WO2001052890A1 - Complexes de proteines de choc thermique/stress utilises en tant que vaccins contre les troubles neurodegeneratifs - Google Patents

Complexes de proteines de choc thermique/stress utilises en tant que vaccins contre les troubles neurodegeneratifs Download PDF

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WO2001052890A1
WO2001052890A1 PCT/US2001/001825 US0101825W WO0152890A1 WO 2001052890 A1 WO2001052890 A1 WO 2001052890A1 US 0101825 W US0101825 W US 0101825W WO 0152890 A1 WO0152890 A1 WO 0152890A1
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fragment
heat shock
complex
protein
antigenic
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PCT/US2001/001825
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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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/52—Isomerases (5)
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/04—Intramolecular oxidoreductases (5.3) transposing S-S bonds (5.3.4)
    • C12Y503/04001—Protein disulfide-isomerase (5.3.4.1), i.e. disufide bond-forming enzyme
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • 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 and methods for the use of 10 complexes of heat shock proteins and antigenic peptides associated with neurodegenerative disorders, which can be used as vaccines against neuropsychiatric disorders, such as Alzheimer's Disease.
  • Neurodegenerative disorders are typically characterized by a number of neuropathological abnormalities, such as neuritic plaques, neurof ⁇ brillary tangles (NFTs), Lewy bodies, and intranuclear inclusions. For example, Alzheimer's Disease, the most neuropathological abnormalities, such as neuritic plaques, neurof ⁇ brillary tangles (NFTs), Lewy bodies, and intranuclear inclusions. For example, Alzheimer's Disease, the most of neuropathological abnormalities, such as neuritic plaques, neurof ⁇ brillary tangles (NFTs), Lewy bodies, and intranuclear inclusions. For example, Alzheimer's Disease, the most of neuropathological abnormalities, such as neuritic plaques, neurof ⁇ brillary tangles (NFTs), Lewy bodies, and intranuclear inclusions. For example, Alzheimer's Disease, the most neuropathological abnormalities, such as neuritic plaques, neurof ⁇ brillary tangles (NFT
  • Alzheimer's disease is characterized pathologically by neurodegeneration, the presence of large numbers of amyloid plaques, and neurons that accumulate tau and ubiquitin reactivities within NFTs.
  • the result is damage to regions of the brain and neural circuits responsible for memory and cognition, including neurons in the neocortex, hippocampus, amygdala, basal forebrain cholinergic system, and brainstem monoaminergic
  • Neuritic plaques are extracellular lesions, consisting largely of a deposit of a peptide called ⁇ -amyloid (A ⁇ ).
  • a ⁇ ⁇ -amyloid
  • the predominant forms of ⁇ -amyloid are the 40 amino-acid form, A ⁇ 40, and the 42 amino acid form, A ⁇ 42.
  • Most secreted forms of A ⁇ peptides are A ⁇ 40, a soluble species, whereas about 10% of secreted A ⁇ peptides are A ⁇ 42 and A ⁇ 43,
  • a ⁇ -(l-40) and A ⁇ -(l-42)] and A ⁇ -(25-35) also form fibrils and are neurotoxic (see, for example, Howlett et al, 1995, Neurodegeneration 4: 23-32; Pike et al, 1993, J. Neuroscience 13: 1676-1687; Yankner et al, 1990, Science 250: 279-
  • Neurofibrillaxy tangles are intracellular lesions consisting of poorly soluble filaments of the protein tau.
  • hyperphosphorylated forms and highly ubiquitinated forms of the tau protein, a microtubule-binding protein are predominant in such NFTs.
  • Tangles are found in frontotemporal dementia associated with Parkinson's disease, Alzheimer's Disease, progressive supranuclear palsy, Guam disease, and some forms of prion disease.
  • Lewy bodies consisting largely of the protein ⁇ -synuclein, are found in Parkinson's disease, some forms of Alzheimer's disease, and Lewy body dementia (for review, see Hardy and Hardy, 1998, Science 282: 1075-1079 and Greefiifield's Neuropatholgy, Graham and Lantos (eds.), Arnold, London, 1997). Intranuclear inclusions are often associated with an unstable triplet repeat mutation (polyCAG) which cause polyglutamine diseases.
  • polyCAG unstable triplet repeat mutation
  • AD amyloid precursor protein
  • APP amyloid precursor protein
  • ApoE4 an allele of apolipoprotein
  • ApoE4 presenilin-1
  • PS2 presenilin-2
  • PS2 Levy-Lahad et /.,1995, Science 269: 973-977
  • APP a single transmembrane domain integral membrane glycoprotein
  • a ⁇ peptides are generated by endoproteolytic cleavage of APP. Mutations in APP can result in the shift of the processing of the APP precurser so that more of the 42 or 43 amino acid form of A ⁇ is produced.
  • PS1 and PS2 are highly homologous 43 to 50 kD proteins with eight transmembrane domains.
  • Presenilin polypeptides acculmulate as 27/28 kDa N-terminal and 16/17 kDa C-terminal kDa derivatives that become stably associated with each other in vivo.
  • Most genetic abnormalities in the presenilin are missense mutations that result in single amino acid substitutions.
  • no specific gene mutations are associated with the inheritance in the case of late onset AD.
  • specific alleles of apolipoprotein E and ⁇ -2 macroglobulin are associated with increased risk for AD (Blacker et al, 1998, Nature Genet. 19: 357-360).
  • the molecular basis of neurodegenerative diseases other than AD are also beginning to be understood.
  • Heat shock proteins also referred to interchangeably as stress proteins, were first identified as proteins 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 Hspl 00, 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 immunological processes in unstressed cells. For example, 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).
  • ER endoplasmic reticulum
  • proteins 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) wliich is related to Hs ⁇ 90, and Grp78 or BiP, which is related to Hs ⁇ 70 (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
  • mice with gp96 or p84/86 isolated from a particular tumor rendered the mice immune to that particular tumor, but not to antigemcally distinct tumors.
  • Isolation and characterization of genes encoding gp96 and p84/86 revealed significant homology between them, and showed that gp96 and p84/86 were, respectively, the endoplasmic reticular and cytosolic counterparts of the same heat shock proteins (Srivastava et al, 1988, Immunogenetics 28: 205-207; Srivastava et al, 1991, Curr. Top. Microbiol. Immunol. 167: 109-123).
  • Hsp70 was shown to elicit immunity to the tumor from which it was isolated but not to antigemcally distinct tumors.
  • Hsp70 depleted of peptides was found to lose its immunogenic activity (Udono and Srivastava, 1993, J. Exp. Med. 178: 1391-1396).
  • the heat shock proteins are not immunogenic per se, but form noncovalent complexes with antigenic peptides, and the complexes can elicit specific immunity to the antigenic peptides (Srivastava, 1993, Adv. Cancer Res. 62: 153-177; Udono et al, 1994, J. Immunol. 152: 5398-5403; Suto et al, 1995, Science 269: 1585-1588).
  • stress protein-peptide complexes can be isolated from pathogen-infected cells and used for the treatment and prevention of infection caused by pathogens, such as viruses and other intracellular pathogens, including bacteria, protozoa, fungi and parasites (see PCT publication WO 95/24923, dated September 21, 1995).
  • Immunogenic stress protein-peptide complexes can also be prepared by in vitro complexing of stress protein and antigenic peptides, and the uses of such complexes for the treatment and prevention of cancer and infectious diseases has been described in PCT publication WO 97/10000, dated March 20, 1997.
  • the use of heat shock proteins in combination with a defined antigen for the treatment of cancer and infectious diseases have also been described in PCT publication WO 97/06821, dated February 27, 1997.
  • the administration of expressible polynucleotides encoding eukaryotic heat shock proteins to mammalian cells for stimulating an immune response, and for treatment of infectious diseases and cancer has been described in PCT publications, WO 97/06685 and WO 97/06828, both dated February 27, 1997.
  • the present invention provides methods for the use of complexes of heat shock proteins and antigenic peptides as vaccines for the treatment and prevention of neurodegenerative disorders, such as Alzheimer's Disease.
  • a purified complex comprising a heat shock protein and an antigenic molecule, which antigenic molecule displays the antigenicity of an antigen associated with a neurodegenerative disorder.
  • the heat shock protein is noncovalently associated with the antigenic molecule.
  • the heat shock protein is noncovalently associated with the antigenic molecule.
  • the complex of a heat shock protein and an antigenic molecule is purified to apparent homogeneity, as viewed on an SDS-PAGE gel.
  • the complex is the product of a method comprising complexing said heat shock protein and said antigenic molecule in vitro.
  • the complex is a fusion protein comprising the heat shock protein fused to the antigenic molecule.
  • the heat shock protein is Hsp70, Hsp90, gp96, PDI, or calreticulin.
  • the heat shock protein is covalently associated with the antigenic molecule.
  • the antigenic molecule is ⁇ -amyloid or a fragment thereof, an oligomeric A ⁇ complex or a fragment thereof, an ApoE4-A ⁇ complex or a fragment thereof, tau protein or a fragment thereof, a mutant amyloid precurser protein or a fragment thereof, a mutant of presenillin or a fragment thereof, -synuclein or a fragment thereof, or a prion protein or a fragment thereof.
  • the antigenic molecule is ⁇ -amyloid, or a fragment thereof.
  • the invention provides a composition comprising a purified population of complexes of heat shock proteins bound to antigenic molecules, said complexes being purified from cells transformed with and expressing a nucleic acid encoding a protein displaying the antigenicity of an antigen associated with a neurodegenerative disorder.
  • the population of complexes of heat shock proteins bound to antigenic molecules is purified to apparent homogeneity, as viewed on an SDS-PAGE gel.
  • 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 pharmaceutical composition comprising an amount of a purified molecular complex effective for treatment or prevention of a neurodegenerative disorder, and a pharmaceutically acceptable carrier, said molecular complex comprising a heat shock protein and an antigemc molecule, which antigenic molecule displays the antigenicity of an antigen associated with a neurodegenerative disorder.
  • the heat shock protein is covalently associated with the antigenic molecule.
