WO2001015655A2 - Peptides et substances, procedes et dispositif les utilisant pour diagnostiquer et traiter les troubles neurodegeneratifs - Google Patents

Peptides et substances, procedes et dispositif les utilisant pour diagnostiquer et traiter les troubles neurodegeneratifs Download PDF

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Publication number
WO2001015655A2
WO2001015655A2 PCT/IL2000/000509 IL0000509W WO0115655A2 WO 2001015655 A2 WO2001015655 A2 WO 2001015655A2 IL 0000509 W IL0000509 W IL 0000509W WO 0115655 A2 WO0115655 A2 WO 0115655A2
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peptides
peptide
neurodegenerative disorder
group
amino acid
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PCT/IL2000/000509
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English (en)
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WO2001015655A3 (fr
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Daniel M. Michaelson
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Ramot University Authority For Applied Research & Industrial Development Ltd.
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Priority to AU67223/00A priority Critical patent/AU6722300A/en
Priority to EP00954874A priority patent/EP1209969A2/fr
Publication of WO2001015655A2 publication Critical patent/WO2001015655A2/fr
Priority to US09/847,586 priority patent/US20030148404A1/en
Publication of WO2001015655A3 publication Critical patent/WO2001015655A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Definitions

  • the present invention relates to peptides derived from protein or proteins associated with a neurodegenerative disorder and to methods, substances and devices utilizing same. More particularly, the present invention relates to peptides representing immunogenic epitopes derived from a protein to which at least one antibody is produced in vivo at onset or during progression of a neurodegenerative disorder, such as, but not limited to, Alzheimer's disease. According to the teachings of the present invention the peptides can be used to (i) diagnose existence, non-existence, type or state of a neurodegenerative disorder; (ii) selectively remove an antibody from the blood of a patient suffering from the neurodegenerative
  • the present invention further relates to a method for identifying peptides useful for identifying an existence, non-existence, type or state of a neurodegenerative disorder in an individual. Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.
  • AD Alzheimer's Disease
  • a common form of neurodegenerative dementia of unknown cause Alzheimer's Disease typically initiates in late middle age and characterized by progressive memory loss and mental deterioration, associated with brain damage, and resulting in relentlessly progressive intellectual and personality decline.
  • the cholinergic changes are manifested by dysfunction and death of neurons in the basal forebrain and by a concomitant reduction in presynaptic cholinergic parameters in the cortex and the hippocampus [Sims, 1983].
  • AD is the commonest of the dementia, at present AD cannot be satisfactorily diagnosed during life and the quest for simple, non-invasive tests for diagnosis of AD is one of the highest priorities in the field.
  • AD neuropsychological testing
  • mini-mental status exam MMSE
  • the Blessed test [McDougall, 1990].
  • the former is a quick test, which is about 5 minutes long, that roughly assesses cognitive skills, reading, writing, orientation, and short-term memory.
  • the latter test in addition to the above mentioned faculties also evaluates activities associated with daily living.
  • the accuracy of the neuropsychological tests is not very impressive [Forstl, 1998] and ranges between 70-90 %, depending on the examiner.
  • apoE apolipoprotein E
  • AD Alzheimer's disease
  • AD markers Amyloid B peptide and Tau in the cerebrospinal fluid (CSF) each used individually have been unreliable as AD markers.
  • predictive reliability is increased and as such these markers are currently inco ⁇ orated in a detection kit marketed by Athena Diagnostics. Nonetheless, the reliability of this marker combination is limited since the results obtained therewith suffer from excessive overlap between AD and non-AD patients, as well as situations where Tau and amyloid -protein levels are both either low or both high in which case determinations are not effective.
  • Neural thread protein is marketed by Nymox Corporation as an early marker for AD [de la Monte, 1992; Monte, 1997]. It is present in neurons, is associated with neural plaques and is selectively upregulated in the AD brain [De La Monte, 1996].
  • the NTP diagnostic kit rely on detecting NTP in urine.
  • p97 is an iron-binding protein which has been shown to be present in the AD brain and to be specifically located in microglia cells in close association with senile plaques [Jefferies, 1996]. Studies have shown that significantly higher levels of p97 are present in AD sera as compared with normal control (NC) sera [Kennard, 1996].
  • Synapse Technologies Inc. is currently developing an AD marker system which utilizes this protein.
  • Immunological mechanisms and AD Several reports indicate the involvement of immunological mechanisms in the etiology of AD. These include the presence of immunoglobulins (Igs) in senile plaques [Ishii, 1976; Eikelenboom, 1982], the presence of antibodies in AD sera which have been shown histochemically to react with neuronal tissue [Ishii, 1976; Eikelenboom, 1982; Nandy, 1978; Watts, 1981; Fillit, 1985], and abnormally increased expression of AHLA-DR antigens in brains of AD patients [Rogers, 1987; Pouplard-Barthelaix, 1987; McGeer, 1987]. Furthermore, the presence of immune complexes in the cerebrospinal fluid (CSF) of AD patients [Cameron, 1985] and defective cellular immune function have also been described [Skias, 1985; Singh, 1986]. AD specific antibodies
  • AD sera contain antibodies that bind to specific constituents of cholinergic neurons [Chapman, 1988]. It was subsequently shown that AD sera contain a repertoire of antibodies directed against the heavy neurofilament subunit (NF-H), and that a subpopulation of these antibodies is specific to AD. It was also shown that this subpopulation of antibodies bind to NF-H epitopes the levels of which are significantly higher in neurofilaments of cholinergic neurons than in those obtained from heterogeneous neuronal preparations [Chapman, 1989; Soussan, 1994].
  • NF-H heavy neurofilament subunit
  • AD is associated with aberrant phosphorylation of neurofilaments and of other cytoskeletal proteins [Sternberger, 1985; Grundke-Iqbal, 1986; Lichtenberg-Kraag, 1992; Masliah, 1993].
  • NF-H from a purely cholinergic neuron preparation contains more than twofold more phosphorylated serine residues than does NF-H extracted from a heterogeneous neuron source [Soussan, 1994].
  • AD specific anti-cholinergic NF-H IgG bind to phosphorylated epitopes, it is possible that the specificity of these antibodies is caused by a cross reaction of antibodies that were generated in vivo against an abnormal antigen such as hyperphosphorylated neurofilaments or Tau.
  • a method of identifying an existence, non-existence, type or state of a neurodegenerative disorder in an individual comprising the steps of (a) immunoreacting with a serum sample derived from the individual at least one peptide representing at least one epitope derived from an endogenous protein to which at least one antibody is produced in vivo at onset or during progression of the neurodegenerative disorder, the at least one peptide being selected such that the at least one antibody being capable of immunobinding with the at least one peptide; and (b) detecting a presence, absence or degree of the immunobinding to thereby identify the existence, non-existence, type or state of the neurodegenerative disorder.
  • a proteinaceous substance useful for identifying an existence, non-existence, type or state of a neurodegenerative disorder in an individual comprising at least one peptide representing at least one epitope derived from an endogenous protein to which at least one antibody is produced in vivo at onset or during progression of the neurodegenerative disorder, the at least one peptide being selected such that the at least one antibody being capable of immunobinding the at least one peptide.
  • a filter for removing at least one antibody generated against an endogenous protein associated with the onset or progression of the neurodegenerative disorder from the blood of a patient suffering from the neurodegenerative disorder comprising a solid support and the proteinaceous substance described hereinabove attached thereto such that filtering the blood of a patient suffering from the neurodegenerative disorder through the filter substantially removes the at least one antibody therefrom.
  • an extracorporeal device for removing at least one antibody generated against an endogenous protein associated with the onset or progression of a neurodegenerative disorder from the blood of a patient suffering from the neurodegenerative disorder, the extracorporeal device comprising (a) the filter described above; and (b) a pump for circulating the blood of the patient suffering from the neurodegenerative disorder through the filter, such that the at least one antibody is substantially removed from the blood of a patient.
  • a peptide comprising an amino acid sequence representing at least one epitope of an endogenous protein to which at least one antibody is produced in vivo at onset or during progression of a neurodegenerative disorder.
