1990, DNA Prot. Eng. Tech. 2: 14-18
  • pDR2 and ⁇ DR2 available from Clontech Laboratories.
  • Retrovirases can efficiently infect and transfer genes to a wide range of cell types including, for example, primary hematopoietic cells.
  • retrovirases such as Moloney murine leukemia virus, most of the viral gene sequences can be removed and replaced with an hsp coding sequence, while the missing viral functions can be supplied in trans.
  • the host range for infection by a retroviral vector can also be manipulated by the choice of envelope used for vector packaging.
  • a retroviral vector can comprise a 5' long terminal repeat (LTR), a 3' LTR, a packaging signal, a bacterial origin of replication, and a selectable marker.
  • the ND-associated antigenic peptide DNA is inserted into a position between the 5' LTR and 3' LTR, such that transcription from the 5' LTR promoter transcribes the cloned DNA.
  • the 5' LTR comprises a promoter, including but not limited to an LTR promoter, an R region, a U5 region and a primer binding site, in that order. Nucleotide sequences of these LTR elements are well known in the art.
  • a heterologous promoter as well as multiple drug selection markers may also be included in the expression vector to facilitate selection of infected cells (see McLauchlin et al, 1990, Prog. Nucleic Acid Res. and Molec. Biol. 38: 91-135; Morgenstern et al, 1990, Nucleic Acid Res. 18: 3587-3596; Choulika et al, 1996, J. Virol 70: 1792-1798; Boesen et al, 1994, Biotherapy 6: 291-302; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3: 110-114).
  • Any cell type that can endogenously produce an ND-associated antigenic polypeptide can be used for recombinant hsp expression.
  • the recombinant cells may be cultured under standard conditions of temperature, incubation time, optical density, and media composition.
  • a cells may be cultured under conditions emulating the nutritional and physiological requirements of a cell in which the ND-associated antigen is endogenously expressed.
  • Modified culture conditions and media may be used to enhance production of hsp-peptide complexes.
  • recombinant cells may be grown under conditions that promote inducible hsp expression. Any technique known in the art may be applied to establish the optimal conditions for producing ND-associated hsp-peptide complexes.
  • ND-associated antigenic peptides may be used as vaccines to elicit an immune response.
  • Hsp-peptide complexes are preferably cross-linked after their purification from cells or tissues as described in Section
  • hsps are covalently coupled to ND-associated antigen by chemical crosslinking.
  • Chemical crosslinking methods are well known in the art.
  • glutaraldehyde crosslinking may be used.
  • Glutaradehyde crosslinking has been used for formation of covalent complexes of peptides and hsps (see Barrios et al, 1992, Eur. J. Immunol. 22: 1365-1372).
  • ND-associated hsp-peptide complex is crosslinked in the presence of 0.002% glutaraldehyde for 2 hours. Glutaraldehyde is removed by dialysis against phosphate buffered saline (PBS) overnight (Lussow et al, 1991, Eur. J. Immunol. 21: 2297-2302).
  • PBS phosphate buffered saline
  • the hsp and ND-associated antigen(s) are crosslinked by ultraviolet (UV) crosslinking.
  • the ND-associated heat shock protein-peptide complexes can be assayed for immunogenicity using any method known in the art.
  • any method known in the art By way of example but not limitation, one of the following three procedures can be used.
  • mice are injected with the hsp ND-associated antigen complex, using any convenient route of administration.
  • other mice are injected with heat shock protein peptide complexes not associated with ND-associated antigens, or cells containing heat shock protein peptide complexes not associated with ND-associated antigens.
  • Cells containing ND-associated antigens may act as a positive control for the assay.
  • the mice are injected twice, 7-10 days apart. Ten days after the last immunization, the spleens are removed and the lymphocytes released. The released lymphocytes may be re-stimulated subsequently in vitro by the addition of dead cells that expressed the antigen of interest.