  • the antigenic molecule is coupled to a universal helper.
  • the heat shock protein is Hsp70, Hsp90, gp96, PDI, or calreticulin.
  • the antigemc molecule is ⁇ -amyloid or a fragment thereof, an oligomeric A ⁇ complex or a fragment thereof, an ApoE4-A ⁇ complex or a fragment thereof, tau protein or a fragment thereof, a mutant amyloid precurser protein or a fragment thereof, a mutant of presenillin or a fragment thereof, c -synuclein or a fragment thereof, or a prion protein or a fragment thereof.
  • the antigenic molecule is ⁇ -amyloid, or a fragment thereof.
  • the invention further provides a recombinant cell transformed with (i) a first nucleic acid comprising a first nucleotide sequence that is operably linked to a first promoter and that encodes a heat shock protein, and (ii) a second nucleic acid comprising a second nucleotide sequence that is operably linked to a second promoter and encodes an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder, such that the heat shock protein and the antigenic molecule are expressed within the cell and non-covalently associate with each other to form a complex that in sufficient amount is capable of eliciting an immune response to the antigenic molecule.
  • the cell is a human cell.
  • the antigenic molecule is ⁇ -amyloid or a fragment thereof, an oligomeric A ⁇ complex or a fragment thereof, an ApoE4-A ⁇ complex or a fragment thereof, tau protein or a fragment thereof, a mutant amyloid precurser protein or a fragment thereof, a mutant of presenillin or a fragment thereof, ⁇ -synuclein or a fragment thereof, or a prion protein or a fragment thereof.
  • the antigenic molecule is ⁇ -amyloid, or a fragment thereof.
  • the invention provides a recombinant cell transformed with a nucleic acid comprising a nucleic acid sequence that is operably linked to a promoter, said nucleic acid sequence encoding a fusion protein that comprises a heat shock protein operatively linked to an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder.
  • the cell is a human cell.
  • the antigenic molecule is ⁇ -amyloid or a fragment thereof, an oligomeric A ⁇ complex or a fragment thereof, an ApoE4-A ⁇ complex or a fragment thereof, tau protein or a fragment thereof, a mutant amyloid precurser protein or a fragment thereof, a mutant of presenillin or a fragment thereof, ⁇ -synuclein or a fragment thereof, or a prion protein or a fragment thereof.
  • the antigenic molecule is ⁇ -amyloid, or a fragment thereof.
  • the invention provides pharmaceutical compositions comprising recombinant cells and pharmaceutically acceptable carriers.
  • the invention further provides a method for preparing a complex of a heat shock protein associated with an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder, comprising: a) culturing a recombinant cell that expresses a heat shock protein and is transformed with a nucleic acid comprising a nucleotide sequence that is operably linked to a promoter and encodes an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder, under conditions such that the antigemc molecule is expressed by the cell and associates with the heat shock protein expressed by the cell; and b) recovering a population of complexes of the heat shock proteins noncovalently associated with the antigenic molecules.
  • the method further comprises the step of c) purifying the complexes.
  • the complexes are purified by affinity chromatography.
  • the method further comprises the step of d) treating the purified complexes with a crosslinking agent such that the hsps become covalently attached to the antigenic molecules.
  • a method for preparing a complex of a heat shock protein associated with an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder comprising: a) culturing a recombinant cell transformed with (i) a first nucleic acid comprising a first nucleotide sequence that is operably linked to a first promoter and that encodes a heat shock protein, and (ii) a second nucleic acid comprising a second nucleotide sequence that is operably linked to a second promoter and encodes an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder, under conditions such that the heat shock protein is expressed by the cell and associates with the antigenic molecule; and b) recovering a population of complexes of the heat shock protein noncovalently associated with antigenic molecules from the cells.
  • the method further comprises the step of c) purifying the complexes.
  • the complexes are purified by affinity chromatography.
  • the method further comprises the step of d) treating the purified complexes with a crosslinking agent such that the hsps become covalently attached to the antigemc molecules.
  • a method for preparing a fusion protein capable of eliciting an immune response against a neurodegenerative disorder comprising: a) culturing a recombinant cell transformed with a nucleic acid comprising a nucleotide sequence that is operably linked to a promoter and that encodes a fusion protein comprising a heat shock protein operatively linked to an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder, under conditions such that the fusion protein is expressed by the cell; and b) recovering the fusion protein from the cells.
  • the invention further provides methods for preparing in vitro complexes of heat shock proteins associated with one or more ND-associated antigenic molecules.
  • a method for preparing in vitro complexes of heat shock proteins associated with one or more ND-associated antigenic molecules comprising: a) incubating a heat shock protein and one or more antigenic molecules under conditions and for a length of time sufficient for the formation of the complexes, wherein said antigenic molecules display the antigenicity of an antigen associated with a neurodegenerative disorder, and b) isolating said complexes.
  • the heat shock protein is purified.
  • the method further comprises the step of c) treating the isolated complexes with a crosslinking agent such that the hsp of the complex becomes covalently attached to one or more antigenic molecule of the complex.
  • the antigenic molecules used in the methods of the invention display the antigemcities of antigens associated with a neurodegenerative disorder.
  • one or more of the antigenic molecules are ⁇ -amyloid or a fragment thereof, an oligomeric A ⁇ complex or a fragment thereof, an A ⁇ oE4-A ⁇ complex or a fragment thereof, tau protein or a fragment thereof, a mutant amyloid precurser protein or a fragment thereof, a mutant of presenillin or a fragment thereof, ⁇ -synuclein or a fragment thereof, or a prion protein or a fragment thereof.
  • one or more of the antigenic molecules is ⁇ -amyloid, or a fragment thereof.
  • a method for eliciting an immune response against an antigen associated with a neurodegenerative disorder in an individual comprising administering to the individual a complex of a heat shock protein and an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder in an amount effective to elicit an immune response.
  • the method further comprises, before, concurrently, or after administration of the immunogenic complex, administering to the individual a composition comprising antigen presenting cells sensitized in vitro with a sensitizing amount of a second complex of a heat shock protein and a second antigenic molecule, in which said second antigenic molecule shares at least one antigenic determinant with the first antigenic molecule.
  • the invention provides a method of treating or protecting against a neurodegenerative disorder in an individual having a neurodegenerative disorder, or in whom prevention of a neurodegenerative disorder is desired, comprising administering to the individual a purified complex of a heat shock protein and an antigenic molecule in an amount effective to treat or protect against said neurodegenerative disorder, wherein said antigenic molecule displays the antigenicity of an antigen associated with said neurodegenerative disorder.
  • the method further comprises, before, concurrently or after administration of the immunogenic complex, administering to the individual a composition comprising antigen presenting cells sensitized in vitro with a sensitizing amount of a second complex of a heat shock protein and a second antigemc molecule, said second antigemc molecule sharing at least one antigenic determinant with the first antigenic molecule.
  • the invention provides a method of treating or protecting against a neurodegenerative disorder in a subject having a neurodegenerative disorder or in whom prevention of a neurodegenerative disorder is desired comprising: a) culturing a recombinant cell that expresses a heat shock protein and is transformed with a nucleic acid comprising a nucleotide sequence that is operably linked to a promoter and encodes an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder, under conditions such that the antigenic molecule is expressed by the cell and associates with the heat shock protein expressed by the cell; and b) recovering a population of complexes of the heat shock proteins noncovalently associated with the antigenic molecules; and c) administering to the subject an amount of the recovered complexes effective to treat or protect against the neurodegenerative disorder.
  • the method further comprises, after step (b) and before step (c), the step of treating the complexes with a crosslinking agent such that the heat shock protein becomes
  • the invention provides a method of treating or protecting against a neurodegenerative disorder in a subject having a neurodegenerative disorder or in whom prevention of a neurodegenerative disorder is desired comprising:a) culturing a recombinant cell transformed with (i) a first nucleic acid encoding a heat shock protein, and (ii) a second nucleic acid encoding an antigemc molecule displaying the antigenicity of an antigen associated with a neurodegenerative disorder; b) recovering complexes of the heat shock protein noncovalently associated with the antigenic molecule; and c) administering to the subject an amount of the recovered complexes effective to treat or protect against the neurodegenerative disorder.
  • the method further comprises, after step (b) and before step (c), the step of treating the complexes with a crosslinking agent such that the heat shock protein becomes covalently associated with the antigenic molecule.
  • the invention provides a method of treating or protecting against a neurodegenerative disorder in a subject having a neurodegenerative disorder or in whom prevention of a neurodegenerative disorder is desired comprising: a) culturing a recombinant cell transformed with a nucleic acid comprising a nucleotide sequence that is operably linked to a promoter and that encodes a fusion protein comprising a heat shock protein operatively linked to an antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder, under conditions such that the fusion protein is expressed by the cell; b) recovering the fusion protein from the cells; and c) administering to the subject an amount of the fusion protein effective to treat or protect against the neurodegenerative disorder.
  • Neurodegenerative disorders that can be prevented or treated using the compositions and methods of the invention include disorders relating to the central nervous system and/or peripheral nervous system including, but not limited to, cognitive and neurodegenerative disorders such as Alzheimer's Disease, age-related loss of cognitive function and senile dementia, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's Disease, cerebral and 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, seizure disorders such as epilepsy and chronic seizure disorder, stroke, brain or spinal cord trauma, ADDS dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic function disorders such as hypertension and sleep disorders,
  • the invention provides compositions of complexes of hsps and antigenic peptides associated with neurodegenerative diseases and disorders, such as Alzheimer's Disease (AD), which can be used as vaccines to protect against and/or treat such diseases and disorders.
  • AD Alzheimer's Disease
  • compositions and formulations of the present invention are administered to a human subject to prevent a neurodegenerative disorder (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).
  • ND neurodegenerative disorder
  • 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. for Alzheimer's Disease, having the E4 allele of Apolipoprotein E or having a mutation in APP, PS1 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 aheady 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.
  • Antigenic peptides that can be used to form such hsp-antigenic peptide complexes are peptides derived from material found in the brains of patients with neurodegenerative disorders, or synthesized based on the known sequences of peptides associated with the pathology of such disorders.