  • a method of removing at least one antibody generated against an endogenous protein associated with the onset or progression of a neurodegenerative disorder from the blood of a patient suffering from the neurodegenerative disorder comprising the step of circulating the blood of the patient through an extracorporeal device including at least one peptide representing at least one epitope derived from an endogenous protein and capable of immunobinding at least one antibody recognizing the endogenous protein and which is associated with the neurodegenerative disorder, the extracorporeal device is configured such that when the blood of the patient is circulated therethrough the at least one peptide immunobinds the at least one antibody to thereby substantially remove antibodies associated with the neurodegenerative disorder from the blood of the patient.
  • an array device useful for identifying an existence, non-existence, type or state of a neurodegenerative disorder in an individual, the array device comprising a plurality of peptides each being attached to a solid support in a regiospecific manner, the plurality of peptides representing epitopes derived from at least one endogenous protein to which a plurality of antibodies are produced in vivo at onset or during progression of the neurodegenerative disorder, each of the plurality of peptides being selected such that each of the plurality of antibodies being capable of immunobinding the at least each of the plurality of peptides.
  • the endogenous protein is selected from the group consisting of NF-H, NF-M, Tau and B-amyloid protein.
  • the at least one epitope is a continuous epitope. According to still further features in the described preferred embodiments the at least one epitope a discontinuous epitope.
  • the at least one peptide includes a number of amino acids selected from the group consisting of at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, between seventeen and twenty five and between twenty five and at least thirty, According to still further features in the described preferred embodiments the at least one peptide includes an amino acid sequence as set forth in SEQ ID NO:23.
  • the at least one peptide includes a plurality of peptides and further wherein the at least one antibody includes a plurality of antibodies, whereas the plurality of peptides are selected such that the plurality of antibodies are capable of respectively immunobinding with the plurality of peptides.
  • each of the plurality of peptides includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 -76.
  • each of the plurality of peptides is of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 29, 32, 36, 38, 42, 44, 46, 54, 59, 62, 68, 70, 77 and 78.
  • each of the plurality of peptides is of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 32, 42, 54, 59, 62 and 77.
  • the neurodegenerative disorder is associated with progressive loss of cognitive functions.
  • the neurodegenerative disorder is associated with progressive loss of control of motoric functions.
  • the neurodegenerative disorder is associated with progressive loss of motoric functions
  • the neurodegenerative disorder is selected from the group consisting of diseases accompanied by dementia, such as, but not limited to, Alzheimer's disease; Multi-infarct Dementia (MID); Pick's disease;
  • Frontotemporal dementias with Parkinsonism linked to chromosome 17 Dementia pugilistica; Parkinson's disease with dementia; Gerstmann-
  • the at least one peptide includes an immobilizing moiety covalently attached thereto.
  • the immobilizing moiety is a member of a binding pair.
  • the member of the binding pair is selected from the group consisting of biotin, avidin, streptavidin, an antibody, a hapten, a receptor, a ligand, Ni and NTA.
  • the immobilizing moiety is covalently attached to a terminal of the at least one peptide, the terminal is selected from the group consisting of a carboxy terminal and an amino terminal.
  • At least one amino acid of the at least one peptide is a modified amino acid.
  • a method of identifying peptides useful for identifying an existence, non-existence, type or state of a neurodegenerative disorder in an individual comprising the steps of (a) preparing a plurality of peptides corresponding to a plurality of continuous or discontinuous sequences derived from an endogenous protein to which at least one antibody is produced in vivo at onset or during progression of the neurodegenerative disorder; (b) screening the plurality of peptides for at least one peptide being immunoreactive with a serum derived from at least one patient suffering from the neurodegenerative disorder, thereby identifying peptides useful of identifying an existence, non-existence, type or state of the neurodegenerative disorder
  • the continuous or discontinuous sequences derived from the endogenous protein include at least one phospho amino acid.
  • the at least one phospho amino acid is selected from the group consisting of phosphoserine, phosphothreonine and phosphotyrosine .
  • the phosphoserine forms a part of a sequence motif selected from the group consisting of sequence motives as set forth in SEQ ID NOs: 3, 4 and 5.
  • the continuous or discontinuous sequences derived from the endogenous protein include at least one repeat of the sequence set forth by SEQ ID NO:2.
  • the continuous or discontinuous sequences derived from the endogenous protein include at least one sequence motif selected from the group consisting of SEQ ID NOs:l, 3 and 4.
  • the step of preparing the plurality of peptides includes covalently attaching to each of the plurality of peptides at least one immobilizing moiety.
  • the immobilizing moiety is a member of a binding pair as further detailed above.
  • a method of generating a peptide combination useful for identifying an existence, non-existence, type or state of a neurodegenerative disorder in an individual comprising the steps of: (a) identifying at least one endogenous protein to which at least one antibody is produced in vivo at onset or during progression of the neurodegenerative disorder; (b) generating a plurality of peptides corresponding to the at least one endogenous protein; (c) reacting specific subsets of the plurality of peptide with serum obtained from: (i) a first population of individuals suffering from the neurodegenerative disorder; and (ii) a second population of individuals not suffering from the neurodegenerative disorder; and (d) identifying subset or subsets of the plurality of peptides being immunoreactive with a high number of said individuals of said first population and a low number of said individuals of said second population to thereby generate the peptide combination useful for identifying an existence, non-existence, type or state of a
  • each of the pfplurality of peptides includes a number of amino acids selected from the group consisting of at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, between seventeen and twenty five and between twenty five and at least thirty,
  • the plurality of peptides are bound in a regiospecific manner to a solid support, such that reactive peptides are identifiable according to their regiospecificity.
  • At least a portion of the plurality of peptides each include at least one phospho amino acid.
  • the at least one phospho amino acid is selected from the group consisting of phosphoserine, phosphothreonine and phosphotyrosine.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing peptides and substances, and methods and devices utilizing these peptides and substances for diagnosing and treating neurodegenerative disorders.
  • FIG. 1 depicts the results obtained from blocked or unblocked plates assayed using a saturating peptide concentration.
  • the plates were either blocked with 0.5 % Gelatin, 1 % Caseinate or not blocked, before the addition of the peptide.
  • the serum was diluted in PBST and the secondary antibody in PBST containing either 0.5 % Gelatin or 1 % Caseinate.
  • FIG. 2 depicts detection results as a response to increasing concentrations of peptide, using either PBS or TBS as the reaction buffer.
  • FIG. 3 depicts detection results as a response to AD serum dilutions.
  • FIG. 4 depicts detection results as a response to secondary antibody-enzyme conjugate dilutions.
  • FIG. 5 represents the amino acid sequence of the Tau protein (SEQ ID NO:79). Regions within the protein which can be used to generate peptides according to the teachings of the present invention include boxed Serine and/or Threonine residue(s), at least one of which is phosphorylated.
  • FIG. 6 is a schematic depiction of an extraco ⁇ oreal device for removing antibody or antibodies associated with a neurodegenerative disorder from the blood of a patient suffering from the disorder, according to the present invention.
  • FIG. 7 depicts an algorithm used to separate AD from normal control (NC) serum samples according to signals obtained using present invention.
  • FIG. 8 is schematic representation of profiles of antibody levels against different peptides, characteristic for AD or NC sera.
  • FIG 9. illustrates various peptide combinations which enable distinction between four different pairs of test subject groups.
  • the present invention is of a peptide or peptides which can be used to diagnose and/or treat a neurodegenerative disorder such as Alzheimer's Disease (AD).
  • AD Alzheimer's Disease
  • the present invention can be used to detect a presence, or absence of an antibody or antibodies produced, in vivo, against an endogenous protein at onset or during progression of the neurodegenerative disorder, to thereby identify the existence, non- existence, type or state of the neurodegenerative disorder.
  • the peptide or peptides according to the present invention can also be used to remove an antibody or antibodies produced, in vivo, against an endogenous protein, from the blood of a patient suffering from a neurodegenerative disorder, to thereby effect treatment of the neurodegenerative disorder.
  • the present invention also provides a method of identifying peptides useful for identifying an existence, non-existence, type or state of a neurodegenerative disorder in an individual.
  • the term "treat” includes substantially inhibiting, slowing or reversing the progression of a disease, substantially ameliorating clinical symptoms of a disease or substantially preventing the appearance of clinical symptoms of a disease.