  • 8x10 ⁇ immune spleen cells may be stimulated with 4x10 4 mitomycin C treated or ⁇ -irradiated (5-10,000 rads) cells containing the antigen of interest (or cells transfected with an appropriate gene, as the case may be) in 3ml RPMI medium containing 10% fetal calf serum. In certain cases 33% secondary mixed lymphocyte culture supernatant may be included in the culture medium as a source of T cell growth factors (See, Glasebrook, et al., 1980, J. Exp. Med. 151:876). To test the primary cytotoxic T cell response after immunization, spleen cells may be cultured without stimulation. In some experiments spleen cells of the immunized mice may also be re-stimulated with antigenically distinct cells, to determine the specificity of the cytotoxic T cell response.
  • the mixed lymphocyte culture is added to a target cell suspension to give different effector:target (E:T) ratios (usually 1:1 to 40:1).
  • E:T effector:target
  • the target cells are prelabelled by incubating lxl O 6 target cells in culture medium containing 20 mCi 51 Cr/ml for one hour at 37 °C. The cells are washed three times following labeling.
  • Each assay point (E:T ratio) is performed in triplicate and the appropriate controls incorporated to measure spontaneous 51 Cr release (no lymphocytes added to assay) and 100% release (cells lysed with detergent). After incubating the cell mixtures for 4 hours, the cells are pelletted by centrifugation at 200g for 5 minutes. The amount of 51 Cr released into the supernatant is measured by a gamma counter. The percent cytotoxicity is measured as cpm in the test sample minus spontaneously released cpm divided by the total detergent released cpm minus spontaneously released cpm.
  • a concentrated hybridoma supernatant derived from K-44 hybridoma cells (an anti-MHC class I hybridoma) is added to the test samples to a final concentration of 12.5%.
  • Primary T cells are obtained from spleen, fresh blood, or CSF and purified by centrifugation using FICOLL-PAQUE PLUS (Pharmacia, Upsalla, Sweden) essentially as described by Kruse and Sebald, 1992, EMBO J. 11 : 3237-3244.
  • the peripheral blood mononuclear cells are incubated for 7-10 days with a lysate of cells expressing an ND- associated antigen.
  • Antigen presenting cells may, optionally be added to the culture 24 to 48 hours prior to the assay, in order to process and present the antigen in the lysate.
  • the cells are then harvested by centrifugation, and washed in RPMI 1640 media (GibcoBRL, Gaithersburg, Md.).
  • PHA-blasts 5x10 4 activated T cells/well (PHA-blasts) are in RPMI 1640 media containing 10% fetal bovine serum, 10 mM HEPES, pH 7.5, 2 mM L-glutamine, 100 units/ml penicillin G, and 100 ⁇ g/ml streptomycin sulphate in 96 well plates for 72 hrs at 37°C, pulsed with 1 ⁇ Ci 3 H-thymidine (DuPont NEN, Boston, Mass.)/well for 6 hrs, harvested, and radioactivity measured in a TOPCOUNT scintillation counter (Packard Instrument Co., Meriden, Conn.).
  • the immunogenicity of a ND- associated hsp-peptide complex is determined by measuring antibodies produced in response to the vaccination with the complex.
  • microtitre plates 96-well Immuno Plate II, Nunc
  • a purified, non-hsp-complexed form of the peptide used in the vaccine e.g. A ⁇ 42
  • PBS-T-BSA PBS containing 0.05% (v/v) TWEEN 20 and 1% (w/v) bovine seram albumin
  • PBS-T Fifty ⁇ l/well of plasma or CSF from a vaccinated animal (such as a model mouse or a human patient) is applied at 20 °C for 1 hour, and the plates are washed 3 times with PBS-T.
  • the anti-peptide antibody activity is then measured calorimetrically after incubating at 20 °C for 1 hour with 50 ⁇ l/well of sheep anti-mouse or anti-human immunoglobulin, as appropriate, conjugated with horseradish peroxidase (Amersham) diluted 1:1,500 in PBS-T-BSA and (after 3 further PBS-T washes as above) with 50 ⁇ l of an o-phenylene diamine (OPD)-H 2 0 2 substrate solution.
