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WO2005116659A2 - Compositions et methodes associees a la maladie d'alzheimer - Google Patents

Compositions et methodes associees a la maladie d'alzheimer Download PDF

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Publication number
WO2005116659A2
WO2005116659A2 PCT/US2005/017263 US2005017263W WO2005116659A2 WO 2005116659 A2 WO2005116659 A2 WO 2005116659A2 US 2005017263 W US2005017263 W US 2005017263W WO 2005116659 A2 WO2005116659 A2 WO 2005116659A2
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Prior art keywords
marker
subject
sample
level
markers
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PCT/US2005/017263
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English (en)
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WO2005116659A3 (fr
Inventor
David Lowe
Howard Schulman
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Neurodx, Llc
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Publication of WO2005116659A2 publication Critical patent/WO2005116659A2/fr
Publication of WO2005116659A3 publication Critical patent/WO2005116659A3/fr

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    • 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
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention provides compositions, methods and kits useful for the diagnosis and treatment of Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • the invention provides polypeptides and metabolites that are markers of AD, polynucleotides that encode the polypeptides and antibodies that specifically bind to the polypeptides.
  • the invention also provides fragments, precursors, successors and modified versions of the foregoing polypeptides, metabolites, polynucleotides and antibodies.
  • the invention also provides compositions comprising the foregoing polypeptides, metabolites, polynucleotides and antibodies.
  • the invention also provides methods for using the polypeptides, metabolites, polynucleotides and antibodies in the diagnosis and treatment of AD, monitoring progression of the disease and screening of candidate therapeutic compounds.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • AD is the fourth most common cause of death in the United States, after heart disease, cancer and stroke. It currently affects more than four million people in the United States, and this number is expected to double during the next forty years as the population ages.
  • AD is also the most common cause of chronic dementia, and it is estimated that ten percent of the population older than 65 years of age has mild to severe dementia. This high prevalence, combined with the rate of growth of the over 65 demographic, make dementia and particularly AD, important public health problems.
  • AD is the third most expensive disease in the United States, costing approximately $100 billion each year. This figure includes both direct costs, such as nursing home care and in-home day care, as well as indirect costs, such as lost patient and caregiver productivity.
  • AD Alzheimer's disease
  • Hallmark pathologies of Alzheimer's disease include granulovascular neuronal degeneration, extracellular neuritic plaques with ⁇ -amyloid deposits, intracellular accumulation of Tau protein as neurofibrillary tangles, neurofibrillary degeneration, synaptic loss, and extensive neuronal cell death. It is now known that these histopathologic lesions of AD correlate with the dementia observed in many elderly people.
  • AD amyloid precursor protein
  • AD Alzheimer's disease
  • a single model of AD satisfactorily accounts for all neuropathologic findings or the requirement of aging for disease onset. The mechanisms of disease progression are equally unclear. Early detection and identification of Alzheimer's disease would allow prompt, appropriate treatment and care to be provided.
  • One aspect of the invention provides polypeptides that have been identified as differentially expressed in samples obtained from AD subjects as compared to samples obtained from non-AD subjects ("polypeptide markers").
  • polypeptide markers polypeptide markers
  • the invention also provides polypeptides that have substantial homology to polypeptide markers, modified polypeptide markers as well as fragments of the polypeptide markers.
  • the invention also includes precursors and successors of the polypeptide markers in biological pathways.
  • the invention also provides molecules that comprise a polypeptide marker, homologous polypeptide, a modified polypeptide marker or a fragment, precursor or successor of a polypeptide marker (e.g., a fusion protein).
  • a polypeptide marker e.g., a fusion protein
  • the term "polypeptides of the invention” shall be understood to include all of the foregoing.
  • Another aspect of the invention provides non-peptide small molecules that have been identified as differentially expressed in samples obtained from AD subjects as compared to samples obtained from non-AD subjects ("metabolite markers").
  • the invention also provides modified metabolite markers as well as fragments of metabolite markers.
  • the invention also includes precursors and successors of the metabolite markers in biological pathways.
  • the invention also provides molecules that comprise a metabolite marker, a modified metabolite marker or a fragment, precursor or successor of a metabolite marker.
  • metabolites of the invention shall be understood to include all of the foregoing.
  • Another aspect of the invention provides polynucleotides encoding polypeptides of the invention ("polynucleotide markers").
  • polynucleotide markers polynucleotide markers
  • the invention also provides polynucleotides that have substantial homology to polynucleotide markers, modified polynucleotide markers, and fragments of polynucleotide markers.
  • the invention also provides molecules that comprise a polynucleotide marker, homologous polynucleotide, a modified polynucleotide marker or a fragment of a polynucleotide marker (e.g., a vector).
  • the "markers" of the present invention are intended to include analogs, compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not alter the differential expression of the marker.
  • the term "polynucleotides of the invention” shall be understood to include all of the foregoing.
  • Another aspect of the invention provides molecules that specifically bind to a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention.
  • the binding molecule may be an antibody, antibody fragment, or other molecule.
  • the invention also provides methods for producing a binding molecule that specifically recognizes a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention.
  • compositions comprising a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention, a binding molecule (e.g., an antibody) that is specific for a polypeptide of the invention, metabolite of the invention or polypeptide of the invention, an inhibitor of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention, or another molecule that can increase or decrease the level or activity of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention.
  • Such compositions may be pharmaceutical compositions formulated for use as therapeutics.
  • Another aspect of the invention provides a method for detecting a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention, h one embodiment, the method comprises contacting a biological sample obtained from a subject with a binding molecule (e.g., an antibody) under conditions that permit the formation of a stable complex, and detecting any stable complexes formed.
  • the method comprises determining the activity of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention.
  • the method comprises dete ⁇ nining the level of a polypeptide of the invention in a cell obtained from the subject by detecting the presence of a polynucleotide that encodes the polypeptide.
  • Another aspect of the invention provides a method for diagnosing AD in a subject by detecting a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention in a biological sample.
  • the method comprises obtaining a sample from a subject suspected of having AD or at risk for AD and comparing the level or activity of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention in the sample with the level of activity in a sample obtained from a non-AD subject or with a reference range or value.
  • the method is used for staging or stratifying subjects with AD, monitoring the progression of the disease or response to therapy.
  • a plurality of polypeptides of the invention, metabolites of the invention, or polynucleotides of the invention are detected.
  • the method comprises detecting known biomarkers or considering other clinical indicia in addition to detecting one or more polypeptides of the invention, metabolites of the invention or polynucleotides of the invention in a biological sample.
  • Another aspect of the invention provides methods for treating AD by administering a therapeutic agent to a subject that increases or decreases the level or activity of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention.
  • the method comprises administering a therapeutic agent that decreases (i.e., bring toward the normal range) the level or activity of the polypeptide, metabolite or polynucleotide.
  • the method comprises administering a therapeutic agent that increases the level or activity of the polypeptide, metabolite or polynucleotide.
  • Another aspect of the present invention provides a method for screening a candidate compound for use as a therapeutic agent for treating AD.
  • the method comprises administering the candidate compound to an AD subject and screening for the ability to modulate the level or activity of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention.