  • peptide includes native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptido- mimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body, or more immunogenic.
  • modifications include, but are not limited to, cyclization, N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-
  • a peptide according to the present invention can be a cyclic peptide.
  • Cyclization can be obtained, for example, through amide bond formation, e.g., by inco ⁇ orating Glu, Asp, Lys, Orn, di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at various positions in the chain (-CO-NH or -NH-CO bonds).
  • Backbone to backbone cyclization can also be obtained through inco ⁇ oration of modified amino acids of the formulas H-N((CH 2 ) n -COOH)-C(R)H-COOH or H-N((CH 2 ) n -COOH)-C(R)H-
  • n 1-4, and further wherein R is any natural or non-natural side chain of an amino acid.
  • Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-
  • Natural aromatic amino acids, T ⁇ , Tyr and Phe may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (Nol), ring- methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl- Tyr.
  • Tables 1-2 below list all the naturally occurring amino acids (Table 1) and non-conventional or modified amino acids (Table 2). TABLE 1
  • a peptide according to the present invention can be used in a self standing form or be a part of moieties such as proteins and display moieties such as display bacteria and phages.
  • a peptide according to the present invention includes at least five, optionally at least six, optionally at least seven, optionally at least eight, optionally at least nine, optionally at least ten, optionally at least eleven, optionally at least twelve, optionally at least thirteen, optionally at least fourteen, optionally at least fifteen, optionally at least sixteen or optionally at least seventeen, optionally between seventeen and twenty five or optionally between twenty five and at least thirty amino acid residues (also referred to herein interchangeably as amino acids).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids.
  • derived from a protein refers to peptides derived from the specified protein or proteins and further to homologous peptides derived from equivalent regions of proteins homologous to the specified proteins of the same or other species, provided that these peptides are effective for the detection of antibodies associated with a neurodegenerative disorder.
  • the term further relates to permissible amino acid alterations and peptido- mimetics designed based on the amino acid sequence of the specified proteins or their homologous proteins.
  • epitope and the phrase “antigenic determinant” both refer to a region of a molecule, such as, for example, the peptide(s) of the present invention, which region is characterized by specific molecular arrangement so as to be recognized and bound by a specific antibody species.
  • an epitope can either be continuous, i.e., defined by a contiguous sequence, or discontinuous, i.e., defined by a combination of at lest two non-contiguous regions of the sequence.
  • antibody also refers to "antibody species" or “monospecific antibody” and is used to define an antibody subset which is of the same clonal origin and which therefore reacts with a single epitope.
  • Antibodies of any Ig class can be targeted by the peptides of the present invention, of preferable targeting are presently antibodies of the IgG and IgM classes which are present in the blood serum.
  • self antibody refers to antibodies produced against epitopes which form a part of a self (endogenous) protein.
  • the production of self antibodies in an individual often results in what is known as an "autoimmune response”.
  • antibody(s) associated with a neurodegenerative disorder refers to antibody or antibodies which are directed against an endogenous protein, which antibodies are produced in vivo at onset or during the progression of a neurodegenerative disorder.
  • neurodegenerative disorder is used to define a disorder characterized by progressive loss of cognitive functions, progressive loss of control of motoric functions and/or progressive loss of motoric functions.
  • disorders can include diseases accompanied by dementia, such as, but not limited to, Alzheimer's disease; Multi-infarct Dementia (MID); Pick's disease; Frontotemporal dementias with Parikinsonism linked to chromosome 17; Dementia pugilistica; Parkinson's disease with dementia; Gerstmann-Straussler-Scheinker disease with tangles; vascular dementia and neurodegenerative diseases not accompanied by dementia such as, but not limited to, Parkinson's disease; Multiple sclerosis; ALS; TIA and stroke without dementia.
  • AD Alzheimer's Disease
  • AD is a common form of neurodegenerative dementia of unknown cause.
  • AD is partially characterized by the presence, in cholinergic neurons, of a variant of the heavy neurofilament subunit (NF-H), which variant contains a significantly higher level of hype ⁇ hosphorylated epitopes than NF-H found in heterogeneous neuronal cells. It has been shown that AD sera contain a repertoire of antibodies directed against these epitopes of NF-H, and that a subpopulation of these antibodies is specific to AD. It has further been shown that a large portion of this antibody subpopulation is specific to the carboxy terminal of this protein.
  • NF-H heavy neurofilament subunit
  • a set of peptides which represent the epitopes of the carboxy terminal of NF-H were generated and screened against sera of AD and non-AD individuals.
  • Candidate peptides were identified, which can be used for diagnosing AD.
  • NF-H has a linear configuration
  • the carboxy domain thereof can be represented with overlapping peptides that span the entire molecule.
  • the peptide approach becomes manageable due to the characteristic sequence and organization of the carboxy terminal domain.
  • This domain is composed of numerous repeats of only three sequences. Each of these sequences is 6 to 8 amino acids long and it contains an AKSP (SEQ ID NO:2) motif, the serine of which when contained within the native NF-H molecule represents a potential phosphorylation site [Soppet, 1992].
  • AKSP SEQ ID NO:2
  • the specific configuration of the NF-H molecule allows to construct a small number of phosphorylated and non-phosphorylated peptides which span the entire length of the relevant NF-H domain.
  • peptides generated according to the teachings of the present invention have been utilized with great success in specifically identifying sera of AD patients.
  • NF-H protein candidates associated with neurodegenerative disorders in which the formation of self antibodies is observed.
  • protein candidates can include, but are not limited to, NF-M, Tau (either in solution or as insoluble tangle), B-amyloid protein or peptides derived from B-amyloid protein (in solution or in the form of insoluble plaques).
  • the method according to this aspect of the present invention is implemented by executing the following method steps, in which, in a first step, a plurality of peptides corresponding to a plurality of continuous or discontinuous sequences derived from an endogenous protein, to which at least one antibody is produced in vivo at onset or during progression of the neurodegenerative disorder, are prepared.
  • the endogenous sequence is first computer analyzed for theoretical antigenic determinants, by for example, the software provided by the Genetic Computer Group package of the Wisconsin University (GCG).
  • GCG Genetic Computer Group package of the Wisconsin University
  • the plurality of peptides are screened for the presence of at least one peptide which is differentially immunoreactive (e.g., not immunoreactive or which is substantially less or more immunoreactive) with a serum derived from a normal control individual, as is compared to a serum derived from a patient suffering from the neurodegenerative disorder.
  • at least one peptide which is differentially immunoreactive e.g., not immunoreactive or which is substantially less or more immunoreactive
  • Peptides thus identified can then be used for identifying an existence, non-existence, type or state of a neurodegenerative disorder.
  • screening approaches include, but are not limited to, enzyme linked immuno-sorbent assay (ELISA), immunopercipitation, western blots, slot and dot blots, magnetic bead separation, solid support arrays, affinity columns and phage or bacterial display.
  • ELISA enzyme linked immuno-sorbent assay
  • Western blots western blots
  • slot and dot blots magnetic bead separation
  • solid support arrays affinity columns and phage or bacterial display.
  • the peptide(s) when a peptide or peptides are used in context with screening methods which include a solid or semisolid support, the peptide(s) preferably include a binding moiety such that the peptide can be immobilized to such supports.
  • the peptide(s) further include an immobilizing moiety covalently attached thereto.
  • an immobilizing moiety can be a charged moiety which can electrostatically bind surface charges provided on the support.
  • the immobilizing moiety is a member of a binding pair which can bind to its co-member when the latter is attached to the solid support.
  • binding pairs include, but are not limited to, biotin-avidin/streptavidin, antibody-antigen/hapten, e.g., a peptide tag such as FLAG c-myc and the like, cellulose binding domain (CBD)-cellulose, receptor-ligand and Ni-NTA.
  • One member of the binding pair can be covalently bound to the peptide, for example, at the amino or carboxy terminal, and the other member of the binding pair can be covalently or otherwise bound to the support, such that the immobilization of the peptide on the support is provided by the interaction between the members of the binding pair.
  • peptides can be attached directly to a solid support by reacting an amino- or carbloxyl group of the peptide with a reactive group which forms a part of the solid support.