  • the reaction is stopped with 150 ⁇ l of 2M H 2 SO 4 after 5 minutes and absorbance is determined in a Kontron SLT-210 photometer (SLT Lab-instr., Zurich, Switzerland) at 492 nm (ref. 620 nm).
  • the methods can also be used to assay the efficacy of the vaccines of the present invention and monitor the progress of AD in those receiving the vaccines.
  • the diagnostic methods for AD to be utilized according to the present invention include but are not limited to testing for molecular indicators of AD or for alterations in neurophysiological function that would be affected by AD. In a preferred embodiment, more than one of the assays described below would be carried out to confirm the diagnosis of the present or extent of AD.
  • phosphorylated Tau protein using a monoclonal antibody specific for the phosphorylated form, as disclosed in U.S. Patent No. 5,733,734, serves as an indicator for AD.
  • the monoclonal antibody is Alz- 50 (U.S. Patent No. 5,811,310).
  • the presence of phosphorylated Tau may be tested in either brain tissue or cerebrospinal fluid or cultures of olfactory neurons from the patient .
  • Tau proteolysis products have been found to be present in the blood or spinal fluids of individuals with AD (U.S. Patent No. 5,492,812). Thus, testing for the presence of tau peptides in blood or spinal fluid samples may provide a diagnostic measure of the presence or progression of AD in patients and other individuals.
  • the presence or extent of AD can also be determined by measuring the relative abundance of A ⁇ 42 and A ⁇ 40. In normal individuals, the amount of A ⁇ 40 far exceeds the amount of A ⁇ 42. In contrast, A ⁇ 42 predominates in AD patients. Additionally, all mutations implicated in FAD, whether in the APP, PSl, or PS2 genes, relate to the processing of APP, and are thought to produce AD through promoting the synthesis of A ⁇ 42. Thus, the relative amount of A ⁇ 40 and A ⁇ 42, for example in a CSF sample or tissue biopsy from the brain or pancreas from an individual, would be an indicator of the presence or progress of AD in the individual.
  • Calcium activated neutral proteases are enzymes that regulate signal transduction by modulating the activities of signaling molecules (e.g. protein kinases and phosphatases) through partial proteolysis. Calcium activated neutral proteases are themselves regulated by partial proteolysis, wherein in the presence of calcium a precursor form of an enzyme undergoes autoproteolytic cleavage to produce a functional enzyme. It has been shown that the ratios of cleaved to uncleaved calcium activated neutral proteases are altered in AD patients. Thus, by measuring the relative amounts of each isoform in a test subject in comparison with a control subject, it is possible to detect AD in an individual (U.S. Patent No. 5,624,807).
  • AD patients have elevated levels of acetylcholinesterase (AChE) activity in ocular fluids.
  • a colorimetric assay for the determination of AChE activity described by Ellman et al. (1961, Biochem. Pharmacol. 7:161-177) may be utilized to measure AchE activity levels in ocular fluid samples, t.e. aqueous humor or vitreous humor samples, the result of which would indicate the presence or absence and possibly the extent of AD (U.S. Patent No. 5,595,883).
  • AD One of the characteristics of AD is an impairment in cytokine secretion, for example IL-1, IL-3 and IL-6. It has been postulated that the impairment is a downstream effect of impaired neural function. Thus it would be possible to assay for blood cytokine levels as indicators of AD (U.S. Patent No. 5,874,312)
  • protems or peptides described supra may be assayed for by a radioimmunoassay, an enzyme-linked immunosorbant assay, a sandwich assay, a gel immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay or an immunoelectrophoresis assay, or any other method known in the art. These methods are well known to those skilled in the art.