  • Such peptides include, but are not limited to, peptides of, or derived from, the following: ⁇ -amyloids found in neurodegenerative plaques, for example, plaques associated with AD; tau protein, which is found in neurofibrillary tangles (NFTs); ⁇ -synuclein, which is found in Lewy bodies; prion protein (PrP), which is associated with spongiform encephalopathies, e.g., Creutzfeldt- Jakob disease, and other peptides associated with such neuropsychiatric disorders.
  • ⁇ -amyloids found in neurodegenerative plaques for example, plaques associated with AD
  • tau protein which is found in neurofibrillary tangles (NFTs)
  • ⁇ -synuclein which is found in Lewy bodies
  • PrP prion protein
  • Such antigenic peptides are described in detail in Section 4.1, below.
  • hsp-ND-associated peptide antigens can be produced by any of a number of methods.
  • antigenic peptides associated with neurodegenerative disorders and hsps can be obtained by recombinant or synthetic methods, or can be isolated and purified from recombinant cells.
  • Complexes between neurodegenerative disorder-associated peptides and hsps can be formed by covalent or non-covalent association of antigenic peptides with hsps.
  • 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.
  • complexes may be formed in vivo or in vitro using a variety of methods, described herein. Methods for preparing such hsp antigenic complexes are described in detail in Sections 4.2 to 4.4, below.
  • Heat shock protein-antigenic peptide complexes may be used as vaccines against neurodegenerative diseases and disorders. Without being bound by any particular theory, such complexes may act by eliciting a B-cell and/or T-cell response in patients with such disorders. As part of the B-cell response, complexes of hsps with ND-associated antigens may generate antibodies specific for neurodegenerative disease-associated peptides, which in turn, may accelerate clearance of antigenic materials that accumulate in the brains of patients with such diseases. As part of the T-cell response, complexes of hsps with antigenic peptides may elicit a cytotoxic T cell response against cells that express neurodegenerative disease-associated peptide sequences. Methods for the use of such hsp- neurodegenerative disease-associated peptide complexes as vaccines against such neurodegenerative disorders are described in Section 4.5 in detail herein. 4.1 Neurodegenerative Disease Antigens
  • ND-associated antigens comprise peptides and polypeptides, and fragments thereof, that are found associated with plaques, tissues or cells of subjects with neurodegenerative diseases and disorders, such as Alzheimer's Disease, and that are specific to subjects with such diseases and disorders.
  • antigenic peptides can be complexed with heat shock proteins and used to elicit an immune response against such disorders.
  • ND- associated antigens include, but are not limited to, ⁇ -amyloid (A ⁇ ) peptides and fragments thereof, oligomeric A ⁇ complexes and fragments thereof, fragments of ApoE4-A ⁇ complexes, hyperphosphorylated tau and fragments thereof, APP mutant proteins and fragments thereof, presenillin mutant proteins and fragments thereof, ⁇ -synuclein of Lewy bodies and fragments thereof, prion protein (PrP) and fragments thereof, as well as other antigenic peptides present in ND plaques, neurofibrillary tangles and lesions, cells, and tissues.
  • a ⁇ ⁇ -amyloid
  • oligomeric A ⁇ complexes and fragments thereof fragments of ApoE4-A ⁇ complexes
  • hyperphosphorylated tau and fragments thereof APP mutant proteins and fragments thereof, presenillin mutant proteins and fragments thereof, ⁇ -synuclein of Lewy bodies and fragments thereof, prion protein (
  • Antigenic peptides associated with nueropsychiatric disease or neurodegenerative disorders, or antigenic portions thereof, can be chosen from among those known in the art to be associated with such diseases and disorders. Alternatively, such antigens can be selected for their antigenicity or their immunogenicity, as determined by immunoassays or by their ability to generate an immune response.
  • antigenic peptides may be derived from ⁇ - amyloid (A ⁇ ).
  • a ⁇ ⁇ - amyloid
  • Peptide fragments of A ⁇ -(l-40) and A ⁇ -(l-42)] and A ⁇ -(25-35) are used.
  • Full-length A ⁇ peptides [A ⁇ -(l-40) and A ⁇ -(l-42)] and A ⁇ -(25-35) form fibrils and are neurotoxic (see, for e.g., Howlett et al, 1995, Neurodegeneration 4: 23-32; Pike et al, 1993, J. Neuroscience 13: 1676-1687; Yankner et al, 1990, Science 250: 279-282).
  • ⁇ -amyloid The predominant forms of ⁇ -amyloid are the 40 amino-acid form, A ⁇ 40, and the 42 amino acid form, A ⁇ 42.
  • amino acids 35 (methionine) and 41-42 appear to play important roles in the process of fibril formation. Therefore, in this embodiment, a peptide antigen comprising A ⁇ 40, or fragments thereof, comprising the amino acid sequence (N)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO:l), or a fragment comprising 5 to 10, 10 to 15, 15 to 20, 20 to 30, or 30 to 40 consecutive amino acids thereof may be used as an antigen.
  • NDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIA (SEQ ID NO:2), or a fragment containing 10, 11, 12, 13, 14, 15 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 consecutive amino-acids thereof, can be used as peptide antigens for use in hsp complexes as vaccines against AD.
  • An allele is one of a group of genes which occur alternatively at a given genetic locus.
  • DNA sequence polymorphisms that result in changes in amino acid sequence of an AD-associated antigen such as A ⁇ 40 and A ⁇ 42 will exist within a population of individual organisms (e.g., within a human population). Such polymorphisms may exist, for example, among individuals within a population due to natural allelic variation.
  • an antigen may comprise a dimeric, oligomeric or multimeric form of a ⁇ -amyloid.
  • fibrils can be formed by in vitro polymerization of A ⁇ 40 and A ⁇ 42 (Harper and Lansbury, 1997, Annu. Rev. Biochem. 66: 385-407).
  • the A ⁇ amyloid protofibril is a discrete intermediate in the in vitro process of A ⁇ fibril formation (Harper et al, 1997, Chem. Biol. 4: 119-125). This stable intermediate, and proteolytic fragments thereof, can also be used as antigenic peptides for complexing with hsps.
  • apolipoproteins, or fragments thereof can be used as antigens.
  • Apolipoprotein E Apolipoprotein E
  • ApoE Apolipoprotein E
  • One isoform of ApoE, ApoE4 is particularly associated with a high risk for AD.
  • complexes of ApoE4, or derivatives, fragments, or analogs thereof can be sources for antigenic peptides.
  • complexes of apolipoproteins and A ⁇ , or fragments such as proteolytic products thereof can also be used as antigens.
  • antigenic peptides may comprise amino acid sequences derived from APP proteins known to be associated with AD, or fragments thereof.
  • peptide fragments of a mutant APP comprising a mutation at codon 717 may be used (Chartier-Harlin et al, 1991, Nature 353: 844-6).
  • antigenic molecules may be peptide fragments of a mutant APP comprising a mutation at codon 670 or 671.
  • antigenic peptides derived from tau protein sequences are used. Hyperphosphorylated and ubiquitinated forms of the microtubule- associated protein tau, as well as tau mutations, are found associated with the pathologies of many neurodegenerative disorders, including AD (Lynch et al, Neurology 44: 1878-1884; Spillantini et al, 1998, Proc. Natl. Acad. Sci. U.S.A. 95: 7737-41). Thus, tau protein- derived antigenic peptides may be used to elicit an immune response against such neurodegenerative disorders.
  • the gene sequence encoding tau protein and the corresponding proteins sequences are available in public databases (GenBank accession no. NM_005910).
  • modified amino acid sequences derived from hyperphosphorylated or ubiquitinated tau protein are also within the scope of the invention.
  • antigenic peptides may comprise amino acid sequences derived from presenilin mutant proteins, PS1 (GenBank accession no. NM_000021; Sherrington et al, 1995, Nature 375: 754-760) and PS2 (GenBank accession no. 000447 ; Levy-Lahad et al, 1995, Science 269: 973-977).
  • antigenic peptides may comprise amino acid sequences derived from ⁇ -synuclein (GenBank accession no. AF044672), which is found in Lewy bodies in various neurodegenerative disorders. Nucleotide sequences encoding ⁇ - synuclein amino acid sequences may be used to for recombinant expression of antigenic ⁇ - synuclein peptide fragments. Such ⁇ -synuclein antigenic molecules may be used to treat a subject with a neurodegenerative disorder having the pathology of the presence of Lewy bodies. Such disorders include, but are not limited to, Parkinson's disease, some forms of Alzheimer's disease, and Lewy body dementia.
  • antigenic peptides may comprise amino acid sequences derived from the known amino acid sequence of prion protein (PrP; GenBank accession no. AF076976; GenBank accession no. NM_000311, Kretzschmar et al, 1986, DNA 5: 315-324).
  • prion antigenic molecules may be used to treat a subject with a neurodegenerative disorder having a prion pathology.
  • Such disorders include, but are not limited to, spongiform encephalopathies, e.g., Creutzfeldt- Jakob disease.
  • antigenic peptides may comprise amino acid sequences derived from the huntingtin protein sequences (GenBank accession no. NM_002111), and mutants, variants or fragments thereof. Such antigenic peptides can be used to form antigenic complexes with hsps to treat subjects with Huntington's Disease, or any other disease associated with altered activity or expression of huntingtin.
  • polyglutamine repeats, as well as proteins containing polyglutamine repeats are used as antigenic peptides.
  • amino acid sequences of an ND-associated antigenic peptide may be determined either by manual or automated amino acid sequencing techniques well known in the art.
  • Amino acid sequences and nucleotide sequences of antigenic molecules described above 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.
  • the ND-associated antigenic peptide may be coupled to a carrier protein comprising one or more T cell epitopes.
  • T helper cell epitopes can enhance immunogenicity of peptide constructs containing a limited number of epitopes.
  • mycobacteria can induce a strong T-cell response
  • mycobacteria derivatives have been used to potentiate immune responses, with or without adjuvants (see Lachmann et al, eds., 1986, 0 in Ciba Foundation Symposium on Synthetic Peptides as Antigens, Wiley, Chichester, Vol. 119, pp.