  • Peptides according to the teachings of the present invention can be synthesized by standard peptide synthesis techniques, for example, using either standard 9-fluorenylmethoxycarbonyl (F-Moc) chemistry [see, for example, Atherton, 1985] or standard butyloxycarbonate (T-Boc) chemistry although it is noted that, more recently, the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl system, developed by Sheppard et al. has found increasingly wide application [Sheppard, 1986]. The correctness of the structure and the level of purity, which will normally be in excess of 85 %, should be carefully checked, and particular attention be given to the correctness of internal disulfide bridging arrangements when present.
  • F-Moc 9-fluorenylmethoxycarbonyl
  • T-Boc butyloxycarbonate
  • chromatographic analyses including high performance liquid chromatography (HPLC), and spectrographic analyses, including Raman spectroscopy, may, for example, be employed for this pu ⁇ ose.
  • HPLC high performance liquid chromatography
  • Raman spectroscopy Raman spectroscopy
  • any suitable synthesis method may also be employed to synthesize peptide(s) directly on a solid support. Methods for synthesizing peptides on solid supports are well known in the art [for further detail see Bodanszky, 1985; Coe, 1998; Sucholeiki, 1998; Albericio, 1997]
  • an immobilizing moiety or any other moiety or modified amino acid can readily be inco ⁇ orated into a synthesized peptide.
  • the peptides according to the present invention may be synthesized by any conventional method, either directly using manual or automated peptide synthesis techniques as mentioned above, or indirectly by RNA or DNA synthesis and conventional techniques of molecular biology and genetic engineering. Such techniques may be used to produce hybrid proteins containing one or more of the polypeptides fused into another polypeptide sequence such as the case of the bacterial or phage display mentioned above in context with screening methods. It should be noted however that inco ⁇ orating modified amino acids cannot be made directly using a recombinant DNA system. As such, since some of the peptides of the present invention include modifications such as phosphorylation, these peptides are preferably chemically synthesized as described hereinabove. It will be appreciated however that since directed phosphorylation can be provided by some cell expression system, such peptides can also be produced by recombinant techniques, although in this case, verification of phosphorylation should be employed prior to use.
  • peptides which are specifically reactive with serum of an individual suffering from a neurodegenerative disorder have been identified using the screening method of the present invention as hereinabove described, such peptides can be used to implement additional aspects of the present invention as further detailed in the following sections.
  • At least one peptide preferably a set of peptides, which are utilizable for diagnosing or treating a neurodegenerative disorder, such as Alzheimer's disease.
  • a neurodegenerative disorder such as Alzheimer's disease.
  • the utilization of such peptide or peptides for the diagnosis and/or treatment of a neurodegenerative disorder is further described hereinbelow.
  • the peptides according to the present invention each include an amino acid sequence representing at least one continuous or discontinuous epitope derived from an endogenous protein to which at least one antibody is produced in vivo at onset or during progression of a neurodegenerative disorder.
  • This endogenous protein is defined as a protein normally expressed in the body of an individual and which, the over expression, specific localization and/or modification thereof is associated with a neurodegenerative disorder.
  • the at least one epitope derived from the endogenous protein is either a continuous epitope or a discontinuous epitope.
  • epitopes present in peptides and proteins are defined by the residues of the amino acid within the sequence of the peptide or protein and/or the modifications, such as the addition of prosthetic groups, to these amino acids.
  • an epitope is determined by either a continuous or discontinuous stretch of amino acids which typically includes at least five to seven amino acids. It will be appreciated that an epitope is determined by either the primary structure (the sequence of amino acids) and/or by the spatial conformation of this sequence which can be determined by the secondary, tertiary, globular (quaternary) structure, or any combinations thereof.
  • peptides derived from the endogenous protein each represent at least one epitope of this protein, such that antibodies reactive with the protein are also reactive with these peptides.
  • peptides encompassing all the possible epitopes, continuous or discontinuous, which are included within an endogenous protein can be generated, according to the teachings of the present invention.
  • generating all or a substantial fraction of such epitopes is preferably effected by phage or bacterial display, whereas generating a smaller fraction can be efficiently effected by peptide libraries, as is further exemplified in the Examples section that follows in context of the NF-H and Tau proteins.
  • the peptide(s) include at least one phospho-amino acid.
  • the phospho-amino acid is phosphoserine. It will be appreciated however, that other phosphorylated amino acids can be used in context with the peptides of the present invention, especially phosphorylated forms of amino acids which have been demonstrated to be associated with motifs found in proteins associated in one way or another with neurodegenerative disorders. Such phosphorylated amino acids include, but are not limited to, phosphotyrosine and phosphothreonine.
  • the phosphoserine forms a part of a sequence motif AKSP as set forth in SEQ ID NO:2.
  • the phosphoserine forms a part of a sequence motif as set forth in SEQ ID NOs: 3, 4 or 5, each of which includes an AKSP core.
  • the endogenous protein is Tau, antibodies to which characterize AD patients.
  • Figure 5 represents the amino acid sequence of Tau (SEQ ID NO:79).
  • Preferred regions within the protein which can be used to generate peptides according to the teachings of the present invention include boxed serine and threonine residues, at least one of which is phosphorylated.
  • the endogenous protein is NF-H, antibodies to which characterize AD patients.
  • SEQ ID NOs:5-76 represent peptides generated according to the teachings of the present invention and which represent epitopes derived from the carboxy terminal of NF-H.
  • a subset including, for example, some of the peptides set forth in SEQ ID NOs:5-76 is utilized for the detection of antibodies associated with AD. More preferably, a subset including some of the peptides set forth in SEQ ID NOs: 21, 29, 32, 36, 38, 42, 44, 46, 54, 59, 62, 68, 70, 77 and 78 or most preferably a subset including the peptides set forth in SEQ ID NOs: 21, 32, 42, 54, 59, 62 and 77 are utilized for the detection of antibodies associated with AD.
  • novel disease associated proteins can be utilized to generate short peptides of 5-25 amino acids in length spanning the novel protein. Single peptides or subsets of peptides can then be tested against serum derived from a population of individuals suffering from a neurodegenerative disorder and serum derived from a healthy population to thereby uncover peptides or subset of peptides which are most accurate in predicting the disease state.
  • the present invention is also applicable to yet uncovered proteins against which antibodies are generated at onset or during the progression of a neurodegenerative disorder.
  • one or more peptides according to the present invention can be presented in context of non-related amino acid sequences.
  • a proteinaceous substance useful for identifying an existence, non-existence, type or state of a neurodegenerative disorder in an individual which includes at least one peptide representing at least one epitope derived from an endogenous protein to which at least one antibody is produced in vivo at onset or during progression of the neurodegenerative disorder.
  • the proteinaceous substance is preferably immobilized.
  • immobilization is preferably effected as described hereinabove, with respect to immobilizing moieties.
  • immobilization can be effected by translationally fusing the peptide DNA sequence to a carrier DNA which codes for a carrier protein.
  • This carrier protein-peptide fusion protein when expressed by specific display systems enables displaying the peptide on the exterior portion of a bacteria or phage. Methods of constructing display libraries are well known in the art.
  • One main advantage of using display libraries, as opposed to peptide libraries, is the ability to dramatically increase the repertoire of sequences displayed because such sequences need not be presented in a regiospecific context as is the case for peptide libraries which are not propagatable.
  • Display libraries according to this aspect of the present invention can be used to detect binding to antibodies associated with a neurodegenerative disorder.
  • screening for suitable peptides and identification of the existence, non-existence, type or state of the neurodegenerative disorder can be effected.
  • Positive isolates, either phages or bacteria, can be thereafter directly employed in the diagnosis of patients in a fashion similar to that described above for self standing peptides.
  • peptides suitable for the specific immunobinding of antibodies which are produced in vivo at onset or during progression of a neurodegenerative disorder such as Alzheimer's disease (AD), Multiple Infarct Dementia (MID) and Parkinson's Disease with Dementia (PwD) are provided.
  • AD Alzheimer's disease
  • MID Multiple Infarct Dementia
  • PwD Parkinson's Disease with Dementia
  • the present invention also provides an approach which can be used to identify new peptides derived from characterized and in the future from yet to be characterized endogenous proteins which are associated with self antibody production in neurodegenerative disorders.