  • the method comprises providing the candidate compound to a cell from an AD subject and screening for the ability to modulate the intracellular level of a polypeptide of the invention, a metabolite of the invention or polynucleotide of the invention.
  • Another aspect of the invention provides a kit for performing the methods described above.
  • the kit is for the diagnosis of AD by detection of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention in a biological sample from a subject.
  • a kit for detecting a polypeptide of the invention, metabolite of the invention or polynucleotide of the present invention may include an antibody capable of binding to the polypeptide, metabolite or polynucleotide.
  • AD subject and "a subject who has AD” are intended to refer to subjects who have been diagnosed with AD or probable AD.
  • non- AD subject and “a subject who does not have AD” are intended to refer to a subject who has not been diagnosed with AD or probable AD.
  • a non-AD subject may be healthy and have no other disease, or they may have a disease other than AD.
  • subject refers to any living organism capable of eliciting an immune response.
  • the term subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the term "antibody” refers to an immunoglobulin molecule capable of binding an epitope present on an antigen.
  • immunoglobulin molecules such as monoclonal and polyclonal antibodies, but also bi-specific antibodies, humanized antibodies, chimeric antibodies, anti-idiopathic (anti-ID) antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fusion proteins and any modifications of the foregoing that comprise an antigen recognition site of the required specificity.
  • biological sample includes a sample from any body fluid or tissue (e.g., serum, plasma, blood, cerebrospinal fluid, urine, sputum, thin cortical slice, brain tissue homogenate).
  • a component e.g., a marker
  • a component is referred to as “differentially expressed” in one sample as compared to another sample when the method used for detecting the component provides a different level or activity when applied to the two samples.
  • a component is referred to as "increased" in the first sample if the method for detecting the component indicates that the level or activity of the component is higher in the first sample than in the second sample (or if the component is detectable in the first sample but not in the second sample).
  • a component is referred to as "decreased" in the first sample if the method for detecting the component indicates that the level or activity of the component is lower in the first sample than in the second sample (or if the component is detectable in the second sample but not in the first sample).
  • marker is referred to as "increased” or “decreased” in a sample (or set of samples) obtained from an AD subject (or a subject who is suspected of having AD, or is at risk of developing AD) if the level or activity of the marker is higher or lower, respectively, compared to the level of the marker in a sample (or set of samples) obtained from a non-AD subject, or a reference value or range.
  • fold increase and “fold decrease” refer to the relative increase or decrease of in the level of a marker in one sample (or set of samples) compared to another sample (or set of samples).
  • a positive fold change indicates an increase in the level of a marker while a negative fold change indicates a decrease in the level of a marker.
  • the increase or decrease may be measured by any method or technique known to those of skill in the art. As will be appreciated by one of skill in the art, the observed increase or decrease may vary depending on the method or technique that is used.
  • the term “marker” includes polypeptide markers, metabolite markers and polynucleotide markers that are differentially expressed in one sample compared to another sample.
  • polypeptide markers For clarity of disclosure, aspects of the invention will be described with respect to “polypeptide markers,” “metabolite markers” and “polynucleotide markers.” However, statements made herein with respect to “polypeptide markers” are intended to apply to other polypeptides of the invention.
  • a polynucleotide described as encoding a "polypeptide marker” is intended to include a polynucleotide that encodes a polypeptide marker, a polypeptide that has substantial homology to a polypeptide marker, modified polypeptide markers, fragments, precursors and successors of a polypeptide marker, and molecules that comprise a polypeptide marker, homologous polypeptide, a modified polypeptide marker or a fragment, precursor or successor of a polypeptide marker (e.g., a fusion protein).
  • polypeptide refers to a single amino acid or a polymer of amino acid residues.
  • a polypeptide may be composed of two or more polypeptide chains.
  • a polypeptide includes a protein, a peptide, an oligopeptide, and an amino acid.
  • a polypeptide can be linear or branched.
  • a polypeptide can comprise modified amino acid residues, amino acid analogs or non-naturally occurring amino acid residues and can be interrupted by non-amino acid residues.
  • amino acid polymers that have been modified, whether naturally or by intervention, e.g., formation of a disulfide bond, glycosylation, lipidation, methylation, acetylation, phosphorylation, or by manipulation, such as conjugation with a labeling component.
  • a "fragment" of a polypeptide refers to a plurality of amino acid residues comprising an amino acid sequence that has at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 20 contiguous amino acid residues or at least 30 contiguous amino acid residues of a sequence of the polypeptide.
  • a "fragment" of polynucleotide refers to a single nucleic acid or to a polymer of nucleic acid residues comprising a nucleic acid sequence that has at least 15 contiguous nucleic acid residues, at least 30 contiguous nucleic acid residues, at least 60 contiguous nucleic acid residues, or at least 90% of a sequence of the polynucleotide.
  • the terms "fragments,” “analogs,” or “derivatives” are used interchangeably to mean a chemical substance that is related to another substance (i.e., marker).
  • the fragment can be, for example, differentially expressed in one sample compared to another sample.
  • the fragment is differentially expressed in samples from AD subjects when compared to non-AD subjects.
  • a compound is referred to as "isolated" when it has been separated from at least one component with which it is naturally associated.
  • a polypeptide can be considered isolated if it is separated from contaminants including metabolites, polynucleotides and other polypeptides.
  • Isolated molecules can be either prepared synthetically or purified from their natural environment. Standard quantification methodologies known in the art can be employed to obtain and isolate the molecules of the invention.
  • polynucleotide refers to a single nucleotide or a polymer of nucleic acid residues of any length.
  • the polynucleotide may contain deoxyribonucleotides, ribonucleotides, and/or their analogs and may be double-stranded or single stranded.
  • a polynucleotide can comprise modified nucleic acids (e.g., methylated), nucleic acid analogs or non-naturally occurring nucleic acids and can be interrupted by non-nucleic acid residues.
  • a polynucleotide includes a gene, a gene fragment, cDNA, isolated DNA, mRNA, tRNA, rRNA, isolated RNA of any sequence, recombinant polynucleotides, primers, probes, plasmids, and vectors.
  • a polypeptide or metabolite marker is a member of a biological pathway.
  • the term "precursor” or “successor” refers to molecules that precede or follow the polypeptide marker, metabolite marker or polynucleotide marker.
  • the present invention can include additional members of the biological pathway that come before or follow the polypeptide marker, metabolite marker or polynucleotide marker. Such identification of biological pathways and their members is within the skill of one in the art.
  • binding pairs e.g., an antibody and an antigen
  • affinity constant of at most 10 "6 moles/liter, at most 10 "7 moles/liter, or at most 10 "8 moles/liter.
  • two polypeptides are "substantially homologous" when there is at least 70% homology, at least 80% homology, at least 90% homology, at least 95% homology or at least 99% homology between their amino acid sequences, or when polynucleotides encoding the polypeptides are capable of forming a stable duplex with each other.
  • two polynucleotides are "substantially homologous" when there is at least 70% homology, at least 80% homology, at least 90% homology, at least 95% homology or at least 99% homology between their amino acid sequences or when the polynucleotides are capable of forming a stable duplex with each other.
  • homology refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100.
  • Readily available computer programs can be used to aid in the analysis of similarity and identity, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, D.C., which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 for peptide analysis.