  • antigens can be conjugated to a T helper cell epitope, or carrier protein, and used to immunize subjects, optionally primed with BCG (bacillus Calmette-Guerin Mycobacterium tuberculosis var. bovis).
  • BCG Bacillus Calmette-Guerin Mycobacterium tuberculosis var. bovis
  • a carrier protein is 5 tuberculin purified protein derivative (PPD).
  • PPD tuberculin purified protein derivative
  • tetanus toxin sequences can be used as carrier proteins, for example, tt830-844 from tetanus toxin (Kumar et al, 1992, J.
  • the circumsporozoite protein CST3 of a human malarial parasite Plasmodiumfalciparum (Kumar et al, supra) may be coupled to an ND-associated antigen to enhance 0 immunogenicity.
  • the ND-associated antigenic peptide coupled to a universal helper comprising one or more T cell epitopes may then be complexed with heat shock proteins, as described in Section 4.3, below. 5
  • the peptide can be produced, either by recombinant techniques or by synthetic methods.
  • the antigenic peptide may be synthesized using conventional peptide synthesis or o any of a number of other protocols well known in the art.
  • a peptide corresponding to a mutant protein associated with AD such as an APP or presenilin mutant, can be synthesized by use of a peptide synthesizer. Either the entire protein can be synthesized, or an antigenic determinant thereof, preferably the portion of the protein that contains the mutant or variant amino acid(s).
  • ND-associated peptide potentially useful for a vaccine protective against neurodegenerative diseases and disorders may be synthesized by using conventional peptide synthesis or other protocols well known in the art.
  • Peptides having the same amino acid sequence as peptides associated with ND-associated diseases and disorders may be synthesized by solid-phase peptide synthesis using procedures similar to those described by Merrifield, 1963, J. Am. Chem. Soc. 85: 2149.
  • N- ⁇ -protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric support i.e., polystyrene beads.
  • the peptides are synthesized by linking an amino group of an N- ⁇ -deprotected amino acid to an ⁇ -carboxyl group of an N- ⁇ -protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide.
  • a reagent such as dicyclohexylcarbodiimide.
  • the attachment of a free amino group to the activated carboxyl leads to peptide bond formation.
  • the most commonly used N- ⁇ - protecting groups include Boc which is acid labile and Fmoc which is base labile.
  • analogs and derivatives of ND-associated antigenic peptides can be chemically synthesized.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the sequence of the ND-associated antigenic peptides.
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, or ithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ - alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
  • ND-associated antigenic peptides and polypeptides can produced by recombinant means.
  • the nucleotide sequence encoding an ND-associated antigen Once the nucleotide sequence encoding an ND-associated antigen has been identified, the nucleotide sequence, or a fragment thereof, 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 antigen. Methods for recombinant production of ND-associated peptide antigens 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 ND- associated antigen 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 ND-associated antigen.
  • 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 ND-associated antigen of any desired length can be generated using PCR primers that flank the nucleotide sequence encoding the peptide-binding domain.
  • an ND-associated antigen gene sequence can be cleaved at appropriate sites with restriction endonuclease(s) if such sites are available, releasing a fragment of DNA encoding the ND-associated antigen gene, or an antigenic derivative thereof. 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. I: 277-278). The DNA fragment that encodes the ND-associated antigen 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 ND-associated antigens 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; Grunstein 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 ND-associated antigen gene can be isolated from cells which express the ND-associated antigen.
  • 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 ND-associated antigen is available, the ND- associated antigen may be identified by binding of a labeled antibody to the ND-associated antigen synthesizing clones.
  • nucleotide sequences encoding an ND-associated antigen protein can be identified and obtained by hybridization with a probe comprising a nucleotide sequence encoding ND-associated antigen 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 32 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.
  • Nucleotide sequences encoding an ND-associated antigenic polypeptide can be inserted into the expression vector for propagation and expression in recombinant cells.
  • An expression construct refers to a nucleotide sequence encoding an ND- associated antigenic polypeptide operably associated with one or more regulatory regions which allows expression of the ND-associated antigenic polypeptide in an appropriate host cell.
  • "Operably-associated” refers to an association in which the regulatory regions and the ND-associated antigenic polypeptide sequence to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation of the ND-associated peptide or polypeptide sequence.
  • a variety of expression vectors may be used for the expression of ND-associated antigenic polypeptides, 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 ND-associated antigenic peptide gene sequence, and one or more selection markers.
  • Non-limiting examples of prokaryotic expression vectors may include the ⁇ gt vector series such as ⁇ gtl 1 (Huynh et al, 1984 in "DNA Cloning Techniques", Vol. I: A Practical Approach (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studier et al, 1990, Methods Enzymol., 185: 60-89).
  • Non-limiting examples of regulatory regions that can be used for expression in E. coli may include but not limited to lac, trp, Ipp, phoA, recA, tac, ⁇ P L> and phage T3 and T7 promoters (Makrides, 1996, supra).
  • a potential drawback of a prokaryotic host- vector system is the inability to perform many of the post-translational processing events of mammalian cells.
  • a eukaryotic host- vector system is preferred, a mammalian host- vector system is more preferred, and a human host- vector system is the most preferred.
  • the regulatory regions necessary for transcription of the ND-associated antigenic peptide or polypeptide can be provided by the expression vector.
  • a translation initiation codon (ATG) may also be provided to express a nucleotide sequence encoding an ND-associated antigenic polypeptide that lacks an initiation codon.
  • RNA polymerase and transcription factors bind to the regulatory regions on the expression construct to effect transcription of the ND- associated antigenic polypeptide sequence in the host organism.
  • the precise nature of the regulatory regions needed for gene expression may vary from host cell to host cell. Generally, a promoter is required which is capable of binding RNA polymerase to initiate the transcription of an operably-associated nucleic acid sequence.
  • Such regulatory regions may include those 5'-non-coding sequences involved with initiation of transcription and translation, such as a TATA box, cap site, a CAAT box, transcription factor binding sites, enhancer elements, and the like.
  • the non-coding region 3' to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.
  • Both constitutive and inducible regulatory regions may be used for expression of the ND-associated antigenic peptide or polypeptide. It may be desirable to use inducible promoters when the conditions optimal for growth of the recombinant cells and the conditions for high level expression of the ND-associated antigenic polypeptide are different. Examples of useful regulatory regions are provided in the next section below.
  • ND-associated antigenic polypeptides 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 (RS V-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.
  • mice mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45: 485-495), albumin gene control region which is active in liver (Pinkert et al, 1987, Genes Dev. 1: 268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al, 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al, 1987, Science 235: 53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes Dev.
  • beta-globin gene control region which is active in myeloid cells (Mogram et al, 1985, Nature 315: 338-340; KoUias 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 ND-associated antigenic peptide or polypeptide 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; Gorman, 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; Gorman, 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 ND-associated antigenic peptide.
  • selectable or screenable marker genes for initially isolating or identifying host cells that contain DNA encoding an ND-associated antigenic peptide.
  • 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 11: 223), hypoxanthine- guanine phosphoribosyltransferase (Szybalski and Szybalski, 1962, Proc. Natl. Acad. Sci. U.S.A.
  • adenine phosphoribosyltransferase genes can be employed in tk " , 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, wliich confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 2072); neomycin phosphotransferase (neo), which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al, 1981, J. Mol. Biol. 150: 1); and hygromycin phosphotransferase (hyg), which confers resistance to hygromycin (Santerre et al, 1984, Gene 30: 147).
  • Other selectable markers such as but not limited to histidinol and ZeocinTM can also be used.
  • DNA sequences with regulatory functions such as promoters
  • linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of cDNA or synthetic DNA encoding an ND-associated antigenic polypeptide, 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.
  • 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 ND-associated antigenic polypeptide sequence operably associated with regulatory regions can be directly introduced into appropriate host cells for expression and production of ND-associated antigenic peptide-hsp complexes 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 ND- associated antigenic polypeptide 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 ND-associated antigenic peptide in the host cells.
  • Expression constructs containing cloned nucleotide sequence encoding ND- associated antigenic polypeptides can be introduced into the host cell by a variety of techniques known in the art, including but not limited to, for prokaryotic cells, bacterial transformation (Hanahan, 1985, in DNA Cloning, A Practical Approach, 1: 109-136), and for eukaryotic cells, calcium phosphate mediated transfection (Wigler et al, 1977, Cell 11; 223-232), liposome-mediated transfection (Schaefer-Ridder et al, 1982, Science 215: 166- 168), electroporation (Wolff et al, 1987, Proc. Natl. Acad. Sci. 84: 3344), and microinjection (Cappechi, 1980, Cell 22: 479-488). Co-expression of an ND-associated antigenic peptide and an hsp in the same host cell can be achieved by essentially the same methods.
  • Cell lines that stably express ND-associated antigenic polypeptides or ND-associated antigenic peptide-hsp complexes 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 ND-associated antigenic polypeptide 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.
  • a number of viral-based expression systems may also be utilized with mammalian cells to produce ND-associated antigens.
  • 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 Shefrk, 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. coli.
  • 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 ND-associated antigenic peptide sequences (Karasuyama et al, Eur. 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 expression of the ND-associated antigenic peptide.
  • 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 Epstein-Barr virus (EBV) origin (OriP) and EBV nuclear antigen 1 (EBNA-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.
  • ND-associated antigenic peptides may also be made with a retrovirus-based expression system.
  • retroviruses can efficiently infect and transfer genes to a wide range of cell types including, for example, primary hematopoietic cells.
  • retroviruses such as Moloney murine leukemia virus, most of the viral gene sequences can be removed and replaced with nucleic acid sequences encoding the ND- associated antigen, 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).
  • yeast a number of vectors containing constitutive or inducible promoters may be used with Saccharomyces cerevisiae (baker's yeast), Schizosaccharomyces pombe (fission yeast), Pichia pastoris, and Hansenula polymorpha (methylotropic yeasts).