  • peptides synthesizable according to the present invention can be utilized as tools with which the identification and treatment of individuals possessing a neurodegenerative disorder can be effected.
  • these peptides can also be used to further characterize neurodegenerative disorders.
  • the method according to this aspect of the present invention is effected by implementing the following method steps, in which, in a first step, a serum sample derived from the individual is immunoreacted with peptide(s) which are prepared according to the present invention.
  • the reaction can be effected through several approaches some of which are listed hereinabove.
  • the term "serum” refers to mammalian blood or any portion or derivative thereof, treated or untreated. Preferably it refers to a blood sample from which hematopoietic cells have been removed.
  • the serum sample is reacted with a plurality of peptides which are arrayed on a solid support, as further detailed hereinunder.
  • a plurality of peptides which are arrayed on a solid support, as further detailed hereinunder.
  • An example to possible reaction conditions and components is given in the Examples section that follows. It will be appreciated in this case, however, that other reaction parameters and components which enable the detection of a reaction can be employed by a skilled artisan while implementing the present invention.
  • a presence, absence or the degree of immunobinding between the at least one peptide and an antibody contained within the serum sample is effected.
  • the detection of binding can be visualized, for example, colorimetrically, fluoroscentically or be otherwise realized by any other method commonly practiced in the art, such as, for example, radioactivity counting and the like. It will be appreciated that these methods can be employed either manually or automatically. For example, it is possible, and it is further exemplified in the Examples section that follows, to use ELISA detection along with automated sample processing to yield detection. Alternatively, technologies for automated detection of microarrayed samples can be employed. Many examples of microarray detection systems exist in the art. The use of peptide loaded microchips is envisaged.
  • peptides synthesizable according to the teachings of the present invention are preferably utilized in an array configuration for identifying an existence, non-existence, type or state of a neurodegenerative disorder in an individual.
  • each of the plurality of peptides is attached to a solid support in a regiospecific manner to form an array device. This enables the recognition of positively reacted peptides according to their regiospecific location or attachment to the solid support.
  • Each of the plurality of peptides can represent a single epitope, or alternatively a plurality of epitopes derived from an endogenous protein to which a plurality of antibodies are produced in vivo at onset or during the progression of a neurodegenerative disorder.
  • a positive immunobinding reaction detected for each of the peptides utilized can be indicative of the existence, non-existence, type or state of a neurodegenerative disorder. It will be appreciated that the use of an array device allows for the co-analysis of multiple immunobinding reactions which enables a more precise identification of the existence, non- existence, type or state of the disorder.
  • a state, type or existence of a neurodegenerative disorder is represented by a specific subset of antibodies, which are reactive to several endogenous proteins
  • co-analysis of multiple immunobinding reactions can enable the more precise identification of a specific state, type or existence of the disorder. This is particularly advantageous in cases where treatment of certain neurodegenerative disorders is most effective when specifically designed according to the state of progression or type of the disorder. As such, the recognition of a specific state or type of a neurodegenerative disorder can potentially enable a more effective treatment thereof.
  • Employing a combination of peptides as herein described enables the detection of neurodegenerative disorders in general. Specific sets which includes various combinations of peptides would enable detection of different neurodegenerative disorders and type and progression states thereof.
  • prior art methods and kits measure single biochemical markers, and as a result, such prior art methods typically and inherently enable the detection of a single subtype of a single neurodegenerative disorder.
  • peptides synthesizable according to the teachings of the present invention can be used for treating a neurodegenerative disorder by removing antibody(s) associated with the neurodegenerative disorder from the blood of a patient suffering from the disorder.
  • a filter for removing antibody(s) associated with a neurodegenerative disorder from the blood of a patient suffering from the neurodegenerative disorder is provided.
  • the filter includes a solid support and an attached proteinaceous substance which includes a peptide or peptides according to the present invention.
  • the filter can include a single type of peptide or alternatively it can include several types representing several epitopes associated with a single or several endogenous protein associated with a single neurodegenerative disorder.
  • the proteinaceous substance is further described hereinabove.
  • filter is used herein to refer to any element which is capable of supporting the attached proteinaceous substance while at the same time allow for the blood of a patient to flow through in a manner which enables intimate contact between the blood components and the peptide(s) included within the proteinaceous substance. As such it is meant to include columns, membranes and the like.
  • an extraco ⁇ oreal device designed or adapted for removing antibody(s) associated with a neurodegenerative disorder from the blood of a patient suffering from the neurodegenerative disorder.
  • An example to such an extraco ⁇ oreal device is shown in Figure 6, and is referred to hereinbelow as device 10.
  • Device 10 includes a pump 12 for circulating the blood of a patient 13 suffering from the neurodegenerative disorder through a filter 14, which includes peptide or peptides as previously described herein above.
  • a pump 12 for circulating the blood of a patient 13 suffering from the neurodegenerative disorder through a filter 14, which includes peptide or peptides as previously described herein above.
  • antibody(s) associated with the neurodegenerative disorder are substantially removed from the blood of patient 13. It will be appreciated that in cases where these antibodies generate an autoimmune response which contributes to the onset or progression of the disorder, the removal of these antibodies would greatly diminish, or abolish the progression of the disorder.
  • the present invention provides peptides with which a neurodegenerative disorder can be diagnosed and treated. Furthermore, the present invention provides a method with which new peptides of characterized and yet to be characterized endogenous proteins associated with self antibody production in neurodegenerative disorders can be identified. In addition, the present invention provides devices for diagnosing and treating neurodegenerative disorders, which devices inco ⁇ orate the peptide(s) according to the present invention. Finally, since the peptides of the present invention represent epitopes of proteins which are associated with neurodegenerative disorders, such peptides can also be used to further investigate and characterized such disorders.
  • Rational Structure and characteristics of NF antigens Since it was shown that a subset of NF-H associated antibodies is present at higher levels in AD than in negative control subjects [Chapman, 1988; Chapman, 1989], one may deduce that the NF-H molecule can be used to detect these antibodies present in blood serum. To do so, one must first characterize the structure of the molecule.
  • neurofilaments a major constituent of the neuronal cytoskeleton, are composed of three different proteins. These subunits are called the heavy (NF-H), the medium (NF-M) and the light (NF-L) proteins, and their approximate molecular masses are 200, 160, and 68 kDa, respectively. All the neurofilament proteins contain a conserved helical rod domain which forms the basis of their polymerization and assembly to 10 nm wide filaments.
  • the remaining carboxy terminal domains of the neurofilament proteins form side arms which extend from the helical core of the neurofilament fiber and cross-bridge it to adjacent neurofilaments or to other cytoskeletal elements [Robinson, 1988; Steinert, 1988].
  • These extended carboxy terminal tail domains contain multiple repeats of the sequence motif Lys-Ser-Pro (KSP, SEQ ID NO:l) which repeat approximately 10 times in NF-M and more than 40 times in NF-H.
  • KSP sequences The serine residues in these repeating KSP sequences are heavily phosphorylated and serve as substrates for second messenger-independent kinases [Julien, 1983; Lee, 1988; Wible, 1989; Roder, 1991].
  • Neurofilament proteins can also be phosphorylated by second messenger- dependent kinases including protein kinase C, cyclic AMP-dependent protein kinase, and Ca 2+ /calmodulin-dependent kinase [Gonda, 1990; Sihag, 1990; Tokui, 1990; Dosemeci, 1992].
  • the sites phosphorylated by the latter kinases are situated in the amino terminal end of the neurofilament subunits and are much less abundant than those of the repeating KSP motif [Nixon, 1991].
  • the extent of phosphorylation of neurofilament proteins is developmentally controlled and varies between different parts of the neuron [Dahl, 1983; Sternberger, 1983; Lee, 1987; Dahl, 1988; Carden, 1987].
  • NF-H has been successfully used as an antigen in antibody capture assays, where it was shown that AD-sera contain markedly and significantly higher levels of anti-NF-H antibody as compared with normal control (NC) sera [Chapman, 1988; Chapman, 1989]. Moreover, when the native NF-H molecule was replaced with the highly phosphorylated carboxy terminal tail of the NF-H as the antigen in the immunoassay, the separation between signals obtained from AD and NC sera was further improved [Soussan, 1994].