  • nucleotide sequence similarity and identity Programs for determining nucleotide sequence similarity and identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent similarity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded- specific nuclease(s), and size determination of the digested fragments.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.
  • the invention is based in part on the discovery that certain polypeptides and metabolites are differentially expressed in serum samples obtained from AD subjects compared to serum samples obtained from non-AD subjects.
  • the markers of the present invention may have diagnostic and/or therapeutic use in other neurological disorders in addition to AD.
  • the terms "neurological disorder” or "CNS disorder,” refer to an impairment or absence of a normal neurological function or presence of an abnormal neurological function in a subject.
  • neurological disorders can be the result of disease, injury, and/or aging.
  • neurological disorder also includes neurodegeneration, which causes morphological and/or functional abnormality of a neural cell or a population of neural cells.
  • Non-limiting examples of morphological and functional abnormalities include physical deterioration and/or death of neural cells, abnormal growth patterns of neural cells, abnormalities in the physical connection between neural cells, under- or over production of a substance or substances, e.g., a neurotransmitter, by neural cells, failure of neural cells to produce a substance or substances which it normally produces, production of substances, e.g., neurotransmitters, and/or transmission of electrical impulses in abnormal patterns or at abnormal times.
  • Neurological disorders include, but are not limited to, memory disorders, dementia, memory loss, epilepsy, and ischemia. Neurological disorders also include neurodegenerative diseases.
  • Neurodegeneration can occur in any area of the brain of a subject and is seen with many disorders including, but not limited to, Amyotrophic Lateral Sclerosis (ALS), multiple sclerosis, Huntington's disease, and Parkinson's disease.
  • Serum samples obtained from AD subjects and non-AD subjects were separated into a high molecular weight fraction, containing proteins with molecular weights greater than about 5-kDa, and a low molecular weight fraction containing free floating peptides and small molecules having a molecular weight of less than about 5-kDa. After removal of high abundance proteins, the high molecular weight fraction was digested with trypsin. Each fraction was separated by chromatographic means and analyzed by mass spectrometry. The high molecular weight fraction was submitted to proteolysis before analysis by mass spectrometry as discussed in the Example. The resulting spectra were compared to identify individual markers that showed significant association with ALS.
  • ALS Amyotrophic Lateral Sclerosis
  • Huntington's disease Huntington's disease
  • Tables 1 and 2 provide polypeptide markers that were identified as significantly (p ⁇ 0.005) increased in serum samples obtained from AD subjects compared with serum samples obtained from non-AD subjects.
  • Tables 3 and 4 provide polypeptide and metabolite markers that were identified as significantly (p ⁇ 0.005) increased in serum samples obtained from AD subjects compared to serum samples obtained from non-AD subjects.
  • Tables 5 and 6 provide polypeptide markers that were identified as significantly (p ⁇ 0.005) decreased in serum samples obtained from AD subjects compared with serum samples obtained from non-AD subjects.
  • Tables 7 and 8 provide polypeptide and metabolite markers that were identified as significantly (p ⁇ 0.005) decreased in serum samples obtained from AD subjects compared to serum samples obtained from non-AD subjects.
  • the markers set forth in the Tables are each identified by a plurality of the following: the mass to charge ratio (m/z), chromatographic retention time (RT), the charge state of a molecular ion (z), protonated parent mass (M+H), expression ratio (exp.
  • ratio which is a ratio of mean group intensities indicating the relative normalized signal for disease group compared to control
  • retention index (RJ) which is a linear measure of a component's elution time in comparison to a straight-chain alkane series injected independent of the sample
  • fold change an expression change factor where positive indicates an intensity increase and negative indicates a decrease versus the control
  • identification number from NCBI's reference sequence database (Accession # and gi #) and additional information (e.g., the name or sequence of the peptide marker as contained in the NCBI queried database and database searching using the TurboQUEST program).
  • the physical and chemical properties presented in the Tables is sufficient to distinguish the component from other materials; e.g., the components are uniquely identified by the mass to charge ratio (m/z) and the retention time (RT).
  • m/z mass to charge ratio
  • RT retention time
  • the magnitude of the variation depends to some extent on the reproductively of the separation means and the specificity and sensitivity of the detection means used to make the measurement.
  • the method and technique used to measure the markers is sensitive and reproducible.
  • the retention time and mass to charge ratio may vary to some extent depending on a number of factors relating to the protocol used for the chromatography and the mass spectrometry parameters (e.g., solvent composition, flow rate).
  • sample preparation and analysis conditions are carefully controlled.
  • the data set forth in the Tables reflects the method that was used to detect the markers.
  • the retention time of the marker is about the value stated for the marker (within about 10% of the value stated, within about 5% of the value stated, within about 1% of the value stated) and has a mass to charge ratio of about the value stated for the marker (within about 10%) of the value stated, within about 5% of the value stated, within about 1% of the value stated).
  • the polypeptide, metabolite and polynucleotide markers of the invention are useful in methods for diagnosing AD, determining the extent and/or severity of the disease, monitoring progression of the disease and/or response to therapy.
  • the markers are also useful in methods for treating AD and for evaluating the efficacy of treatment for the disease.
  • the markers may also be used as pharmaceutical compositions or in kits.
  • the markers may also be used to screen candidate compounds that modulate their expression.
  • the markers may also be used to screen candidate drugs for treatment of
  • the invention provides polypeptides and metabolites.
  • the invention provides an isolated component described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8.
  • the invention provides an isolated molecule that comprises a foregoing polypeptide or metabolite.
  • the invention provides a polypeptide or metabolite having substantial homology with a component set forth in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8.
  • the invention provides a molecule that comprises a foregoing polypeptide.
  • the invention provides a polypeptide or metabolite having (i) a mass-to-charge value and (ii) an RT value of about the values stated, respectively, for a component described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8.
  • the invention provides a molecule that comprises a foregoing polypeptide or metabolite.
  • the invention provides a polypeptide or metabolite having (i) a mass-to-charge value within 10% (more particularly within 5%, more particularly within 1%) and (ii) an RT value within 10% (more particularly within 5%, more particularly within 1%) of the m/z and RT values stated, respectively, for a component described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or
  • the invention provides a molecule that comprises a foregoing polypeptide or metabolite.
  • the invention provides a polypeptide that is a fragment, precursor, successor or modified version of a marker described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8.
  • the invention provides a molecule that comprises the foregoing polypeptide.
  • the invention provides a polypeptide or metabolite that is structurally different from the components specifically identified in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8 but has the same (or nearly the same) function or properties.
  • such a polypeptide may have amino acid sequence that is changed only in nonessential amino acid residues.
  • the invention provides a molecule that comprises a foregoing polypeptide or metabolite.
  • Polypeptide and metabolite markers may be isolated by any suitable method known in the art. Native polypeptide and metabolite markers can be purified from natural sources by standard methods known in the art (e.g., chromatography, centrifugation, differential solubility, immunoassay). In one embodiment, polypeptide and metabolite markers may be isolated from a serum sample using the chromatographic methods disclosed herein. In another embodiment, polypeptide and metabolite markers may be isolated from a sample by contacting the sample with substrate-bound antibodies that specifically bind to the marker. Metabolite makers may be synthesized using the techniques of organic and inorganic chemistry.