  • U.S. Patent No.5,778,893 discloses methods of diagnosing AD, including the extent of AD, in an individual by applying agents that agonize or antagonize neuromuscular signaling and determining the response of the individual to said agents in comparison to control individuals.
  • a cholinergic antagonist is administered to the eye of an individual, the pupil allowed to dilate in response to the cholinergic antagonist, and the rate of which the pupil returns to its normal diameter measured and compared to the corresponding rates in control individuals.
  • a light source illuminates the eye of an individual suspected to have AD.
  • the response of the individual's pupils to the light is measured by a computer system connected to a video camera that records the response of the pupils (U.S. Patent 5,883,691).
  • the hsp-based compositions and formulations described in Section 4.3 above and Section 4.8 below are administered to human patients with NDs.
  • the compositions and formulations are administered to human subjects that do not have a ND as a preventative measure from developing the disease.
  • the methods of the present invention can be applied to the preparation of hsp-peptide complexes associated with any neurodegenerative disorder, and compositions comprising the same.
  • Such disorders relate to the central nervous system and/or peripheral nervous system and include, but are not limited to, cognitive and neurodegenerative disorders such as senile dementia, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's Disease, progressive supranuclear palsy, Guam disease, Lewy body dementia, prion diseases, such as spongiform encephalopathies, e.g., Creutzfeldt- Jakob disease, polyglutamine diseases, such as Huntington's disease, myotonic dystrophy, Freidrich's ataxia and other ataxias, well as Gilles de la Tourette's syndrome, autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders that include, but are not limited to schizophrenia, schizoaffective disorder, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, panic disorder, as well as unipolar and bipolar affective disorders.
  • the purified ND associated hsp-peptide complex vaccines may have particular utility in the treatment of human neurodegenerative disorders. It is appreciated, however, that the vaccines developed using the principles described herein will be useful in treating diseases of other mammals, for example, farm animals including: cattle; horses; sheep; goats; and pigs, and household pets including: cats; and dogs, that have similar pathologies.
  • compositions and formulations of the present invention are administered to a human subject to prevent ND, including inhibiting the progression of a disease in an asymptomatic patient, for example a patient having the molecular landmarks of AD (e.g., above normal levels of phosphorylated Tau and/or A ⁇ 42).
  • the human subject to which the vaccines of the invention are administered is one having a genetic background that increases the likelihood of a given ND (e.g. having the e4 allele of Apolipoprotein E or having a mutation in APP, PS 1 or PS2 which gives rises to FAD).
  • the human subject to which the preventative vaccines of the invention are administered is a non-senile adult above the age of 60.
  • compositions and formulations of the present invention are administered to a human subject that has been diagnosed with a ND or suspected of having a ND.
  • treatment of a ND encompasses the treatment of patients already diagnosed as having ND at any clinical stage; the prevention of the disease in the patients with early symptoms and signs; the delay of the onset or evolution or aggravation or deterioration of the symptoms or signs of a ND; and/or promoting regression of a ND in symptomatic patients.
  • Adoptive immunotherapy refers to a therapeutic approach for treating neurodegenerative diseases in which immune cells are administered to a host with the aim that the cells mediate specific immunity, either directly or indirectly, to cells that express ND-associated antigens and/or antigenic components, and result in treatment of the neurodegenerative disorder, or prevention of the neurodegenerative disorder, as the case may be (see U.S. Patent Application Serial No. 08/527,546, filed September 13, 1995, which is incorporated by reference herein in its entirety).
  • the use of hsp-peptide complexes for sensitizing antigen presenting cells in vitro for use in adoptive immunotherapy is described in PCT publication WO 97/10002, dated March 20, 1997, which is incorporated by reference herein in its entirety.
  • Methods for sensitizing antigen presenting cells (APC) using hsps in complexes with antigenic (or immunogenic) molecules, for adoptive immunotherapy are described in detail herein.
  • therapy by administration of hsp-peptide complexes is combined with adoptive immunotherapy using APC sensitized with hsp-antigenic molecule complexes.