  • a synthetic peptide approach becomes manageable in this case due to the characteristic sequence and organization of the carboxy terminal domain.
  • This domain is composed of numerous repeats of only three sequences. Each of these sequences is 6 to 8 amino acids long and it contains an AKSP (SEQ ID NO:2) motif, the serine of which when contained within the native NF-H molecule represents a potential phosphorylation site [Soppet, 1992].
  • AKSP SEQ ID NO:2
  • the specific configuration of the NF-H molecule allows to construct a small number of phosphorylated and non-phosphorylated peptides which span the entire length of the relevant NF-H domain. These peptides can then be used for a systematic "epitope walk" along the molecule.
  • AKSPVKEEAKSPAEAKSP SEQ ID NO: 12
  • each of these peptides has a potential serine phosphorylation site in the middle, which is part of a KSP, and which is flanked by two additional KSP (SEQ ID NO:l) moieties.
  • each peptide can exist in eight different states of phosphorylation. Accordingly a total of 64 such peptides covers all the possible states of phosphorylation of the NF-H carboxy terminal domain, because motif C (SEQ ID NO:5) does not occur in tandem in the naturally occuring protein.
  • the peptide sequences selected are as follows (all peptides are biotinylated at the N-terminal):
  • Tau - an alternative protein candidate The Tau protein, which physiologically stabilizes microtubules in the neuronal axon, is an integral constituent of paired helical filaments (PHF), which form neurofibrillary tangles. Hyperphosphorylation of Tau has been considered the main cause of PHF assembly [Goedert, 1992] although alternatively, this protein could be involved in a secondary event in PHF formation.
  • PHF paired helical filaments
  • FIG. 5 presents the amino acid sequence of Tau.
  • Peptides of 6-30 amino acid residues containing at least one phosphorylated serine or threonine among the boxed serines and threonines can serve as peptides for implementing the present invention.
  • Binding peptides to a solid support Binding peptides to a solid support:
  • ELISA enzyme linked immunosorbent assay
  • the polystyrene can be treated to modify the electrostatic and hydrophobic binding forces of the plastic surface.
  • the antibody- containing solution is then added and after binding of the antibody to the protein, the non-specific antibodies are washed out.
  • an anti-antibody antibody labeled with an enzyme which catalyzes a color reaction is added and the complex and detection is effected by adding the chromogenic substrate to the enzyme.
  • the non-covalent binding forces between the short peptide and the plastic surface are usually too weak to prevent the short peptide from being washed out. If the peptide is negatively charged at working pH, this problem can be traversed by precoating the wells with poly-L-lysine which is positively charged, and as such binds the peptide to the plate with electrostatic bonds. It is also common practice to add the peptide-solution to the well, evaporate all the liquid and immobilize the peptide to the surface by fixation with Methanol.
  • plates made of plastic containing reactive groups that specifically bind amino- or carboxyl groups, enabling covalent binding of the peptides to the plate surface can be used.
  • these plates are useful only for peptides which contain only a single amino- or carboxyl group.
  • peptides rich in Lysine such as the peptides employed herein, can be used in conjunction with such plates, such use is not preferred since these peptides can bind to the plate by means of the internal Lysine residues, and not necessarily through the terminal group. This prevents the binding of the peptide in a specific configuration, causes a large fraction of the peptides to bind parallel to the surface and as a consequence renders these peptides inaccessible to the antibodies in the serum. All the above mentioned methods were attempted in context of the present invention yet produced unreliable results. The OD obtained from wells to which a peptide had been added, was not significantly higher than the OD values of control wells to which the peptide has not been added.
  • Bead-based detection of antibodies An alternative strategy was to use peptides conjugated to micron sized beads. In this assay the serum is added to the beads in a reaction tube. At the end of the incubation period, the beads are collected (spun down) and the supernatant removed. The beads are washed and detection is performed. This system worked far better than the plate-based methods used previously. However, this method is extremely cumbersome and time consuming, variations between identical samples are quite big, and reproducibility between experiments can be problematic.
  • Streptavidin-biotin based method The method that proved most reliable and successful is based on peptides biotinylated at the amino- terminal end and streptavidin coated multi-well plates. As further detailed hereinunder, this method gave very good signal-to-noise ratio, it proved to be very sensitive in a wide range of serum concentrations, and provided good reproducibility between samples, plates and repeated experiments.
  • Tris Buffered Saline (TBS): 50 mM Tris- HCl, pH 7.4 (Tris (Sigma, T-1378); HCl (Merck 1.00319.1000)) 200mM NaCl (Merck, 1.06404.1000).
  • TBST 0.05 % (w/v) Tween-20 (Sigma, P- 7949) in TBS.
  • Phosphate Buffered Saline PBS: 0.1 M phosphate buffer, pH 7.2 (Sodium phosphate, monobasic (Sigma. S-8182) and Sodium phosphate, dibasic (Sigma, S-7907)). 200 mM NaCl (Merck, 1.06404.1000).
  • PBST 0.05 % (w/v) Tween-20 (Sigma, P-7949) in PBS.
  • Dilution/ Blocking buffer 0.5 % Gelatin (Difco, 0143-17-9) in TBST.
  • Streptavidin stock solution 2 mg/ml Streptavidin (Sigma, S-4762) in purified water. Aliquoted and kept at -20°C, this solution is stable indefinitely.
  • Multiwell Plates Nunc Maxiso ⁇ (Cat. No. 442404) 96 well C-shaped microplates.
  • Secondary antibody Goat anti-human IgG Horse radish peroxidase conjugate (Jackson, 109-035-088).
  • Substrate solution 1 mg/ml OPD (Sigma, P-8412) + 0.005% H202 (Merck, 1.07210.0250) in 50mM Sodium Citrate buffer, pH 5 (Merck, 1.00 244.1000).
  • a Streptavidin stock solution (2 mg/ml Streptavidin prepared in purified water, aliquoted, and stored at -20 °C) was diluted 1 :400 in ddH 2 0 and a 100 ⁇ l aliquot was added to each well of a multiwell plate (96 well C-shaped microplates). The plates were incubated at 37 °C overnight until the liquid was entirely evaporated and stored until use at 4 °C in plastic bags containing a desiccating material. Such coated plates retained their activity for at least few days. The plates were washed 4 times by immersing in TBST. 200 ⁇ l of blocking solution were added to each of the wells. The plates were then incubated for 1 hour at room temperature. 100 ⁇ l of the biotinylated peptide (1 ⁇ g/ml in TBST) were added to each of the wells with the exception of the control wells. The plates were then incubated for 1 hour at room temperature.
  • AD is a highly heterogenic neurodegenerative disorder, and it is therefor unlikely that one single biomarker will be able to detect all cases. Furthermore, preliminary results suggest that some peptides detect antibodies in the blood of certain cases, while other peptides are more efficient for the detection of antibodies of other cases.
  • this apparent characteristic of a single peptide can be turned into an advantage. For example, if a certain serum sample analyzed with a given set of peptides produces a combined signal larger than an empirically set cut-off value, this serum sample is considered positive.
  • the relative proportions attributed to individual peptides, when used in combination do not have to be proportional to the signal, as such, freedom in the optimization of the inclusion criteria can be obtained.
  • the peptide used for optimization was peptide 3M (SEQ ID NO:31). Serum was pooled from 10 AD patients and pretreated with chloroform.
  • Blocking The plates were either blocked with 0.5 % Gelatin, 1 % Caseinate or not blocked, before the addition of peptide.
  • the serum was diluted in PBST and the secondary antibody in PBST containing either 0.5 % Gelatin or 1 % Caseinate.
  • a saturating concentration of the peptide was reached at 0.4-0.8 ⁇ g/ml. Accordingly, 1 ⁇ g/ml was chosen as a standard working concentration.
  • the different blocking agents used did not substantially effect the signal, but improved reproducibility (see Table 1 hereinbelow). As such, 0.5 % Gelatin was chosen as the blocking agent for subsequent experiments.