  • polypeptides markers may be synthesized using recombinant or chemical methods.
  • polypeptide markers can be produced by transforming a host cell with a nucleotide sequence encoding the polypeptide marker and cultured under conditions suitable for expression and recovery of the encoded protein from the cell culture. (See, e.g., Hunkapiller et al., 1984 Nature 310:105-111).
  • the invention provides polynucleotides that encode the polypeptides of the invention.
  • the polynucleotide may be genomic DNA, cDNA, or mRNA transcripts that encode the polypeptides of the invention.
  • the invention provides polynucleotides that encode a polypeptide described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8, or a molecule that comprises such a polypeptide.
  • the invention provides polynucleotides that encode a polypeptide having substantial homology with a component set forth in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8, or a molecule that comprises such a polypeptide.
  • the invention provides polynucleotides that encode a polypeptide having (i) a mass-to-charge value and (ii) an RT value of about the values stated, respectively, for a marker described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8, or a molecule that comprises such a polypeptide.
  • the invention provides polynucleotides that encode a polypeptide having (i) a mass-to-charge value within 10%) (more particularly within 5%, more particularly within 1%) and (ii) an RT value within 10% (more particularly within
  • the invention provides polynucleotides that encode a polypeptide that is a fragment, precursor, successor or modified version of a marker described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8, or a molecule that comprises such polypeptide.
  • the invention provides polynucleotides that encode a polypeptide that is structurally different from a polypeptide specifically identified in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8 but has about the same (or nearly the same) function or properties, or a molecule that comprises such polypeptide.
  • the polynucleotides described may be used as surrogate markers of AD. Thus, for example, if the level of a polypeptide marker is increased in AD-patients, an increase in the mRNA that encodes the polypeptide marker may be interrogated rather than the polypeptide marker (e.g., to diagnose AD in a subject).
  • polypeptides listed in Tables 1-8 are fragments of complete proteins ("parent proteins"), either because they were present as fragments in the sample or as a result of the trypsin digestion that was performed during the processing of certain fractions of the sample (see Example).
  • the parent proteins are included as polypeptide markers.
  • the sequence of the parent protein can be ascertained from the amino acid sequence of the fragment by searching a protein sequence database.
  • Polynucleotides encoding the polypeptides markers listed in Tables 1-8 can be used to screen existing genomic, cDNA or expression libraries to find the gene that encodes the polynucleotide of the invention.
  • a library is typically screened using a probe that is complementary either to the polynucleotide that encodes a polypeptide in Tables 1-8, or to its complement, under hybridization conditions. Hybridization is monitored by any suitable method known in the art. Once located, the gene can be cloned. The protein product of a gene that encodes a fragment of a polynucleotide marker is also included as a polypeptide marker. Alternatively, the sequence of the polynucleotide that encode a polypeptide listed in Tables 1-8 can be used to search databases such as SWISS-PROT and GenBank, which will provide the gene sequence(s) comprising the nucleic acid sequence, and the amino acid sequence of the gene product.
  • Polynucleotide markers may be isolated by any suitable method known in the art. Native polynucleotide markers may be purified from natural sources by standard methods known in the art (e.g., chromatography, centrifugation, differential solubility, immunoassay). In one embodiment, a polynucleotide marker may be isolated from a mixture by contacting the mixture with substrate bound probes that are complementary to the polynucleotide marker under hybridization conditions. Alternatively, polynucleotide markers may be synthesized by any suitable chemical or recombinant method known in the art. In one embodiment, for example, the makers can be synthesized using the methods and techniques of organic chemistry.
  • a polynucleotide marker can be produced by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the invention provides molecules that specifically bind to a polypeptide marker, a metabolite marker or a polynucleotide marker.
  • the binding molecules include antibodies and antibody fragments.
  • the invention provides antibodies that specifically bind to a component described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8, or to a molecule that comprises a foregoing component.
  • the invention provides antibodies that specifically bind to a polypeptide having substantial homology with a component set forth in Table 1,
  • the invention provides antibodies that specifically bind to a component having (i) a mass-to-charge value and (ii) an RT value of about the values stated, respectively, for a marker described in Table 1, Table 2, Table 3, Table 4,
  • the invention provides antibodies that specifically bind to a component having (i) a mass-to-charge value within 10% (more particularly within 5%, more particularly within 1%) and (ii) an RT value within 10% (more particularly within 5%, more particularly within 1%) of the m/z and RT values stated, respectively, for a component described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8, or to a molecule that comprises a foregoing component.
  • the invention provides antibodies that specifically bind to a component that is a fragment, modification, precursor or successor of a marker described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8, or to a molecule that comprises a foregoing component.
  • the invention provides antibodies that specifically bind to a polypeptide marker, a metabolite marker or a polynucleotide marker that is structurally different from a component specifically identified in Table 1, Table 2,
  • antibodies that specifically bind polypeptide markers, metabolite markers or polynucleotide markers of the invention already may be known and/or available for purchase from commercial sources.
  • the antibodies of the invention may be prepared by any suitable means known in the art.
  • antibodies may be prepared by immunizing an animal host with a marker or an immunogenic fragment thereof (conjugated to a carrier, if necessary).
  • Adjuvants e.g., Freund's adjuvant
  • optionally may be used to increase the immunological response.
  • Sera containing polyclonal antibodies with high affinity for the antigenic determinant can then be isolated from the immunized animal and purified.
  • antibody-producing tissue from the immunized host can be harvested and a cellular homogenate prepared from the organ can be fused to cultured cancer cells.
  • Hybrid cells which produce monoclonal antibodies specific for a marker can be selected.
  • the antibodies of the invention can be produced by chemical synthesis or by recombinant expression.
  • a polynucleotide that encodes the antibody can be used to construct an expression vector for the production of the antibody.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • Antibodies that specifically bind markers of the invention can be used, for example, in methods for detecting components described in Table 1, Table 2, Table 3,
  • the antibodies are conjugated to a detection molecule or moiety (e.g., a dye, and enzyme) and can be used in ELISA or sandwich assays to detect markers of the invention.
  • a detection molecule or moiety e.g., a dye, and enzyme
  • antibodies against a polypeptide marker, metabolite marker or polynucleotide marker of the invention can be used to assay a tissue sample (e.g., a thin cortical slice) for the marker.
  • the antibodies can specifically bind to the marker, if any, present in the tissue sections and allow the localization of the marker in the tissue.
  • compositions comprising a polypeptide, metabolite or polynucleotide marker of the invention, a binding molecule that is specific for a polypeptide, metabolite or polynucleotide marker (e.g., an antibody), an inhibitor of a polypeptide, metabolite or polynucleotide marker, or other molecule that can increase or decrease the level or activity of a polypeptide marker, metabolite marker or polynucleotide marker.
  • Such compositions may be pharmaceutical compositions formulated for use as a therapeutic.
  • the invention provides a composition that comprises a polypeptide, metabolite or polynucleotide marker of the invention, such as a component described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8, a polypeptide having substantial homology with a component or having (i) a mass-to- charge value and (ii) an RT value of about the values, respectively, for a component, or a molecule comprising such a component.
  • the invention provides a composition that comprises a component that is a fragment, modification, precursor or successor of a marker described in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8, or to a molecule that comprises a foregoing component.