  • Saccharomyces cerevisiae bakeer's yeast
  • Schizosaccharomyces pombe Schizosaccharomyces pombe
  • Pichia pastoris Pichia pastoris
  • Hansenula polymorpha methylotropic yeasts
  • a baculovirus Autographa californica nuclear polyhidrosis virus (AcNPV) can be used as a vector to express an ND-associated antigenic peptide in Spodoptera frugiperda cells.
  • the ND-associated antigenic peptide DNA may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). These recombinant viruses are then used to infect host cells in which the inserted DNA is expressed (see, e.g., Smith et al, 1983, J. Virol. 46: 584; Smith, U.S. Patent No. 4,215,051).
  • the expression vector must be used with a compatible host cell which may be derived from a prokaryotic or an eukaryotic organism, including, but not limited to bacteria, yeasts, insects, mammals, and humans. Any cell type that can produce ND- associated antigenic polypeptides and is compatible with the expression vector may be used, including those that have been cultured in vitro or genetically engineered. Host cells may be obtained from normal or affected subjects, including healthy humans, patients with neurodegenerative disorders or disease, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers.
  • Preferred mammalian host cells include but are not limited to those derived from humans, monkeys and rodents, (see, for example, Kriegler M. in “Gene Transfer and Expression: A Laboratory Manual", New York, Freeman & Co. 1990), such as monkey kidney cell line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293, 293-EBNA), or 293 cells subcloned for growth in suspension culture (Graham et al, 1977, J. Gen. Virol. 36: 59), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin. Proc. Natl. Acad. Sci.
  • monkey kidney cell line transformed by SV40 COS-7, ATCC CRL 1651
  • human embryonic kidney line (293, 293-EBNA
  • 293 cells subcloned for growth in suspension culture Graham et al, 1977, J. Gen. Virol. 36:
  • mice sertoli cells mouse sertoli cells (Mather, 1980, Biol. Reprod. 23: 243-251), mouse fibroblast cells (NIH-3T3), monkey kidney cells (CVI ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), and mouse mammary tumor cells (MMT 060562, ATCC CCL51).
  • Exemplary cancer cell types used for demonstrating the utility of recombinant cells (producing ND-associated antigenic peptide-peptide complexes) as a cancer vaccine are provided as follows: mouse fibroblast cell line, NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin fransfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7.
  • 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.
  • the ND-associated antigenic protein, or an antigenic portion thereof can be purified by any methods appropriate for the protein, and then used to form complexes with hsps in vivo or in vitro as described in Section 4.3, below.
  • recombinant cells may be used to co-express ND-associated antigens together with hsps for formation of hsp-ND-associated antigen complexes in vivo.
  • conditions for growth of recombinant cells may be different from those for expression of ND-associated antigenic polypeptides and hsps. Modified culture conditions and media may be used to enhance production of hsp-peptide complexes.
  • recombinant cells containing ND-associated antigenic polypeptides with their cognate promoters may be exposed to heat or other environmental stress, or chemical stress. Any technique known in the art may be applied to establish the optimal conditions for producing ND-associated antigenic polypeptide or hsp-peptide complexes.
  • the recombinant ND-associated antigenic peptides and polypeptides of the invention can be recovered and purified from recombinant cell cultures by known methods, including ammonium sulfate precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, immunoaffinity chromatography, hydroxyapatite chromatography, and lectin chromatography.
  • the invention provides methods for purification of recombinant ND-associated antigenic peptides and polypeptides by affinity purification.
  • affinity chromatography well known in the art. One approach is based on specific molecular interactions between an affinity label present on the ND-associated antigenic polypeptide and its binding partner.
  • immunoaffinity chromatography relies on the immunospecific binding of an antibody to an epitope present on the tag. Described below are several methods based on specific molecular interactions of a tag and its binding partner.
  • Protein A affinity chromatography a method that is generally applicable to purifying recombinant ND-associated antigens that are fused to the constant regions of immunoglobulin, is a well known technique in the art.
  • Staphylococcus protein A is a 42 kD polypeptide that binds specifically to a region located between the second and third constant regions of heavy chain immunoglobulins. Because of the Fc domains of different classes, subclasses and species of immunoglobulins, affinity of protein A for human Fc regions is strong, but may vary with other species. Subclasses that are less preferred include human IgG-3, and most rat subclasses. For certain subclasses, protein G (of Streptococci) may be used in place of protein A in the purification.
  • Protein-A sepharose (Pharmacia or Biorad) is a commonly used solid phase for affinity purification of antibodies, and can be used essentially in the same manner for the purification of ND-associated antigenic polypeptide fused to an immunoglobulin Fc fragment.
  • Bound ND-associated antigenic polypeptide can be eluted by various buffer systems known in the art, including a succession of citrate, acetate and glycine-HCl buffers which gradually lowers the pH.
  • a polyhistidine tag may be used, in which case, the ND- associated antigenic polypeptide can be purified by metal chelate chromatography.
  • the polyhistidine tag usually a sequence of six histidines, has a high affinity for divalent metal ions, such as nickel ions (Ni 2+ ), which can be immobilized on a solid phase, such as nitrilotriacetic acid matrices.
  • Polyhistidine has a well characterized affinity for Ni 2+ -NTA- agarose, and can be eluted with either of two mild treatments: imidazole (0.1-0.2 M) will effectively compete with the resin for binding sites; or lowering the pH just below 6.0 will protonate the histidine side-chains and disrupt the binding.
  • the purification method comprises loading the cell culture supernatant onto the Ni 2+ -NTA-agarose column, washing the contaminants through, and eluting the ND-associated antigenic polypeptide with imidazole or weak acid.
  • Ni 2+ -NTA-agarose can be obtained from commercial suppliers such as Sigma (St. Louis) and Qiagen.
  • Antibodies that recognize the polyhistidine tag are also available which can be used to detect and quantify the ND-associated antigenic polypeptide.
  • Another exemplary affinity label that can be used is the glutathione-S- transferase (GST) sequence, originally cloned from the helminth, Schistosomajaponicum.
  • GST glutathione-S- transferase
  • an ND-associated antigenic polypeptide-GST fusion expressed in a prokaryotic host cell, such as E. coli can be purified from the cell culture supernatant by absorption with glutathione agarose beads, followed by elution in the presence of free reduced glutathione at neutral pH.
  • Denaturing conditions are not required at any stage during purification, and therefore, it may be desirable co-purification of hsps and ND-associated polypeptides, for use in the loading of immobilized hsps with ND-associated antigenic peptides.
  • GST is known to form dimers under certain conditions, dimeric ND-associated antigenic peptides may be obtained. See, Smith, 1993, Methods Mol. Cell Bio. 4: 220-229.
  • MBP maltose binding protein
  • E. coli Another useful affinity label that can be used is the maltose binding protein (MBP) of E. coli, which is encoded by the ma E gene.
  • MBP maltose binding protein
  • the secreted ND-associated polypeptide-MBP present in the cell supernatant binds to amylose resin while contaminants are washed away.
  • the bound ND-associated polypeptide-MBP is eluted from the amylose resin by maltose. See, for example, Guan et al, 1987, Gene 67: 21-30.
  • the second approach for purifying ND-associated antigenic peptides is applicable to affinity labels that contain an epitope for which polyclonal or monoclonal antibodies are available.
  • Various methods known in the art for purification of protein by immunospecific binding such as immunoaffinity chromatography, and immunoprecipitation, can be used. See, for example, Chapter 13 in Antibodies A Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor laboratory, 1988; and Chapter 8, Sections I and II, in Current Protocols in Immunology, ed. by Coligan et al, John Wiley, 1991; the disclosure of which are both incorporated by reference herein.
  • the embodiments described above may also be used to recover and purify hsp -ND-associated antigenic peptide complexes from the cell culture medium of mammalian cells, such as human cells expressing an hsp-ND-associated peptide complex of the invention.
  • the methods can be adapted to perform medium and large scale purification of an ND-associated antigenic peptide and or hsp -ND-associated antigenic peptide complexes. Methods that do not require lowering pH or denaturing conditions are most preferred for purification of hsp-antigenic peptide complexes.
  • the methods may be used to isolate ND-associated antigenic peptides from eukaryotic cells, for example, cells of neuronal origin or cells isolated from a subject with a neurodegenerative disorder. 4.2 Sources of Heat Shock Proteins
  • 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.
  • Such 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.
  • mouse Hsp70 Genbank Accession No. M35021, Hunt et al, 1990, Gene 87: 199-204
  • mouse gp96 Genbank Accession No. M16370, Srivastava et al, 1987, Proc. Natl. Acad. Sci. U.S.A. 85: 3807-3811
  • mouse BiP Genbank Accession No. U16277, Haas et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 2250-2254. Due to the degeneracy of the genetic code, the term "hsp gene", as used herein, refers not only to the naturally occurring nucleotide sequence but also encompasses all the other degenerate DNA sequences that encode the hsp.
  • hsps can be produced by any method known in the art, such as the recombinant methods described above (Section 4.1.3) for the production of ND-associated antigens, hsp-encoding cDNA or genomic DNA may be obtained from any species, for example, by PCR amplification.
  • Oligonucleotide primers representing known nucleic acid sequences of related hsps can be used as PCR primers.
  • the oligonucleotide primers represent at least part of the hsp gene that is highly conserved between hsps of different species.
  • the size of the coding region of the hsp gene being amplified is too large to be amplified in a single PCR, several PCR covering the entire gene, preferably with overlapping regions, may be carried out, and the products of the PCR ligated together to form the entire coding sequence.
  • a segment of an hsp gene is amplified, that segment may be cloned, and utilized as a probe to isolate a complete cDNA or genomic clone.
  • the hsp gene may then be cloned into an appropriate expression vector, and be produced, propogated, isolated and purified according to methods for recombinant production of proteins, such as those described for recombinant production of ND-associated antigens described in Section 4.1.2, above.
  • an hsp or a fragment thereof, can be synthesized using methods known in the art, such as those described for the synthesis of ND-associated antigens, in Section 4.1.2, above, h this case, conventional peptide synthesis may be used, or other synthetic protocols well known in the art.