  • Buffers The original protocol was based on phosphate buffered solutions (PBS). Since the specificity of IgG binding to the peptide is dependent on the phosphorylation state of the epitopes, presence of phosphate in the solution may, or may not, interfere with the assay. As such, Tris buffered solutions (TBS) were preferably employed. Figure 2, depicts the response to increasing concentrations of the peptide, employed in either PBS or TBS. It is evident from this Figure that TBS functions at least as well if not better than PBS. Therefore, TBS was used as the preferred buffer for all subsequent experiments.
  • PBS phosphate buffered solutions
  • Serum concentration range As seen in Figure 3, detection is most sensitive to changes in serum concentration between 1 :80 and 1 :320, although detection can also be seen at concentrations between 1 :20 and 1 :40. Signal to noise ratios were maximal ( ⁇ 3) at 1 :20 and decreased below 2 at 1 :160 ( Figure 2).
  • ⁇ 3 Signal to noise ratio
  • Figure 2 When eight AD and eight normal control (NC) samples were analyzed at different dilutions (1 :10 - 1 : 640), it was clear that concentrations below 1 :40 gave poor separation between AD and NC, while using concentration higher than 1 :40 did significantly improve the detection (not shown). Based on these results, a working dilution of 1 :40 was used for all subsequent experiments.
  • Serum incubation time and temperature Three different conditions for serum incubation were tested on eight AD and eight NC samples. In addition to the original protocol's one hour at room temperature, over-night incubations at room temperature or 4°C were tested. The longer incubation times did not significantly improve signals from weakly positive samples, the same samples were generally identified as positive in all three conditions employed (not shown). Based on these results, the serum was incubated for one hour at room temperature in all subsequent experiments. Concentration of the secondary antibody: Five different serum dilutions were probed with six different secondary antibody-enzyme conjugate concentrations. As seen in Figure 4, the highest sensitivity was reached when dilutions 1 :1250 and 1 :2500 were employed.
  • Preliminary experiments included a lipid removal step. This step employed the addition of chloroform (5 % chloroform, v/v) to the sera followed by gentle agitation for 30 minutes at room temperature, and 30 minutes of centrifugation at 20,000 x g at 4°C. The sera (supernatant) was then separated from the chloroform-lipid pellet. However, in subsequent experiments it was shown that no significant difference were observed between signals obtained from the "treated” or "untreated” sera (not shown). As a result all subsequent experimentations were preferably conducted without employing this lipid removal step. Preliminary comparison between AD and NC sera:
  • AD and eight NC sera were analyzed using peptide 3M (SEQ ID NO:31). The experiment was repeated over a period of four days, employing four different plates each day. Two plates were preblocked with Gelatin, while two were not. In one experiment chloroform treated serum was analyzed on two additional plates. Three AD sera consistently gave rise to high signals in all experiments. One AD serum caused high signals in 10 out of the 13 plates and four AD sera were consistently negative. One NC sera caused high signals in some of the plates in the experiments conducted on all four days. Two additional NC sera gave rise to high signals in some of the plates in the experiments conducted on three of the four days.
  • Sample numbers 862, 871, 861, and 874 acquired from AD patients, are separated by more than two standard deviation points from sample number 465.
  • Sample number 485 is separated by one standard deviation point from sample number 465.
  • peptide 3M detected four out of eight AD cases while generating one false positive signal (sample No. 485). It will be appreciated that these results are based on the employment of a single peptide. However, by combining several peptides both the specificity and sensitivity can be improved.
  • Each peptide within a group is compared to a theoretical "ideal" peptide profile which was given the optimal value of 1. The closer the value calculated for each peptide is to 1, the more similar is the peptide' s profile of serum sample signals to the "ideal" peptide profile, and therefor can be regarded as a representative for it's group. According to table 4, therefore, four peptides were identified as representing all 17 peptides: 8LMR; 3LM; 4LM and 8LMR-V. Using these peptides in an algorithm similar to the one described above (which uses 9 peptides), a scheme separating between AD and NC samples is obtained (See Figure 7).
  • Tables 5a-b and 6a-b Summarizing the number of AD and NC samples found in every terminal node results in Tables 5a-b and 6a-b (see below).
  • the terminal nodes are first designated as “AD-nodes” or “NC-nodes” according to what kind of sera is in majority in that node. The serum sample found in minority in such a node is therefore miss- diagnosed.
  • terminal node No. 4 is designated an "AD-node” since there are 10 AD sera and only one NC serum sample in this node. That single NC serum is a false positive sample.
  • Table 5b shows that the 9-peptide algorithm results in a sensitivity of 94 % and a specificity of 98 %. Similarly, the 4-peptide algorithm results in a sensitivity of 85 % and specificity of 92 % (Table
  • the path to the terminal nodes of the four- peptide algorithm could be summarized as limited ranges of antibody levels against each of the peptides ( Figure 8).
  • the level of antibodies in the sera against peptide 4LM should be above a certain cut-off value, but below another one, and above a cut-off value for peptide 3LM ( Figure 7). Since there are six different characteristic groups defining AD samples, this may be a novel way to define new sub-types of AD. Similarly, the 5 different groups defining NC samples may represent a normal or a pre-pathological variation in the normal population.
  • AD results from a complex syndrome rather than a singular cellular event
  • the fact that the peptides employed can measure a variety of antibodies, and not a single biochemical marker, will broaden the range of subtypes of AD detectable, and as a result, increase the general sensitivity of the present invention.
  • Prior art methods and kits measure single biochemical markers, and as such cannot detect all the biochemical markers associated with the various AD subtypes.
  • the present invention enables to investigate the role of the
  • AD specific antibodies in the etiology of AD. It is likely that the antibodies are not only markers for the presence of the disorder, but rather participate in an autoimmune reaction in the central nervous system. In such a case, the peptides found to have the highest affinity for the AD specific antibodies could be used to bind and remove the antibodies from the blood stream of AD patients. This could potentially stop the progression of the disorder. Thus, the present invention can ultimately lead to a strategy for treating the disorder.
  • EXAMPLE 4 Use of specific peptide combinations for identifying specific disorders A large set of experiments was performed in efforts to both reduce the number of potentially beneficial peptides and to examine the possibility of using specific peptide subsets for diagnosing various neurodegenerative diseases.
  • the above 15 peptides were used to screen 96 Alzheimer's disease (AD) and Normal Control (NC) samples.
  • Classification and Regression Tree (CART) statistics were employed on the results obtained from all possible four peptide combinations of the fifteen peptides utilized.
  • Approximately 1800 algorithms were obtained, of which twelve algorithms (not shown) gave results which were comparable in sensitivity and specificity to that obtained in Example 3 ( Figure 7, Tables 6a and 6b).
  • Each algorithm was assigned a rank order number which was the sum of the cross-validation specificity rank order and the cross-validation sensitivity rank order of the algorithm. For example, if an algorithm received 2 for specificity and 11 for sensitivity, its overall rank-order would be 13. The lower the rank order number, the more suited the algorithm was for screening.
  • Cross-validation was performed by constructing the algorithm from 90% of the samples and verifying it against the remaining 10 % of the samples.
  • the assay of the present invention can be utilized to identify AD (identifying 78 % of the clinically diagnosed AD individuals) and MID (95%).
  • the assay of the present invention provided accurate, identifying 86% of the clinically diagnosed NC individuals and 90% of the clinically diagnosed MID individuals.
  • use of different combinations of these seven peptides can serve as a tool for specifically distinguishing between NC, AD, MID, and PwD. Stroke:
  • TIA transient ischemic attack
  • TIA symptoms also referred to as a 'mini- stroke'
  • TIA symptoms are similar to those manifested during a major stroke although TIA symptoms are weaker and of shorter duration.
  • Individuals suffering from TIA are more likely to experience a major stroke [Adams, RD. 1993]; in addition, multiple TIAs can lead to MID.
  • various peptide combinations of the present invention enable distinction between NC and MID.
  • TIA is an underlying cause for MID and a major risk factor for stroke
  • teachings of the present invention can be used to find a combination of peptides suitable for the detection of TIA.
  • Recent reports suggest that the level of antibodies against NF rise with time in most stroke victims.
  • the screening methodology and peptide combinations of the present invention can be utilized to detect and categorize subgroups of stroke-specific autoantibodies thereby enabling the detection of TIA.