  • the invention provides a composition that comprises a polypeptide or metabolite that is structurally different from a component specifically identified in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8 but has the same function or properties, or a molecule that comprises a foregoing component.
  • the invention provides a composition that comprises a polynucleotide that binds to a polypeptide or metabolmic marker, or a molecule that comprises a foregoing polynucleotide.
  • the invention provides a composition that comprises an antibody that specifically binds to a polypeptide or metabolomic marker, or a molecule that comprises a foregoing antibody.
  • the invention provides a composition that comprises a modulator of the level or activity of a polypeptide marker (e.g., an inhibitor of a polypeptide marker, an antisense polynucleotide which is complementary to a polynucleotide that encodes a polypeptide marker), or a molecule that comprises a foregoing modulator.
  • a polypeptide marker e.g., an inhibitor of a polypeptide marker, an antisense polynucleotide which is complementary to a polynucleotide that encodes a polypeptide marker
  • a molecule that comprises a foregoing modulator may be pharmaceutical compositions.
  • a pharmaceutical composition comprises a therapeutically effective amount of an active agent and is formulated with a suitable excipient or carrier.
  • the invention also provides pharmaceutical compositions for the treatinent of AD.
  • compositions may include a marker protein and/or nucleic acid of the invention (e.g., for those markers which are decreased in quantity or activity in AD samples versus non-AD samples), and can be formulated as described herein.
  • these compositions may include an antibody which specifically binds to a marker protein of the invention and/or an antisense polynucleotide which is complementary to a polynucleotide marker of the invention (e.g., for those markers which are increased in quantity or activity in AD samples versus non-AD samples), and can be formulated as described herein.
  • the pharmaceutical compositions of the invention can be prepared in any suitable manner known in the pharmaceutical art.
  • the carrier or excipient may be a solid, semisolid, or liquid material that can serve as a vehicle or medium for the active ingredient.
  • Suitable carriers or excipients are well known in the art and include, but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical compositions may be adapted for oral, inhalation, parenteral, or topical use and may be administered to the patient in the form of tablets, capsules, aerosols, inhalants, suppositories, solutions, suspensions, powders, syrups, and the like.
  • the term "pharmaceutical carrier” may encompass one or more excipients. In preparing formulations of the compounds of the invention, care should be taken to ensure bioavailability of an effective amount of the agent.
  • Suitable pharmaceutical carriers and formulation techniques are found in standard texts, such as Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania.
  • the markers of the invention may be detected by any method known to those of skill in the art, including without limitation LC-MS, GC-MS, immunoassays, hybridization and enzyme assays.
  • the detection may be quantitative or qualitative.
  • a wide variety of conventional techniques are available, including mass spectrometry, chromatographic separations, 2-D gel separations, binding assays (e.g., immunoassays), competitive inhibition assays, and so on.
  • Any effective method in the art for measuring the present/absence, level or activity of a metabolite, polypeptide or polynucleotide is included in the invention. It is within the ability of one of ordinary skill in the art to determine which method would be most appropriate for measuring a specific marker.
  • a ELISA assay may be best suited for use in a physician's office while a measurement requiring more sophisticated instrumentation may be best suited for use in a clinical laboratory.
  • the markers of the invention can be measured by mass spectrometry, which allows direct measurements of analytes with high sensitivity and reproducibility.
  • mass spectrometric methods are available.
  • Electrospray ionization (ESI) allows quantification of differences in relative concentration of various species in one sample against another; absolute quantification is possible by normalization techniques (e.g., using an internal standard).
  • Matrix-assisted laser desorption ionization or the related SELDI® technology (Ciphergen, Inc.) also could be used to make a determination of whether a marker was present, and the relative or absolute level of the marker.
  • Mass spectrometers that allow time-of-flight (TOF) measurements have high accuracy and resolution and are able to measure low abundant species, even in complex matrices like serum or CSF.
  • TOF time-of-flight
  • quantification can be based on derivatization in combination with isotopic labeling, referred to as isotope coded affinity tags ("ICAT").
  • a specific amino acid in two samples is differentially and isotopically labeled and subsequently separated from peptide background by solid phase capture, wash and release.
  • the intensities of the molecules from the two sources with different isotopic labels can then be accurately quantified with respect to one another.
  • one- and two-dimensional gels have been used to separate proteins and quantify gels spots by silver staining, fluorescence or radioactive labeling. These differently stained spots have been detected using mass spectrometry, and identified by tandem mass spectrometry techniques.
  • the markers are measured using mass spectrometry in connection with a separation technology, such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry.
  • a separation technology such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry.
  • TOF mass spectroscopy allows spectral intensity measurement of a large number of biomolecules from a relatively small amount of any complex biological material. Analyzing a sample in this manner allows the marker (characterized by a specific RT and m/z) to be determined and quantified.
  • a sample characterized by a specific RT and m/z
  • many other separation technologies may be used in connection with mass spectrometry. For example, a wide selection of separation columns is commercially available.
  • separations may be performed using custom chromatographic surfaces (e.g., a bead on which a marker specific reagent has been immobilized). Molecules retained on the media subsequently may be eluted for analysis by mass spectrometry.
  • Analysis by liquid chromatography-mass spectrometry produces a mass intensity spectrum, the peaks of which represent various components of the sample, each component having a characteristic mass-to-charge ratio (m/z) and retention time (RT).
  • m/z characteristic mass-to-charge ratio
  • RT retention time
  • the presence of a peak with the m/z and RT of a marker indicates that the marker is present.
  • the peak representing a marker may be compared to a corresponding peak from another spectrum (e.g., from a control sample) to obtain a relative measurement.
  • Any normalization technique in the art e.g., an internal standard
  • Deconvoluting software is available to separate overlapping peaks.
  • the retention time depends to some degree on the conditions employed in performing the liquid chromatography separation.
  • the preferred conditions those used to obtain the retention times that appear in the Tables, are set forth in the Example.
  • the mass spectrometer preferably provides high mass accuracy and high mass resolution.
  • the mass accuracy of a well-calibrated Micromass TOF instrument is reported to be approximately 2 mDa, with resolution m/ ⁇ m exceeding 5000.
  • the level of the markers may be determined using a standard immunoassay, such as sandwiched ELISA using matched antibody pairs and chemiluminescent detection. Commercially available or custom monoclonal or polyclonal antibodies are typically used. However, the assay can be adapted for use with other reagents that specifically bind to the marker.
  • Standard protocols and data analysis are used to determine the marker concentrations from the assay data.
  • a number of the assays discussed above employ a reagent that specifically binds to the marker.
  • Any molecule that is capable of specifically binding to a marker is included within the invention.
  • the binding molecules are antibodies or antibody fragments.
  • the binding molecules are non- antibody species.
  • the binding molecule may be an enzyme for which the marker is a substrate.
  • the binding molecules may recognize any epitope of the targeted markers.
  • the binding molecules may be identified and produced by any method accepted in the art. Methods for identifying and producing antibodies and antibody fragments specific for an analyte are well known.
  • binding assays with random peptide libraries e.g., phage display
  • design methods based on an analysis of the structure of the marker include binding assays with random peptide libraries (e.g., phage display) and design methods based on an analysis of the structure of the marker.