  • Purification of the resulting hsp, or fragment thereof, may then be accomplished using conventional procedures, such as preparative HPLC using gel permeation, partition and/or ion exchange chromatography.
  • preparative HPLC using gel permeation, partition and/or ion exchange chromatography.
  • the choice of appropriate matrices and buffers are well known in the art.
  • hsps associated with peptides can elicit an immune response, by stimulating CD4 helper T lymphocytes and/or CD8 cytotoxic T lymphocytes (CTL) that recognize the hsp-associated peptides and by stimulating antibody production.
  • CTL cytotoxic T lymphocytes
  • antigenic peptides associated with hsps can enter the MHC class I presenting pathway of professional antigen presenting cells (APCs).
  • APCs professional antigen presenting cells
  • Hsp70-peptide complexes from cell lysates has been described previously, see, for example, Udono et al, 1993, J. Exp. Med.178: 1391-1396.
  • These methods can be used to purify the hsp-ND- associated antigenic peptide complexes of the invention from the recombinant cells and/or the cell culture.
  • the invention provides methods for purification of hsp-ND-associated antigenic peptide complexes by affinity purification, based on the properties of the affinity label present on the hsp or the ND-associated antigenic peptide.
  • One approach is based on specific molecular interactions between a tag and its binding partner.
  • the other approach relies on the immunospecific binding of an antibody to an epitope present on the tag.
  • the principle of affinity chromatography well known in the art is generally applicable to both of these approaches.
  • a nucleotide sequence encoding an hsp can be introduced into cells including, but not limited to, epithelial cells, endothelial cells, keratmocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
  • the choice of cell type depends on the type of ND- associated antigen being expressed, and can be determined by one of skill in the art.
  • the cell chosen produces the ND-associated antigen of interest.
  • the ND-associated antigen can be engineered to be expressed in a cell type that does not normally express the ND-associated antigen.
  • an expression construct comprising a nucleic acid sequence encoding the hsp is introduced into an antigenic cell.
  • antigenic cells may include cells that are of neural origin, or may include cells transformed with a nucleic acid such that they express an ND-associated antigenic peptide.
  • the cells e.g., neural cells
  • the cells used in the methods of the invention are of mammalian origin.
  • Mammals contemplated by this aspect of the invention include humans, companion animals (e.g., dogs and cats), livestock animals (e.g., sheep, cattle, goats, pigs and horses), laboratory animals (e.g., mice, rats and rabbits), and captive or free wild animals.
  • any neural cells preferably human neural cells
  • the cells provide the antigenic peptides which become associated non-covalently with the expressed hsp.
  • Introduction of gene constructs encoding the hsp and ND- associated antigen can be carried out by any method known in the art, including gene therapy art, such as but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequence encoding the hsp and ND-associated antigen, cell fusion, chromosome-mediated gene transfer, microcell- mediated gene transfer, spheroplast fusion, etc.
  • hsp70-peptide complexes The purification of hsp70-peptide complexes has been described previously, see, for example, Udono et al, 1993, J. Exp. Med. 178: 1391-1396.
  • the following procedure may be used, presented by way of example but not limitation, to purify hsp70 complexes from cells. Initially, cells are suspended in 3 volumes of IX Lysis buffer consisting of 5mM sodium phosphate buffer, pH 7, 150mM NaCl, 2mM CaCl 2 , 2mM MgCl 2 and ImM phenyl methyl sulfonyl fluoride (PMSF). Then, the pellet is sonicated, on ice, until >99% cells are lysed as determined by microscopic examination.
  • IX Lysis buffer consisting of 5mM sodium phosphate buffer, pH 7, 150mM NaCl, 2mM CaCl 2 , 2mM MgCl 2 and ImM phenyl
  • the cells may be lysed by mechanical shearing and in this approach the cells typically are resuspended in 30mM sodium bicarbonate pH 7.5, ImM PMSF, incubated on ice for 20 minutes and then homogenized in a Dounce homogenizer until >95% cells are lysed.
  • the lysate is then centrifuged at l,000g for 10 minutes to remove unbroken cells, nuclei and other cellular debris.
  • the resulting supernatant is recentrifuged at 100,000g 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, ImM 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, 2mM NaCl, O.lmM EDTA and 15mM 2-mercapruethanol).
  • the column is then developed with a 20mM to 500mM NaCl gradient and then 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). 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.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • 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 R G25 column (Pharmacia). If necessary the hsp70 preparation thus obtained can be repurified through the MonoQ FPLC Column as described above.
  • the hsp70-peptide complex can be purified to apparent homogeneity using this method. Typically 1 mg of hsp70-peptide complex can be purified from 1 g of cells/tissue.
  • An alternative method for purification of hsp70-peptide complexes 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 nonhydrolyzable 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 non-specifically bound 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.
  • purification of hsp70-pe ⁇ tide complexes by ADP-agarose chromatography can be carried out as follows: cells (500 million cells) are homogenized in hypotonic buffer and the lysate is centrifuged at 100,000 g 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 hsp70-peptide complexes can be purified to apparent homogeneity using this procedure.
  • hsp90-peptide complexes from cell lysates has been described, for example, in WO 95/24923, dated September 21, 1995, and WO 97/10000, dated March 20, 1997.
  • a procedure that can be used, presented by way of example and not limitation, is as follows: Initially, neural cells are suspended in 3 volumes of IX Lysis buffer consisting of 5mM sodium phosphate buffer (pH7), 150mM NaCl, 2mM CaCl 2 , 2mM MgCl 2 and ImM phenyl methyl sulfonyl fluoride (PMSF). Then, the pellet is sonicated, on ice, until >99% cells are lysed as determined by microscopic examination.
  • IX Lysis buffer consisting of 5mM sodium phosphate buffer (pH7), 150mM NaCl, 2mM CaCl 2 , 2mM MgCl 2 and ImM phenyl methyl sulfonyl fluoride (PM
  • the cells may be lysed by mechanical shearing and in this approach the cells typically are resuspended in 30mM sodium bicarbonate pH 7.5, ImM 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,000g for 10 minutes to remove unbroken cells, nuclei and other cellular debris.
  • the resulting supernatant is recentrifuged at 100,000g 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 20mM Sodium phosphate pH 7.4, ImM EDTA, 250mM NaCl, ImM 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 dialysis buffer. The proteins are then eluted with a salt gradient of 200mM to 600mM NaCl.
  • Hsp90-peptide complexes can be purified to apparent homogeneity using this procedure. Typically, 150-200 ⁇ g of hsp90-peptide complex can be purified from lg of cells/tissue.
  • a procedure that can be used, presented by way of example and not limitation, is as follows: A cell pellet is resuspended in 3 volumes of buffer consisting of 30mM sodium bicarbonate buffer (pH 7.5) and ImM PMSF and the cells allowed to swell on ice 20 minutes. The cell pellet is then 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,000g for 10 minutes to remove unbroken cells, nuclei and other debris.
  • the supernatant from this centrifugation step is then recentrifuged at 100,000g for 90 minutes.
  • the gp96-peptide complex can be purified either from the 100,000 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 ConA Sepharose 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 2g0 drops to baseline. Then, 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.
  • ⁇ - mannoside ⁇ -methyl mannoside
  • the column is cooled to room temperature and the parafilm removed from the bottom of the column.
  • Five column volumes of the ⁇ -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 column (Pharmacia) equilibrated with a buffer containing 20mM sodium phosphate, pH 7.4. The proteins are then eluted from the column with a 0-1M NaCl gradient and the gp96 fraction elutes between 400n ⁇ M and 550mM NaCl.
  • the procedure may 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 100,000g 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 2+ 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.
  • 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 100,000g 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 20,000g for 30 minutes and the resulting supernatant dialyzed against several changes of PBS (also without the Mg 2+ and Ca 24 to remove the detergent. The dialysate is centrifuged at 100,000g 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 100,000g supernatant, see above.
  • the gp96-peptide complexes can be purified to apparent homogeneity using this procedure. About 10-20 ⁇ g of gp96 can be isolated from lg cells/tissue.
  • complexes of hsps with ND-associated antigenic molecules are produced in vitro.
  • Immunogenic hsp-ND-associated peptide complexes can be generated in vitro by covalent or non-covalent coupling of an hsp with an ND-associated antigenic peptide.
  • antigenic molecules may be isolated from various sources, chemically synthesized, or produced recombinantly.
  • Hsps may also be prepared by a variety of methods, as described in Section 4.2, above. After isolation of hsps and antigenics molecules, complexes are produced in vitro.
  • This section describes a preferred method for preparing non-covalent immunogenic complexes of hsps and ND-associated peptides.
  • Hsps are prepared, as described in Section 4.2, above. Prior to complexing, the hsps may be pretreated with ATP or low pH to remove any peptides that may be associated with the hsp of interest. It may be advantageous to use hsps that are reversibly bound to a solid phase to facilitate buffer exchange, washings and isolation of the complexes before or after the complexing reaction.
  • ATP ATP
  • excess ATP is removed from the preparation by the addition of apyranase as described by Levy et al, 1991, Cell 67: 265-274.
  • the buffer is readjusted to neutral pH by the addition of pH modifying reagents.
  • the ND-associated antigenic molecules (I ⁇ g) and the pretreated hsp (9 ⁇ g) are mixed together to give an approximately 5:1 antigenic molecule : hsp molar ratio.
  • the mixture is then incubated for 15 minutes to 3 hours at 4° to 45° C in a suitable binding buffer such as one containing 20mM sodium phosphate, pH 7.2, 350mM NaCl, 3mM MgCl 2 and ImM phenyl methyl sulfonyl fluoride (PMSF).
  • the preparations are centrifuged through a Centricon 10 assembly (Millipore) to remove any unbound peptide.
  • the hsp-ND-associated peptide complexes formed can be washed free of unbound peptide prior to eluting the hsp-peptide complex off the solid phase.
  • the association of the peptides with the hsp can be assayed by SDS-PAGE.