  • the hsp-peptide complex-sensitized APC can be administered concurrently with hsp-peptide complexes, or before or after administration of hsp-peptide complexes.
  • the mode of administration can be varied, including but not limited to, e.g., subcutaneously, intravenously, intraperitoneally, intramuscularly, intradermally or mucosally.
  • the antigen-presenting cells including but not limited to macrophages, dendritic cells and B-cells, are preferably obtained by production in vitro from stem and progenitor cells from human peripheral blood or bone marrow as described by Inaba et al, 1992, J. Exp. Med. 176:1693-1702.
  • APC can be obtained by any of various methods known in the art.
  • human macrophages are used, obtained from human blood cells.
  • macrophages can be obtained as follows: Mononuclear cells are isolated from peripheral blood of a patient (preferably the patient to be treated), by Ficoll-Hypaque gradient centrifugation and are seeded on tissue culture dishes which are pre-coated with the patient's own seram or with other AB+ human serum. The cells are incubated at 37°C for 1 hr, then non-adherent cells are removed by pipetting. To the adherent cells left in the dish, is added cold (4°C) 1 mM EDTA in phosphate-buffered saline and the dishes are left at room temperature for 15 minutes. The cells are harvested, washed with RPMI buffer and suspended in RPMI buffer.
  • Increased numbers of macrophages may be obtained by incubating at 37°C with macrophage-colony stimulating factor (M-CSF); increased numbers of dendritic cells may be obtained by incubating with granulocyte-macrophage-colony stimulating factor (GM-CSF) as described in detail by Inaba, K., et al, 1992, J. Exp. Med. 176:1693-1702.
  • M-CSF macrophage-colony stimulating factor
  • GM-CSF granulocyte-macrophage-colony stimulating factor
  • APC are sensitized with hsps bound to ND-associated antigenic molecules by incubating the cells in vitro with the complexes.
  • the APC are sensitized with complexes of hsps and antigenic molecules preferably by incubating in vitro with the hsp-complex at 37°C for 15 minutes to 24 hours.
  • 4x10 ⁇ macrophages can be incubated with 10 microgram gp96-peptide complexes per ml or 100 microgram hsp90-peptide complexes per ml at 37°C for 15 minutes-24 hours in 1 ml plain RPMI medium.
  • the cells are washed three times and resuspended in a physiological medium preferably sterile, at a convenient concentration (e.g., lxlOHml) for injection in a patient.
  • a physiological medium preferably sterile
  • the antigen presenting cells are autologous to the patient, that is, the patient into which the sensitized APCs are injected is the patient from which the APC were originally isolated,.
  • the ability of sensitized APC to stimulate, for example, the antigen-specific, class I-restricted cytotoxic T-lymphocytes (CTL) can be monitored by their ability to stimulate CTLs to release tumor necrosis factor, and by their ability to act as targets of such CTLs.
  • CTL cytotoxic T-lymphocytes
  • the hsp-antigenic molecule-sensitized APC are reinfused into the patient systemically, preferably intravenously, by conventional clinical procedures. These activated cells are reinfused, preferentially by systemic administration into the autologous patient. Patients generally receive from about 10 ⁇ to about 10 ⁇ sensitized macrophages, depending on the condition of the patient. In some regimens, patients may optionally receive in addition a suitable dosage of a biological response modifier including but not limited to the cytokines IFN- , IFN- ⁇ , IL-2, IL-4, IL-6, TNF or other cytokine growth factor.
  • a biological response modifier including but not limited to the cytokines IFN- , IFN- ⁇ , IL-2, IL-4, IL-6, TNF or other cytokine growth factor.
  • ND associated hsp-peptide complexes can also be used for passive immunotherapy against neurodegenerative disorders. Passive immunity is the short-term protection of a host, achieved by the administration of pre- formed antibody directed against a heterologous organism.