  • the markers of the invention, especially the metabolite markers also may be detected or measured using a number of chemical derivatization or reaction techniques known in the art. Reagents for use in such techniques are known in the art, and are commercially available for certain classes of target molecules.
  • the chromatographic separation techniques described above also may be coupled to an analytical technique other than mass spectrometry such as fluorescence detection of tagged molecules, NMR, capillary UV, evaporative light scattering or electrochemical detection.
  • Measurement of the relative amount of an RNA or protein marker of the invention may be by any method known in the art (see, e.g., Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley &
  • RNA detection include RNA extraction from a cell or tissue sample, followed by hybridization of a labeled probe (e.g., a complementary polynucleotide) specific for the target RNA to the extracted RNA, and detection of the probe (e.g., Northern blotting).
  • a labeled probe e.g., a complementary polynucleotide
  • protein detection include protein extraction from a cell or tissue sample, followed by hybridization of a labeled probe (e.g., an antibody) specific for the target protein to the protein sample, and detection of the probe.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Detection of specific protein and polynucleotides may also be assessed by gel electrophoresis, column chromatography, direct sequencing, or quantitative PCR (in the case of polynucleotides) among many other techniques well known to those skilled in the art. Detection of the presence or number of copies of all or a part of a marker gene of the invention may be performed using any method known in the art. Typically, it is convenient to assess the presence and/or quantity of a DNA or cDNA by Southern analysis, in which total DNA from a cell or tissue sample is extracted, is hybridized with a labeled probe (e.g., a complementary DNA molecule), and the probe is detected.
  • a labeled probe e.g., a complementary DNA molecule
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • Other useful methods of DNA detection and/or quantification include direct sequencing, gel electrophoresis, column chromatography, and quantitative PCR, as is known by one skilled in the art. Polynucleotide similarity can be evaluated by hybridization between single stranded nucleic acids with complementary or partially complementary sequences. Such experiments are well known in the art.
  • the invention provides methods for diagnosing AD in a subject.
  • the invention provides a method for determining whether a subject has AD. These methods comprise obtaining a biological sample from a subject suspected of having AD, or at risk for developing AD, detecting the level or activity of a marker of the invention in the sample, and comparing the result to the level or activity of the marker in a sample obtained from a non-AD subject, or to a reference range or value, such that a significant difference exists. A "significant difference" is one that permits the other protein to be resolved.
  • an increased level or activity of the marker in a sample obtained from a subject suspected of having AD, or at risk for developing AD is indicative that the subject has or is at risk for developing AD.
  • Markers appropriate for this embodiment include those that have been identified as increased in samples obtained from AD subjects compared with samples from non-AD subjects (e.g., markers described in Table 1, Table 2, Table 3, and Table 4).
  • Other markers appropriate for this embodiment include fragments, precursors, successors and modified versions of such markers, polypeptides having substantial homology to such markers, components having an m/z value and RT value of about the values set forth for the markers described in
  • Table 1, Table 2, Table 3, and Table 4, and molecules comprise one of the foregoing.
  • Other appropriate markers for this embodiment will be apparent to one of skill in the art in light of the disclosure herein.
  • a decreased level or activity of the marker in a sample obtained from a subject suspected of having AD, or at risk for developing AD is indicative that the subject has or is at risk for developing AD.
  • Markers appropriate for this embodiment include those that have been identified as decreased in samples obtained from AD subjects compared with samples from non-AD subjects (e.g., markers described in Table 5, Table 6, Table 7, and Table 8).
  • markers appropriate for this embodiment include fragments, precursors, successor or modified versions of such markers, polypeptides having substantial homology to such markers, components having an m/z value and RT value of about the values set forth for the markers described in Table 5, Table 6, Table 7, and Table 8, and molecules comprise one of the foregoing.
  • Other appropriate markers for this embodiment will be apparent to one of skill in the art in light of the disclosure herein
  • the foregoing methods can be used to determine whether a subject is more likely than not to have AD, that the subject is more likely to have AD than another disease, or that the subject has an increased likelihood of having AD.
  • the foregoing methods can also be used to confirm a diagnosis of AD.
  • the invention also provides a method for determining a subject's risk of developing AD, the method comprising obtaining a biological sample from a subject, detecting the level or activity of a marker in the sample, and comparing the result to the level or activity of the marker in a sample obtained from a non-AD subject, or to a reference range or value wherein an increase or decrease of the marker is correlated with the risk of developing AD.
  • the invention also provides methods for determining the stage or severity of AD, the method comprising obtaining a biological sample from a subject, detecting the level or activity of a marker in the sample, and comparing the result to the level or activity of the marker in a sample obtained from a non-AD subject, or to a reference range or value wherein an increase or decrease of the marker is correlated with the stage or severity of the disease.
  • the invention provides methods for monitoring the progression of the disease in a subject who has AD, the method comprising obtaining a first biological sample from a subject, detecting the level or activity of a marker in the sample, and comparing the result to the level or activity of the marker in a second sample obtained from the subject at a later time, or to a reference range or value wherein an increase or decrease of the marker is correlated with progression of the disease.
  • Each marker may be considered individually, although it is within the scope of the invention to provide combinations of two or more markers for use in the methods and compositions of the invention. The use of such combinations typically will increase the confidence of the analysis.
  • a panel of markers may include markers that are increased in level or activity in AD subject samples as compared to non-AD subject samples, markers that are decreased in level or activity in AD subject samples as compared to non-AD subject samples, or a combination thereof.
  • a panel of makers may include one or more markers of the invention as well as one or more known biomarkers of AD (e.g., A ⁇ ).
  • the panel of markers may also be evaluated with other clinical indicia of AD (e.g., ADAS-Cog, MRI/CT imaging).
  • the marker may be detected in any biological sample obtained from the subject, or in some cases, from a relative of the subject, by any suitable method known in the art (e.g., immunoassays, hybridization assay) see supra.
  • the invention also provides methods for treating AD, as well as other diseases or conditions, by providing a therapeutic agent to a subject that increases or decreases the level or activity of at least one marker of the invention.
  • the method comprises administering a therapeutic agent to a subject that increases level or activity of at least one polypeptide, metabolite or polynucleotide marker of the invention that is decreased in samples obtained from AD subjects compared to samples obtained from non-AD subjects or to a reference range or value.
  • the method comprises administering a therapeutic agent to a subject that decreases the level of at least one polypeptide, metabolite or polynucleotide marker of the invention that is increased in samples obtained from AD subjects compared to samples obtained from non-AD subjects or to a reference range or value.
  • the method further comprises first obtaining a sample from an AD subject, determining the presence, level or activity of at least one marker of the invention in the sample compared to samples obtained from a non-AD subject or to a reference range or value. If the marker is increased in the sample obtained from the AD subject, a therapeutic agent that decreases the level of the marker is administered to the patient.
  • a therapeutic agent that increases the level of the marker is administered to the subject.
  • Therapeutic agents include but are not limited to polypeptide markers, metabolite markers, polynucleotide markers, molecules comprising a polypeptide marker, metabolite marker or polynucleotide marker, antibodies to polypeptide marker, metabolite marker or polynucleotide marker, modulators of the level or activity a polypeptide or polynucleotide marker (e.g., an inhibitor, anti-sense polynucleotides) or compositions comprising one or more of the foregoing.