  • hsp 70 is incubated with equimolar quantities of the antigenic molecule in 20mM sodium phosphate buffer pH 7.5, 0.5M NaCl, 3mM MgCl 2 and ImM ADP in a volume of 100 microliter at 37°C for 1 hr. This incubation mixture is further diluted to 1 ml in phosphate-buffered saline.
  • gp96 to peptides 5-10 micrograms of gp96 immobilized by its affinity tag to a solid phase is incubated with equimolar or excess quantities of the antigenic peptide in a suitable buffer, such as one containing 20mM sodium phosphate buffer pH 7.5, 0.5M NaCl, 3nM MgC12 at about 50 °C for about 10 minutes.
  • a suitable buffer such as one containing 20mM sodium phosphate buffer pH 7.5, 0.5M NaCl, 3nM MgC12 at about 50 °C for about 10 minutes.
  • modified gp96 containing the Ig tag can be immobilized to protein A-Sepharose for this procedure. This incubation mixture is then further incubated for about 30 minutes at room temperature.
  • the solid phase with the bound hsp-peptide complexes is washed several times to remove any unbound peptide.
  • the hsp-peptide complexes is then eluted from the solid phase by the appropriate technique.
  • the immunogenic hsp-ND-associated peptide complexes can optionally be assayed in vitro using, for example, the methods described in Section 4.4.3, below, and optionally characterized further in animal models using the preferred administration protocols and excipients discussed in Section 4.6.1 below
  • ND-associated antigenic peptides covalently attached to hsps may be used as vaccines to elicit an immune response.
  • hsps and ND-associated antigenic peptides are prepared, as described in Sections 4.1.2 and 4.1.3, and Section 4.2, respectively.
  • the hsps Prior to complexing, the hsps may be pretreated with ATP or low pH to remove any peptides that may be associated with the hsp of interest.
  • Non-covalent hsps and ND-associated antigenic peptide complexes can then be prepared, as described in Section 4.3.2.1, above.
  • the hsp and the ND-associated peptide within such complexes are covalently coupled.
  • hsps may be 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).
  • 1 mg of hsp is crosslinked to 1 mg of ND-associated peptide 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
  • Other methods for chemical crosslinking may also be used, in addition other methods for covalent attachment of proteins, such as photocrosslinking (See Current Protocols in Molecular Biology, Ausubel et al. (eds.), Greene Publishing Associates and Wiley Interscience, New York).
  • recombinant fusion proteins comprised of a heat shock protein sequence and an ND-associated antigenic peptide sequence, may be used to treat or prevent neurodegenerative diseases and disorders.
  • an expression vector is constructed using nucleic acid sequences encoding a heat shock protein fused to sequences encoding an ND-associated antigen, using recombinant methods known in the art, such as those described in Section 4.1.2, above.
  • Hsp-antigenic peptide fusions are then expressed and isolated.
  • Such fusion proteins can be used to elicit an immune response (Suzue et al, 1997, Proc. Natl. Acad. Sci. U.S.A. 94: 13146-51).
  • such fusion proteins can be used to elicit an immune response and in immunotherapy against target neurodegenerative disorders.
  • the present invention encompasses the use of hsps in methods for treatment and prevention of neurodegenerative disorders, hi various embodiments described in detail herein, an effective amount of an hsp in a complex with antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder is administered to a patient for therapeutic purposes.
  • hsp-ND- associated antigen complexes are prepared from a cell that displays the antigenicity of an antigen of an neurodegenerative disease or disorder, and used as vaccines against the disease or disorder.
  • the protocols described herein may be used to isolate hsp-peptide complexes from any cell that displays the antigenicity of an antigen associated with the neurodegenerative disorder.
  • cells may express the ND-associated antigen itself, or alternatively, cells may be infected by or engineered to express a fragment or non-pathogenic form of the ND-associated antigen.
  • a method for treatment or prevention of an neuropsychiatric disorder comprises introducing into a cell that displays the antigenicity of an ND-associated antigen an expressible hsp gene sequence, preferably as an expression gene construct.
  • the hsp gene sequence is manipulated by methods described above in Section 4.2.3, so that the hsp gene sequence, in the form of an expression construct, located extrachromosomally or integrated in the chromosome, is suitable for expression of the hsp in the recombinant cells.
  • the recombinant cells containing the expression gene constructs are cultured under conditions such that hsps encoded by the expression gene construct are expressed.
  • Complexes of hsps associated with the antigenic molecule that displays the antigenicity of an antigen associated with a neurodegenerative disorder are purified from the cell culture or culture medium by the methods described in Section 4.3.
  • hsp-peptide complexes are prepared from a cell genetically manipulated to express an hsp, for example, tissues, isolated cells or immortalized eukaryotic cell lines transformed with an ND-associated antigen.
  • an hsp for example, tissues, isolated cells or immortalized eukaryotic cell lines transformed with an ND-associated antigen.
  • immortalized animal cell lines are used as a source of the hsp-peptide complex, it is important to use cell lines that can express the ND-associated antigen of interest.
  • cells that are derived from the same species as the intended recipient of the vaccine Techniques for introducing an expressible form of the hsp gene sequences into these cell lines are described above in Section 4.2.3.
  • Neurodegenerative disorders include disorders relating to the central nervous system and/or peripheral nervous system including, but not limited to, cognitive and neurodegenerative disorders such as Alzheimer's Disease, age-related loss of cognitive function and senile dementia, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's Disease, cerebral and 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, seizure disorders such as epilepsy and chronic seizure disorder, stroke, brain or spinal cord trauma, AIDS dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders that include, but are not limited to
  • the purified hsp-ND-associated antigen 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.
  • 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 serum 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.
  • 4xl0 7 macrophages can be incubated with 10 microgram gp96-pepti.de 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., Ixl0 7 /ml) 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 6 to about 10 12 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.
  • Hsp-ND associated antigen 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.
  • hsp-ND associated antigen 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.
  • the heat shock protein ND-associated antigen 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. 4.5.1 MLTC Assay
  • 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 6 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 antigemcally 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 effecto ⁇ target (E:T) ratios (usually 1:1 to 40:1).
  • E:T effecto ⁇ target
  • the target cells are prelabelled by incubating lxl 0 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%.
  • 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.
  • RPMI 1640 media GibcoBRL, Gaithersburg, Md.
  • 5xl0 4 activated T cells/well PHA-blasts
  • PHA-blasts 5xl0 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 an hsp ND- associated antigen complex is determined by measuring antibodies produced in response to the vaccination with the complex, by an antibody response assay, such as an enzyme-linked immunosorbent assay (ELISA) assay.
  • an antibody response assay such as an enzyme-linked immunosorbent assay (ELISA) assay.
  • ELISA enzyme-linked immunosorbent assay
  • microtitre plates (96-well hnmuno Plate II, Nunc) are coated with 50 ⁇ l/well of a 0.75 ⁇ g/ml solution of a purified, non-hsp- complexed form of the ND-associated antigen used in the vaccine (e.g. A ⁇ 42) in PBS at 4°C for 16 hours and at 20°C for 1 hour.
  • the wells are emptied and blocked with 200 ⁇ l PBS-T-BSA (PBS containing 0.05% (v/v) TWEEN 20 and 1% (w/v) bovine serum albumin) per well at 20 °C for 1 hour, then washed 3 times with PBS-T.
  • PBS-T-BSA PBS containing 0.05% (v/v) TWEEN 20 and 1% (w/v) bovine serum albumin
  • 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-ND associated antigen 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 O 2 substrate solution.
  • OPD o-phenylene diamine
  • the methods can be used to determine whether an asymptomatic human subject displays any of the molecular hallmarks of a specific neurodegenerative disorder.
  • the methods may also be used to diagnose a neurodegenerative disorder in a human subject who exhibits symptoms of the early stages of the neurodegenerative disorder. These methods are useful for identifying individuals at risk of a neurodegenerative disorder who would benefit from the methods of the invention of treatment and prevention of neurodegenerative disorders.
  • the methods can also be used to assay the efficacy of the vaccines of the present invention and monitor the progress of a neurodegenerative disorder in those receiving the vaccines.
  • the diagnostic methods to be utilized according to the present invention include but are not limited to testing for molecular indicators of a particular neurodegenerative disorder, such as AD, or for alterations in neurophysiological function that would be affected by such a neurodegenerative disorder.
  • more than one of the assays described below would be carried out to confirm the diagnosis of the presence or the extent of progession of the neurodegenerative disorder.
  • a monoclonal antibody specific for the phosphorylated form serves as an indicator for neurodegenerative disorders, such as 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, PS1, 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 neurodegenerative disorders
  • 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, i.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 neurodegenerative disorders
  • the proteins or peptides described supra may be assayed for by a radioimmunoassay, an enzyme-linked immunosorbant assay (ELISA), 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).
  • Drug doses are also given in milligrams per square meter of body surface area because this method rather than body weight achieves a good correlation to certain metabolic and excretionary functions (Shirkey, 1965, JAMA 193: 443).
  • body surface area can be used as a common denominator for drug dosage in adults and children as well as in different animal species as indicated below in Table 1 (Freireich et al, 1966, Cancer Chemotherap. Rep. 50: 219-244).
  • the present invention provides dosages of the purified complexes of hsps and
  • an amount of Hsp70- ND-associated antigenic molecule complexes and or gp96 ND-associated -antigenic molecule complexes is administered that is in the range of about 2 microgram to about 5000 micrograms for a human patient, the preferred human dosage being the same as used in a 25g mouse.
  • ⁇ dosage for Hsp-90 peptide complexes in a human patient provided by the present invention is in the range of about 10 to 1,000 micrograms, the preferred dosage being 20 micrograms.
  • the doses recited above are preferably given once weekly for a period of about 4-6 weeks, and the mode or site of administration is preferably varied with each adminisfration.
  • intradermal administrations are given, with each
  • the first injection may be given subcutaneously on the left arm, the second on the right arm, the third on the left belly, the fourth on the right belly, the fifth on the left thigh, the sixth on the right thigh, etc.
  • the same site may be repeated after a gap of one or more injections.
  • split injections may be given.
  • half the dose may be given in one site and the other half on an other site on the same day.