  • ND associated hsp-peptide complexes may used to elicit an immune response in a subject, the sera removed from the subject and used for treatment or prevention of a neurodegerative disorder in a subject having a disorder caused by the presence of a common antigen.
  • Hsp-antigenic molecule complexes are administered to human subjects in doses in a range of about 1 ⁇ g to about 5000 ⁇ g, preferably in a range of about 1 ⁇ g to about 1500 ⁇ g.
  • Methods of introduction include but are not limited to intrathecal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the hsps-peptide complexes may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • Administration can be systemic or local; this may be achieved, for example and not by way of limitation, by topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • the hsp compositions of the present invention are administered intrathecally by an implant be placed in or near the lesioned area of the nervous system.
  • Suitable implants include, but are not limited to, gelfoam, wax, liposome or microparticle-based implants. Such compositions are preferably used when it is desired to achieve sustained release of the hsp-peptide complexes.
  • the hsp compositions of the present invention are administered into the cerebrospinal fluid (CSF) of a patient by means of injection.
  • CSF cerebrospinal fluid
  • administration is in or near the lesioned area of the nervous system.
  • the hsp compositions are administered, either intradermally or subcutaneously, with sites of administration varied sequentially.
  • the doses recited above are given once weekly for a period of about 4 to 6 weeks, and the mode of administration is varied with each administration.
  • Each site of administration may be varied sequentially.
  • the injections can be given, either intradermally or subcutaneously, locally or at a site distant from the brain or central nervous system. The same site can be repeated after a gap of one or more injections.
  • split injections can be given. Thus, for example, half the dose can be given in one site and the other half in another site on the same day.
  • the ND-associated hsp-peptide complexes in accordance with the invention, can be formulated into pharmaceutical preparations for admimsfration to mammals, preferably humans, as preventative or therapeutic vaccines for a ND.
  • Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier can be prepared, packaged, and labeled for prevention and/or therapy of the ND for which the ND-associated hsp-peptide complexes are prepared.
  • the complex is water-soluble, then it can be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions. Alternatively, if the resulting complex has poor solubility in aqueous solvents, then it can be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol.
  • a non-ionic surfactant such as Tween, or polyethylene glycol.
  • the compounds and their physiologically acceptable solvates can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration.
  • the pharmaceutical preparation can be in liquid forai, for example, solutions, syrups or suspensions, or can be presented as a drug product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, or fractionated vegetable oils
  • preservatives
  • the pharmaceutical compositions can 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, macrocrystalline 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, macrocrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato
  • Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
  • the compositions can take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichloroflu
  • the compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds can be formulated into creams, lotions, ointments or tinctures, e.g., containing conventional bases, such as hydrocarbons, petrolatum, lanolin, waxes, glycerin, or alcohol.
  • bases such as hydrocarbons, petrolatum, lanolin, waxes, glycerin, or alcohol.
  • the compounds can 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.
  • the compounds can also be formulated as a depot preparation.
  • Such long acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials e.g., as an emulsion in an acceptable oil
  • ion exchange resins e.g., as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
  • compositions can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient.
  • the pack can for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device can be accompanied by instructions for administration. 4.11. KITS
  • kits for carrying out the therapeutic regimens of the invention comprise in one or more containers therapeutically effective amounts of the ND-associated hsp-peptide complexes in pharmaceutically acceptable form.
  • the ND-derived hsp-peptide complex in a vial of a kit of the invention can be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid.
  • the complex can be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the complex to form a solution for injection purposes.
  • a pharmaceutically acceptable solution e.g., saline, dextrose solution, etc.
  • kits of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad. Instructions are optionally included for administration of ND-derived hsp- antigenic molecule complexes by a clinician or by the patient.