  • the therapeutic agents used in the invention are administered to the subject in an effective amount.
  • an "effective amount” is typically the amount that is sufficient to obtain beneficial or desired clinical results.
  • the effective amount is generally determined by a physician with respect to a specific patient and is within the skill of one in the art. Factors that may be taken into account in determining an effective amount include those relating to the condition being treated (e.g., type, stage, severity) as well as those relating to the subject (e.g., age, sex, weight).
  • the level or activity of a polypeptide marker may be increased or decreased by any suitable technique or method known in the art.
  • the level of a polypeptide marker may be increased by providing the polypeptide marker to a subject.
  • the level of a polypeptide marker may be increased by providing a polynucleotide that encodes the polypeptide marker (e.g., gene therapy).
  • a polynucleotide that encodes the polypeptide marker e.g., gene therapy
  • compounds or molecules known to increase that activity may be provided to the subject.
  • the level of a polypeptide marker may be decreased by providing antibodies specific for the polypeptide marker to the subject.
  • the level of a polypeptide marker may be decreased by providing a polynucleotide that is "anti-sense" to the polynucleotide that encodes the polypeptide marker, or that encodes dysfunctional proteins.
  • compounds or molecules known to decrease that activity e.g., inhibitor or antagonist.
  • the level of a metabolite marker may be increased or decreased by any suitable technique or method known in the art.
  • the level of a metabolite marker may be increase by providing the metabolite marker to the patient.
  • the level of a metabolite marker may be decreased by providing antibodies specific for the metabolite marker to the subject.
  • the therapeutic compounds described herein may be administered alone or in combination with another therapeutic compound, or other form of treatment.
  • the compounds may be administered to the subjects in any suitable manner known in the art (e.g., orally, topically, subcutaneously, intradermally, intramuscularly, intravenously, intra-arterially, intrathecally). Metabolites may be combined with an excipient and formulated as tablets or capsules for oral administration.
  • Polypeptides may be formulated for parenteral administration to avoid denaturation by stomach acids.
  • vectors may be constructed for administration to the subject by a virus or other carrier.
  • cDNA is delivered to target cells (e.g., bone marrow cells) that are later reintroduced into the subject for expression of the encoded protein.
  • a therapeutic composition can be administered in a variety of unit dosage forms depending upon the method of administration.
  • LX. Methods for Screening Candidate Compounds provides methods for screening candidate compounds for use as therapeutic compounds.
  • the method comprises screening candidate compounds for those that bind to a polypeptide, metabolite or polynucleotide molecule of the invention.
  • Candidate compounds that bind to markers can be identified using any suitable method or technique known in the art.
  • a candidate compound or a control is contacted with marker and the ability of the candidate compound to form stable complexes is determined (e.g., flow cytometry, immunoprecipitation).
  • the candidate compound, the marker, or an antibody that specifically binds either may be labeled to facilitate detection.
  • the candidate molecule or marker may be immobilized on a solid support (e.g., a bead).
  • cells expressing a polypeptide marker are contacted with a candidate compound or a control and the ability of the candidate compound to form stable complexes with the cells is determined.
  • the candidate compound or the marker may be labeled to facilitate detection.
  • the method comprises screening candidate compounds for those that have a stimulatory or inhibitory effect on the activity of a marker comprising comparing the activity of the marker in the presence of the candidate molecule with the activity of the marker in the absence of the candidate molecule (e.g., in the presence of a control).
  • the method comprises screening candidate compounds for those that have the ability to increase or decrease the level of a polypeptide, metabolite or polynucleotide marker in a biological sample obtained from a subject, obtaining a first sample from the subject, providing the candidate compound or a control to the subject, at a later time obtaining a second sample from the subject, and comparing the respective activities, and then comparing the respective activities or levels of the marker in the first and second sample.
  • Candidate compounds for which the level or activity of the marker is changed are selected. This embodiment can be used in a clinical trial where a plurality of subjects is evaluated and the results are statistically significant.
  • Kits In another aspect, the invention provides a kit for detecting a polypeptide, metabolite, or polynucleotide marker. In another aspect, the invention provides a kit for diagnosing AD in a patient by detecting at least one polypeptide, metabolite or polynucleotide marker in a biological sample from the subject. In another aspect, the invention provides a kit for screening candidate compounds by detecting stable complexes between the candidate compound and a polynucleotide, metabolite or polynucleotide marker.
  • kits of the invention may comprise one or more of the following: an antibody, wherein the antibody specifically binds with a polypeptide or metabolite marker, a labeled binding partner to the antibody, a solid phase upon which is immobilized the antibody or its binding partner, a polynucleotide probe that can hybridize to a polynucleotide marker, pairs of primers that under appropriate reaction conditions can prime amplification of at least a portion of a polynucleotide marker or a polynucleotide encoding a polypeptide marker (e.g., by PCR), instructions on how to use the kit, and a label or insert indicating regulatory approval for diagnostic or therapeutic use.
  • an antibody wherein the antibody specifically binds with a polypeptide or metabolite marker, a labeled binding partner to the antibody, a solid phase upon which is immobilized the antibody or its binding partner
  • a polynucleotide probe that can hybridize to a polynucleotide marker
  • Serum samples were obtained from AD and non-AD subjects in accordance with a clinical protocol and informed consent that were approved by an institutional review board (IRB) and with procedures that adhere to Good Clinical Practice.
  • Serum Proteome A high molecular weight fraction (“serum proteome") was separated from the serum samples using a 5-kDa molecular weight cut-off spin filter
  • the serum proteome was diluted with PBS buffer (pH 6.0). To increase the effective dynamic range of the measurements, the two most abundant proteins (human serum albumin and IgG) were substantially depleted by an affinity resin (ProMetic Biosciences, Cambridge, UK). The remaining proteins were denatured using guanidine hydrochloride, disulfide bonds were reduced using dithioreitol, and sulfhydryl groups were carboxymethylated using iodoacetic acid/NaOH. The denaturant and reduction-alkylation reagents were removed by buffer exchange.
  • LC-ESI-MS liquid chromatography-electrospray ionization-mass spectrometry
  • TOF time-of-flight
  • proteome sample was fractionated by the cation exchange chromatography into eight fractions.
  • a water/acetonitrile mobile phase was used with increasing KC1 salt gradient. After collection, each fraction was desalted prior to injection onto the LC-ESI-MS (1-D) configuration (described above). Individual molecules were tracked across samples and their differential expression determined.
  • Serum Metabolome A low molecular weight fraction (“serum metabolome") was obtained from the serum sample by removing proteins by precipitation with the addition of an organic solution. The supernatant containing the serum metabolome was divided into two portions. A first portion of the metabolome was analyzed for volatile
  • GC-EI-MS gas chromatography-electron-impact ionization-mass spectrometry
  • the second portion was analyzed for nonvolatile components using the LC-ESI-MS (1-D) configuration (described above).
  • LC-ESI-MS high-resolution (R > 5,000) time-of-flight mass spectrometers were used for profiling.
  • the first portion of the serum metabolome was combined with triethylammonium trifluoroacetate (ETA-TFA) to convert keto groups to derivatizable enols.