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Abstract

Cette invention se rapporte à un procédé servant à identifier l'existence, la non-existence, le type ou l'état d'un trouble neurodégénératif. Ce procédé consiste (a) à réaliser l'immunoréaction d'un échantillon de sérum dérivé d'un individu avec au moins un peptide représentant au moins un épitope dérivé d'une protéine endogène contre laquelle au moins un anticorps est produit in vivo lors de l'apparition ou pendant la progression du trouble neurodégénératif, le ou les peptides en question étant choisis pour que le ou les anticorps en question soient capables d'immunofixation avec le ou les peptides en question; et (b) à détecter la présence, l'absence ou le degré d'immunofixation, afin d'identifier l'existence, la non-existence, le type ou l'état du trouble neurodégénératif en question.
PCT/IL2000/000509 1999-08-31 2000-08-27 Peptides et substances, procedes et dispositif les utilisant pour diagnostiquer et traiter les troubles neurodegeneratifs WO2001015655A2 (fr)

Priority Applications (3)

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AU67223/00A AU6722300A (en) 1999-08-31 2000-08-27 Peptides and substances, methods and devices using same for diagnosing and treating neurodegenerative disorders
EP00954874A EP1209969A2 (fr) 1999-08-31 2000-08-27 Peptides et substances, procedes et dispositif les utilisant pour diagnostiquer et traiter les troubles neurodegeneratifs
US09/847,586 US20030148404A1 (en) 2000-07-27 2001-05-03 Peptides and substances, methods and devices using same for diagnosing and treating neurodegenerative disorders

Applications Claiming Priority (4)

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US38634799A 1999-08-31 1999-08-31
US09/386,347 1999-08-31
US22115000P 2000-07-27 2000-07-27
US60/221,150 2000-07-27

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1180938A2 (fr) * 1999-09-03 2002-02-27 RAMOT UNIVERSITY AUTHORITY FOR APPLIED RESEARCH & INDUSTRIAL DEVELOPMENT LTD. Agents et compositions et procedes utilisant lesdits agents et lesdites compositions pour le diagnostic et/ou le traitement ou la prevention des maladies formant des plaques.
WO2003070760A2 (fr) * 2002-02-20 2003-08-28 F. Hoffmann-La Roche Ag Anticorps anti-a$g(b) et leur utilisation
EP1393073A2 (fr) * 2001-05-03 2004-03-03 Sigma-Genosys, Ltd. Procede d'assemblage de microreseaux de proteines
US8906370B2 (en) 2005-12-12 2014-12-09 Hoffmann-La Roche Inc. Antibodies against amyloid beta 4 with glycosylation in the variable region
US20150051097A1 (en) * 2003-06-25 2015-02-19 Proteome Sciences Plc Methods for Screening Inhibitors of Tau Phosphorylation By Casein Kinase I

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5541294A (en) * 1992-05-28 1996-07-30 New York Blood Center, Inc. Removal of antibodies from blood-derived compositions while retaining coagulation factors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5541294A (en) * 1992-05-28 1996-07-30 New York Blood Center, Inc. Removal of antibodies from blood-derived compositions while retaining coagulation factors

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
ELIZAN T.S. ET AL.: 'Antineurofilament antibodies in postencephalitic and idiopathic Parkinson's disease' J. OF NEUROL. SCI. vol. 59, 1983, pages 341 - 347, XP002943983 *
GRUNDKE-IQBAL I. ET AL.: 'Microtubule-associated polypeptides tau are altered in Alzheimer paired helical filaments' MOLECULAR BR. RES. vol. 4, 1988, pages 43 - 52, XP002943977 *
HASSIN-BAER S. ET AL.: 'Antibodies from Down's syndrome patients bind to the same cholinergic neurofilament protein recognized by Alzheimer's disease antibodies' NEUROLOGY vol. 42, 1992, pages 551 - 555, XP002943980 *
KUMAR M. ET AL.: 'Serum IgG brain reactive antibodies in Alzheimer disease and down syndrome' ALZ. DIS. & ASSOC. DISORD. vol. 2, no. 1, 1988, pages 50 - 55, XP002943979 *
LOPEZ O.L. ET AL.: 'Serum auto-antibodies in Alzheimer's disease' ACTA NEUROL. SCAND. vol. 84, 1991, pages 441 - 444, XP002943984 *
MICHAELSON D.M. ET AL.: 'Serum antibodies to cholinergic neurons in Alzheimer's disease' PROG. IN CLIN. & BIOL. RES. vol. 317, 1989, pages 689 - 694, XP002943982 *
MICHAELSON D.M.: 'Decreased density of forebrain cholinergic neurons and disintegration of the spatial organization of behavior in experimental autoimmune demientia (EAD)' ANN. NY ACAD. SCI. vol. 695, 1993, pages 244 - 248, XP002943985 *
ORON L. ET AL.: 'Animal model and in vitro studies of anti-neurofilament antibodies mediated neurodegeneration in Alzheimer's disease' J. NEURAL TRANSM. vol. 49, 1997, pages 77 - 84, XP002943978 *
SALIH A.M. ET AL.: 'Prevalence of antibodies to neurofilament polypeptides in patients with rheumatoid arthritis complicated by peripheral neuropathy' CLIN. & EXP. RHEUMAT. vol. 16, 1998, pages 689 - 694, XP002943976 *
SOUSSAN L. ET AL.: 'Antibodies to different isoforms of the heavy neurofilament protein (NF-H) in normal aging and Alzheimer's disease' MOL. NEUROBIOL. vol. 9, no. 1-3, 1994, pages 83 - 91, XP002943981 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1180938A4 (fr) * 1999-09-03 2005-03-16 Univ Ramot Agents et compositions et procedes utilisant lesdits agents et lesdites compositions pour le diagnostic et/ou le traitement ou la prevention des maladies formant des plaques.
EP1180938A2 (fr) * 1999-09-03 2002-02-27 RAMOT UNIVERSITY AUTHORITY FOR APPLIED RESEARCH & INDUSTRIAL DEVELOPMENT LTD. Agents et compositions et procedes utilisant lesdits agents et lesdites compositions pour le diagnostic et/ou le traitement ou la prevention des maladies formant des plaques.
EP1393073A2 (fr) * 2001-05-03 2004-03-03 Sigma-Genosys, Ltd. Procede d'assemblage de microreseaux de proteines
EP1393073A4 (fr) * 2001-05-03 2007-03-28 Sigma Genosys Ltd Procede d'assemblage de microreseaux de proteines
US7794719B2 (en) 2002-02-20 2010-09-14 F. Hoffmann-La Roche Ag Anti-amyloid β antibodies
WO2003070760A3 (fr) * 2002-02-20 2004-03-04 Hoffmann La Roche Anticorps anti-a$g(b) et leur utilisation
WO2003070760A2 (fr) * 2002-02-20 2003-08-28 F. Hoffmann-La Roche Ag Anticorps anti-a$g(b) et leur utilisation
KR101038828B1 (ko) * 2002-02-20 2011-06-03 모르포시스 아게 항-Αβ 항체 및 이의 용도
EP2368907A3 (fr) * 2002-02-20 2012-03-14 F. Hoffmann-La Roche AG Anticorps anti-Abeta et leur utilisation
US8216577B2 (en) 2002-02-20 2012-07-10 F. Hoffmann-La Roche Ag Anti-Aβ antibodies and their use
US8329886B2 (en) 2002-02-20 2012-12-11 Hoffman-La Roche Inc. Nucleic acid molecules encoding anti-amyloid beta antibodies
HRP20040712B1 (hr) * 2002-02-20 2013-03-31 F. Hoffmann - La Roche Ag Anti-amiloidna beta antitijela i njihova uporaba
US20150051097A1 (en) * 2003-06-25 2015-02-19 Proteome Sciences Plc Methods for Screening Inhibitors of Tau Phosphorylation By Casein Kinase I
US8906370B2 (en) 2005-12-12 2014-12-09 Hoffmann-La Roche Inc. Antibodies against amyloid beta 4 with glycosylation in the variable region

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WO2001015655A3 (fr) 2001-11-22
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