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Abstract

La présente invention concerne des compositions renferment des complexes de protéines de choc thermique liées de façon covalente ou non à des antigènes qui se caractérisent par l'antigénicité des antigènes présents dans les cellules et les tissus associés à la pathologie de troubles ou de maladies à caractère neurovégétatif (tels que la maladie d'Alzheimer). Ces compositions peuvent être isolées à partir de tout tissu dans lesquelles elles sont présentes, tel que des cellules humaines malades, des modèles non humains pour la maladie ou des cellules cultivées in vitro qui expriment des antigènes associés à des troubles neurodégénératifs. De plus, l'invention concerne des méthodes de prévention et de traitement de maladies et de troubles d'ordre neurovégétatif reposant sur l'emploi des compositions selon l'invention. L'invention concerne également des kits renfermant lesdites compositions.
PCT/US2001/001671 2000-01-21 2001-01-18 Compositions et techniques pour traiter des troubles neurodegeneratifs WO2001052877A1 (fr)

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US11707456B2 (en) 2020-11-19 2023-07-25 Kempharm Denmark A/S Processes for preparing arimoclomol citrate and intermediates thereof

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6495347B1 (en) 1999-07-08 2002-12-17 Stressgen Biotechnologies Corporation Induction of a Th1-like response in vitro
US6657055B2 (en) 1999-07-08 2003-12-02 Stressgen Biotechnologies Corporation Induction of a Th1-like response in vitro
US6797491B2 (en) 2000-06-26 2004-09-28 Stressgen Biotechnologies Corporation Human papilloma virus treatment
US7211411B2 (en) 2000-06-26 2007-05-01 Nventa Biopharmaceuticals Corporation Human papilloma virus treatment
US7754449B2 (en) 2000-06-26 2010-07-13 Nventa Biopharmaceuticals Corporation Human papilloma virus treatment
WO2002034777A1 (fr) * 2000-10-24 2002-05-02 Chiesi Farmaceutici S.P.A. Proteines de fusion utilisees comme traitements d'immunisation contre la maladie d'alzheimer
US6921534B2 (en) 2001-02-05 2005-07-26 Stressgen Biotechnologies Corporation Hepatitis B virus treatment
US9884058B2 (en) 2008-06-26 2018-02-06 Orphazyme Aps Use of Hsp70 as a regulator of enzymatic activity
US11304941B2 (en) 2008-06-26 2022-04-19 Orphazyme A/S Use of HSP70 as a regulator of enzymatic activity
US8540985B2 (en) 2008-06-26 2013-09-24 Orphazyme Aps Use of Hsp70 as a regulator of enzymatic activity
US10543204B2 (en) 2008-06-26 2020-01-28 Orphazyme A/S Use of Hsp70 as a regulator of enzymatic activity
US9289472B2 (en) 2008-06-26 2016-03-22 Orphazyme Aps Use of HSP70 as a regulator of enzymatic activity
US11045460B2 (en) 2008-06-26 2021-06-29 Orphazyme A/S Use of Hsp70 as a regulator of enzymatic activity
US11938125B2 (en) 2008-06-26 2024-03-26 Zevra Denmark A/S Use of Hsp70 as a regulator of enzymatic activity
US9662375B2 (en) 2010-11-30 2017-05-30 Orphazyme Aps Methods for increasing intracellular activity of Hsp70
US10532085B2 (en) 2010-11-30 2020-01-14 Orphazyme A/S Methods for increasing intracellular activity of Hsp70
US10709700B2 (en) 2014-09-15 2020-07-14 Orphazyme A/S Arimoclomol formulation
US11229633B2 (en) 2014-09-15 2022-01-25 Orphazyme A/S Arimoclomol formulation
US10898476B2 (en) 2016-04-13 2021-01-26 Orphazyme A/S Heat shock proteins and cholesterol homeostasis
US11253505B2 (en) 2016-04-29 2022-02-22 Orphazyme A/S Arimoclomol for treating glucocerebrosidase associated disorders
US11707456B2 (en) 2020-11-19 2023-07-25 Kempharm Denmark A/S Processes for preparing arimoclomol citrate and intermediates thereof

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