  • ETA-TFA triethylammonium trifluoroacetate
  • the mixture containing volatile components, was injected into the GC-EI-MS TOF with a 60 minute temperature gradient from 80 to 300 °C and electron-impact ionization.
  • the carrier gas was helium.
  • Individual molecules were tracked across samples and their differential expression determined.
  • the second portion of the serum metabolome was subjected to organic precipitation and dried to a solid using a lyophilizer.
  • the solid material was dissolved in aqueous formic acid, desalted, dried again and redissolved in 0.1 % formic acid for injection onto the column of the LC-ESI-MS configuration (described above).
  • a 100 minute gradient was used from 0% to 90% acetonitrile in water. Individual molecules were tracked across samples and their differential expression determined. Tandem MS.
  • LC/MS/MS was used for separation and identification of differentially expressed components.
  • the eluate from the LC configuration (described above for 1-D MS) flowed into the ion trap mass spectrometer (LCQTM Deca, ThermoFinnigan).
  • Identification of polypeptides and metabolites from the MS/MS spectra was performed using the TurboSEQUEST (ThermoFinnigan) or Mascot (Matrix Science) search program.
  • TurboSEQUEST was able to provide information on up to three post-translational modifications.
  • MS/MS spectra obtained from the LCQ-Deca (ThermoFinnigan) was used to identify intact peptides (free in serum, not trypsinized).
  • a more accurate parent ion molecular weight is obtained from a parallel analysis using the LCT orthogonal-injection ESI-TOF (Micromass).
  • Accuracy of the LCT detection is -10 ppm using the natural internal calibration of known peptides.
  • accuracy of the LCQ-Deca is ⁇ 0.5 Da (-1000 ppm). This data is then examined by a database searching approach (described below).
  • de novo amino acid sequence analysis programs can be used to obtain at least partial sequence analysis.
  • the in silico digestion can include a small number of PTMs.
  • database approaches such as TurboSEQUEST will not work to identify peptides or proteins that are not already in the database. In that case, de novo peptide sequencing software and BLAST searching can be used.
  • Post-Translational Modification PTM.
  • a number of methods were used to detect PTM of the identified polypeptides. Using the known fixed mass that a PTM adds, TurboSEQUEST software can identify up to three PTMs on a peptide. In addition to such informatics approaches, a number of biochemical methods can be used to detect PTMs.
  • mixtures of phosphopeptides can be extracted using anti-phospho- tyrosine and anti-phospho-serine/threonine antibodies.
  • Another approach is the use of an activated metal surface or column to capture phosphorylated proteins.
  • a third tool is the use of dephosphorylating enzyme alkaline phosphatase and a re-analysis to examine changes.
  • glycosylation may also play a role in AD.
  • the analysis of the O- and N-glycosylation can be performed by tryptic digestion of the protein, isolation of glycopeptides by lectin chromatography and mass measurement before and after enzymatic deglycosylation.
  • Carbohydrate structures are calculated from the mass difference between glycosylated and deglycosylated peptide plus the use of tandem MS (MS/MS). Volatile and nonvolatile metabolites.
  • electron-impact ionization provides characteristic fingerprint patterns that can lead to identification if the molecule has been analyzed previously in pure form and entered into a database such as the database provided by National Institute of Standards and Technology (NIST). Otherwise, use of accurate mass to constrain the elemental composition is also useful.
  • tandem mass spectrometry is available with a triple-quadrupole instrument.
  • MS/MS can be used to corroborate identity or provide insight into an unknown's structure.
  • Quantification Proteins, peptides or small molecules were quantified relative to the same, corresponding molecules in a different sample, usually a control or normal sample.
  • This approach relies on the assumption that biological samples consist of complex mixtures of multiple biological components, of which only a minority are relevant to the comparison. The majority of components are relatively constant for the same individual over time or across subject populations. The majority of components whose concentrations do not vary across samples are used as an intrinsic internal standard to normalize the concentrations of components that do vary.
  • the method also relies on the inherent reproducibility of ionization for ESI. The high reproducibility is measured by the coefficient of variation. The majority of peaks have a CV less then 20%>. The validity of this approach is discussed in more detail in Wang W. et al.

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  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des compositions, des trousses et des méthodes pour diagnostiquer, caractériser et traiter la maladie d'Alzheimer (AD). L'invention concerne des polypeptides et de petites molécules non-peptidiques, identifiés comme différentiellement exprimés dans des échantillons obtenus à partir de sujets AD en comparaison à des échantillons obtenus à partir de sujets non-AD, pouvant être utilisés respectivement en tant que marqueurs polypeptidiques et que marqueurs de métabolite. Ces marqueurs peuvent être utilisés seuls ou combinés.
PCT/US2005/017263 2004-05-18 2005-05-18 Compositions et methodes associees a la maladie d'alzheimer WO2005116659A2 (fr)

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US60/572,258 2004-05-18

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WO2005116659A3 WO2005116659A3 (fr) 2006-09-08

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009528517A (ja) * 2006-02-28 2009-08-06 フェノメノーム ディスカバリーズ インク 認知症及び他の神経障害の診断方法
US9034923B2 (en) 2007-02-08 2015-05-19 Phenomenome Discoveries Inc. Methods for the treatment of senile dementia of the alzheimer's type
AU2016206360B2 (en) * 2006-02-28 2018-03-08 Med-Life Discoveries Lp Methods for the diagnosis of dementia and other neurological disorders
CN113049696A (zh) * 2021-03-04 2021-06-29 首都医科大学宣武医院 一种诊断受试者是否患阿尔茨海默病的代谢产物及其用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4414336A (en) * 1981-01-28 1983-11-08 The Green Cross Corporation Method for preparing sample for use in endotoxin test
US6717031B2 (en) * 1995-06-07 2004-04-06 Kate Dora Games Method for selecting a transgenic mouse model of alzheimer's disease

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009528517A (ja) * 2006-02-28 2009-08-06 フェノメノーム ディスカバリーズ インク 認知症及び他の神経障害の診断方法
EP2322531A3 (fr) * 2006-02-28 2011-09-07 Phenomenome Discoveries Inc. Méthodes permettant de diagnostiquer la démence et autres troubles neurologiques
US8304246B2 (en) 2006-02-28 2012-11-06 Phenomenome Discoveries, Inc. Methods for the diagnosis of dementia and other neurological disorders
JP2012255793A (ja) * 2006-02-28 2012-12-27 Phenomenome Discoveries Inc 認知症及び他の神経障害の診断方法
JP2014197018A (ja) * 2006-02-28 2014-10-16 フェノメノーム ディスカバリーズ インク 認知症及び他の神経障害の診断方法
AU2016206360B2 (en) * 2006-02-28 2018-03-08 Med-Life Discoveries Lp Methods for the diagnosis of dementia and other neurological disorders
US9034923B2 (en) 2007-02-08 2015-05-19 Phenomenome Discoveries Inc. Methods for the treatment of senile dementia of the alzheimer's type
US9517222B2 (en) 2007-02-08 2016-12-13 Phenomenome Discoveries Inc. Method for the treatment of senile dementia of the Alzheimer's type
CN113049696A (zh) * 2021-03-04 2021-06-29 首都医科大学宣武医院 一种诊断受试者是否患阿尔茨海默病的代谢产物及其用途

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