  • the mode of administration is sequentially varied, e.g., weekly injections are given in sequence intradermally, intramuscularly, intravenously or infraperitoneally.
  • injections are preferably given at two-week intervals over a period of time of one month. Later injections may be given monthly. The pace of later injections may be modified, depending upon the patient's clinical progress and responsiveness to the immunotherapy.
  • Hsp-ND-associated antigenic molecule complexes of the invention may be formulated into pharmaceutical preparations for administration to mammals for treatment or prevention of neurodegenerative disorders and diseases. Drug solubility and the site of absorption are factors which should be considered when choosing the route of adminisfration of a therapeutic agent.
  • Hsp-ND-associated antigenic molecule complexes of the invention may be administered using any desired route of administration, including but not limited to, e.g., intradermally, subcutaneously, intravenously or intramuscularly, although intradermally is prefe ⁇ ed. Advantages of intradermal administration include use of lower doses and rapid absorption, respectively. Mucosal routes of administration include, but are not limited to, oral, rectal and nasal administration.
  • Preparations for mucosal administrations are suitable in various formulations as described below.
  • the route of administration can be varied during a course of treatment.
  • Prefe ⁇ ed dosages, routes of adminisfration and therapeutic regimens for complexes of peptides and naturally occurring HSPs are described in PCT International patent applications published as WO 96/10411 and WO 97/10001, which are incorporated by reference herein in their entireties.
  • compositions comprising hsp-ND-associated antigenic complexes formulated in a compatible pharmaceutical carrier may be prepared, packaged, and labeled for treatment of the indicated neurodegenerative disorder.
  • an amount of hsp-ND-associated antigenic complex is administered to a human that is in the range of about 2 to 150 ⁇ g, preferably 20 to 20 ⁇ g, most preferably about 5 ⁇ g, given once weekly for about 4-6 weeks, intradermally with the site of administration varied sequentially.
  • the complex may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions.
  • an appropriate buffer for example, phosphate buffered saline or other physiologically compatible solutions.
  • the resulting complex may be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol.
  • the hsp complexes and their physiologically acceptable solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration.
  • the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with phannaceutically 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 may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl py ⁇ olidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinyl py ⁇ olidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g.
  • compositions for oral administration may be suitably formulated to give controlled release of the complexes.
  • Such compositions may take the form of tablets or lozenges formulated in conventional manner.
  • 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 complexes may be 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, dichlorotefrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotefrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotefrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the complexes may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the complexes may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the complexes may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the complexes may be formulated with suitable polymeric or hydrophobic materials (for example, 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 for example, as an emulsion in an acceptable oil
  • ion exchange resins for example, 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 may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • kits for carrying out the therapeutic regimens of the invention comprise in one or more containers therapeutically or prophylactically effective amounts of hsp-ND associated peptide complexes in pharmaceutically acceptable form.
  • the hsp-ND associated peptide complexes in a vial of a kit of the invention may be in the form of a pha ⁇ naceutically acceptable solution, e.g., in combination with sterile saline, dexfrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid.
  • the complex may be lyophihzed or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dexfrose solution, etc.), preferably sterile, to reconstitute the complex to form a solution for injection purposes.
  • a pharmaceutically acceptable solution e.g., saline, dexfrose 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 hsp - ND-associated antigen complexes by a clinician or by the patient.
  • the immunopotency of ND-associated antigens can be determined by monitoring the immune response in test animals following immunization with the ND- associated antigen, or by use of any immunoassay known in the art. Generation of a humoral (antibody) response and/or cell-mediated immunity, may be taken as an indication of an immune response.
  • Test animals may include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., and eventually human subjects.
  • Methods of introducing the vaccine may include oral, infracerebral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or any other standard routes of immunization.
  • the immune response of the test subjects can be analyzed by various approaches such as: the reactivity of the resultant immune serum to the neurodegenerative disorder antigen, as assayed by known techniques, e.g., immunosorbant assay (ELISA), immunoblots, radioimmunoprecipitations, etc., or by protection of the immunized host against the neurodegenerative disorder.
  • ELISA immunosorbant assay
  • the vaccine of the invention may be tested in rabbits for the ability to induce an antibody response to the ND-associated antigen.
  • Male specific-pathogen-free (SPF) young adult New Zealand White rabbits may be used.
  • the test group each receives a fixed concentration of the vaccine.
  • a control group receives an injection of 1 mM Tris-HCl pH 9.0 without the ND-associated antigen.
  • Blood samples may be drawn from the rabbits every one or two weeks, and serum analyzed for antibodies to the ND-associated protein.
  • the presence of antibodies specific for the antigen may be assayed, e.g., using an ELISA assay.
  • cellular immunity may be momtored by methods including but not limited to measuring: a) delayed hypersensitivity as an assessment of cellular immunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels of ND-associated antigen, e.g., 42/43 ⁇ -amyloid.
  • Delayed hypersensitivity skin tests are also of great value in the overall immunocompetence and cellular immunity to an antigen. Inability to react to a battery of common skin antigens is termed anergy (Sato et al, 1995, Clin. Immunol. Pathol. 74: 35- 43). Proper technique of skin testing requires that the antigens be stored sterile at
  • a 25- or 27-gauge needle ensures intradermal, rather than subcutaneous, administration of antigen. Twenty-four and 48 hours after intradermal adminisfration of the antigen, the largest dimensions of both erythema and induration are measured with a ruler. Hypoactivity to any given antigen or group of antigens is confirmed by testing with higher concentrations of antigen or, in ambiguous circumstances, by a repeat test with an intermediate test.
  • the activity of cytolytic T-lymphocytes can be assessed in vitro using the following method.
  • 33% secondary mixed lymphocyte culture supernatant or IL-2 is included in the culture medium as a source of T cell growth factors.
  • T cells are cultured without the stimulator cells. In other experiments, T cells are restimulated with antigenically distinct cells. After six days, the cultures are tested for cytotoxicity in a 4 hour 5I Cr-release assay. The spontaneous 51 Cr-release of the targets should reach a level less than 20%.
  • a tenfold concentrated supernatant of W6/32 hybridoma is added to the test at a final concentration of 12.5% (Heike et al, J. Immunotherapy 15: 165-174).
  • the amount of immunogen to be used and the immunization schedule will be determined by a physician skilled in the art and will be administered by reference to the immune response and antibody titers of the subject.

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Abstract

L'invention concerne des préparations pharmaceutiques comprenant des complexes de protéines de choc thermique (hsp) associés à des molécules antigènes, à utiliser dans le traitement et la prévention de troubles et de maladies neurodégénératives. L'invention porte également sur des procédés d'utilisation desdites préparations pharmaceutiques en tant qu'agents immunothérapeutiques pour le traitement et la prévention de troubles et de maladies neurodégénératifs.
PCT/US2001/001825 2000-01-21 2001-01-18 Complexes de proteines de choc thermique/stress utilises en tant que vaccins contre les troubles neurodegeneratifs WO2001052890A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US6495347B1 (en) 1999-07-08 2002-12-17 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
US6921534B2 (en) 2001-02-05 2005-07-26 Stressgen Biotechnologies Corporation Hepatitis B virus treatment
US20110076323A1 (en) * 2008-01-16 2011-03-31 Alon Monsonego Vaccine for alzheimer's disease
US8540985B2 (en) 2008-06-26 2013-09-24 Orphazyme Aps Use of Hsp70 as a regulator of enzymatic activity
CN106163540A (zh) * 2013-09-13 2016-11-23 安达卢西亚进步与健康公共基金会 用于治疗蛋白病或构象病的聚集蛋白和分子伴侣的组合
US9662375B2 (en) 2010-11-30 2017-05-30 Orphazyme Aps Methods for increasing intracellular activity of Hsp70
US10709700B2 (en) 2014-09-15 2020-07-14 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

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5840540A (en) * 1995-04-28 1998-11-24 The Hospital For Sick Children Nucleic acids encoding presenilin II
US5851996A (en) * 1990-04-27 1998-12-22 Milkhaus Laboratory, Inc. Materials and methods for treatment of plaquing diseases
US5958919A (en) * 1996-09-20 1999-09-28 Washington University Treatment of presymptomatic alzheimer's disease to prevent neuronal degeneration

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5851996A (en) * 1990-04-27 1998-12-22 Milkhaus Laboratory, Inc. Materials and methods for treatment of plaquing diseases
US5840540A (en) * 1995-04-28 1998-11-24 The Hospital For Sick Children Nucleic acids encoding presenilin II
US5958919A (en) * 1996-09-20 1999-09-28 Washington University Treatment of presymptomatic alzheimer's disease to prevent neuronal degeneration

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BROWN I.R.: "Stress genes in the nervous system during development and aging", ANN. N.Y. ACAD. SCI., vol. 851, 1998, pages 123 - 128, XP002940039 *

Cited By (25)

* 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
US7754449B2 (en) 2000-06-26 2010-07-13 Nventa Biopharmaceuticals Corporation Human papilloma virus treatment
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
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
US20110076323A1 (en) * 2008-01-16 2011-03-31 Alon Monsonego Vaccine for alzheimer's disease
US9345753B2 (en) * 2008-01-16 2016-05-24 Yeda Research And Development Co. Ltd. At The Weizmann Institute Of Science Vaccine for alzheimer's disease
US8540985B2 (en) 2008-06-26 2013-09-24 Orphazyme Aps 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
US11938125B2 (en) 2008-06-26 2024-03-26 Zevra Denmark A/S 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
US11304941B2 (en) 2008-06-26 2022-04-19 Orphazyme A/S Use of HSP70 as a regulator of enzymatic activity
US9884058B2 (en) 2008-06-26 2018-02-06 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
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
EP3045180A4 (fr) * 2013-09-13 2017-06-28 Fundación Pública Andaluza Progreso Y Salud Combinaisons de protéines agrégantes et de chaperons moléculaires pour le traitement de protéinopathies ou de maladies confromationnelles
CN106163540A (zh) * 2013-09-13 2016-11-23 安达卢西亚进步与健康公共基金会 用于治疗蛋白病或构象病的聚集蛋白和分子伴侣的组合
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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