WO2009051599A1 - Utilisation de biomarqueurs de la maladie d'alzheimer pour des tests de diagnostic et pour le criblage de médicaments - Google Patents

Utilisation de biomarqueurs de la maladie d'alzheimer pour des tests de diagnostic et pour le criblage de médicaments Download PDF

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WO2009051599A1
WO2009051599A1 PCT/US2007/081989 US2007081989W WO2009051599A1 WO 2009051599 A1 WO2009051599 A1 WO 2009051599A1 US 2007081989 W US2007081989 W US 2007081989W WO 2009051599 A1 WO2009051599 A1 WO 2009051599A1
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amyloid beta
beta polypeptide
disease
labeled
alzheimer
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PCT/US2007/081989
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English (en)
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Milan Fiala
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The Regents Of The University Of California
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Priority to PCT/US2007/081989 priority Critical patent/WO2009051599A1/fr
Publication of WO2009051599A1 publication Critical patent/WO2009051599A1/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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5055—Cells of the immune system involving macrophages
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70585—CD44
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relate to use of a biomarker of Alzheimer's disease for diagnostic tests as well as screening assays for agents that may be useful in treating Alzheimer's disease.
  • AD Alzheimer's disease
  • CSF cerebrospinal fluid
  • Plasma proteome shows changes in multiple proteins implicated in the disease pathology, in particular inflammation, but the test is only 56% sensitive and 80% specific (Hye et al. 2006. Brain 129:3042-3050).
  • CSF- and blood-based tests are gaining entry into clinical use.
  • Low CSF level of Ap 1 . 42 and a high phosphorylated- ⁇ and total ⁇ levels are widely used in Europe to differentiate mild cognitive impairment (MCI) and early AD from normal aging, depression and alcohol abuse (Blennow K & Hampel H. 2003 Lancet Neurol 2:605-613.).
  • Blood tests are more suitable than CSF tests for screening use.
  • the association of plasma A ⁇ i -40 and levels with dementia has been evaluated in several longitudinal studies (Motter et al. 1995. Ann Neurol 38:643-648.; Motter et al. 1995. Ann Neurol 38:643-648; Pomara et al. 2005.
  • Brain amyloidosis of sporadic AD has been attributed to defective A ⁇ clearance (Motter et al. 1995. Ann Neurol 38:643-648). Peripheral clearance of A ⁇ . by complement C3 -adherence to erythrocytes is lower in Alzheimer's disease patients compared to control subjects (Rogers et al. 2006. Neurobiol Aging 27:1733-1739). We have searched for peripheral biomarkers of AD based on the presence of defective phagocytosis of A ⁇ in patients with sporadic Alzheimer's disease. Macrophages of patients with sporadic Alzheimer's disease generally show defective phagocytosis of A ⁇ .
  • monocytes freshly isolated from peripheral blood mononuclear cells also show robust differences between patients and controls in their interactions with FITC-A ⁇ .
  • control monocytes express several molecules that may serve as biomarkers; in contrast, Alzheimer's disease monocytes are susceptible to apoptosis.
  • Amyloid-precursor protein is the precursor protein from which the pathogenic amyloid-beta is cut out. APP is expressed from a single gene in a wide variety of tissue and cell types. At least ten isoforms of the protein have been described, derived by alternative splicing; the 695, 751 and 770 amino acid isoforms appear to be most common. APP695 is exclusive to neurons while the other forms are present in many cell types.
  • the protein has a transmembrane region near the c-terminus, undergoes O-linked and N-linked glycosylation, and contains a Kunitz-type protease inhibitor (KPI) domain in its c-terminal portion"
  • KPI Kunitz-type protease inhibitor
  • Sequential proteolytic processing of APP by ⁇ - and ⁇ - secretase after amino acids 671 and 711/713/714, respectively, give rise to the A ⁇ peptides.
  • the A ⁇ peptides known as A ⁇ x-40, x-42 and x-43 for the number of amino acids they contain, are deposited into the hallmark amyloid plaques of AD. It is these peptides, especially A ⁇ x-42 and x-43, that are thought to be causal in AD.
  • a method for diagnosing Alzheimer's disease. The method comprises: providing a test cell sample from a patient; exposing the test cell sample to a labeled amyloid beta polypeptide; and determining an intracellular level of the labeled amyloid beta polypeptide in the test cell sample, wherein a lower level of labeled amyloid beta polypeptide in the test cell sample compared to a control cell sample is indicative of Alzheimer's disease.
  • the test and control cell samples are macrophages.
  • the labeled amyloid beta polypeptide is a full- length amyloid precursor protein, a peptide fragment thereof, including in particular APi -4O and A ⁇ 1-42 fragments (SEQ ID NO: 1 and SEQ ID NO: 2).
  • the test cell sample may be exposed to the labeled amyloid beta polypeptide in a cell culture media.
  • the labeled amyloid beta polypeptide is preferably fluorescently labeled, e.g., FITC, but in one embodiment, the labeled amyloid beta polypeptide is radiolabeled. In another embodiment, the labeled amyloid beta polypeptide is chromatogenically labeled.
  • the level of labeled amyloid beta polypeptide may be determined by fluorescence microscopy imaging. Alternatively, the level of labeled amyloid beta polypeptide may be determined by flow cytometry. In other embodiments, the level of labeled amyloid beta polypeptide may be determined by radioassay or chromogenic or absorbance based assays, or scintillation counting (for example with 14C-labeled amyloid beta) or by use of fluorogenic labeling.
  • kits for the diagnosis of Alzheimer's disease comprises: a labeled amyloid beta polypeptide; and instructions for isolating and using cell samples for determining relative levels of amyloid beta polypeptide uptake.
  • the amyloid beta polypeptide is labeled with a fluorescent dye, e.g., FITC.
  • the kit may further comprise a chamber slide.
  • a method for detecting a predisposition to Alzheimer's disease in a subject comprises: providing a test cell sample from the subject; exposing the test cell sample to a fluorescently labeled amyloid beta polypeptide; and observing a pattern of cellular distribution of fluorescense, wherein an abnormal pattern of fluorescently labeled amyloid beta polypeptide in the test cell sample compared to a control sample is indicative of a predisposition to Alzheimer's disease.
  • a screening method for identifying a substance useful for treating and/or preventing Alzheimer's disease comprises: contacting a cell sample with the substance; contacting the cell sample with a labeled amyloid beta polypeptide; and determining if the substance effects the level of labeled amyloid beta polypeptide in the sample.
  • macrophages in 8-well chambers or multiwell (96-) well chambers are treated with the test substances at appropriate concentration for specified time and phagocytosis assay with fluorescent amyloid-beta is done by the standard technique
  • a method of diagnosing Alzheimer's disease in a subject comprises: providing a test cell sample from the subject; exposing the test cell sample to an amyloid beta polypeptide; labeling the amyloid beta polypeptide by binding a labeled antibody or fragment thereof to the amyloid beta polypeptide; and determining the level of the labeled amyloid beta polypeptide in the test cell sample, wherein lower levels of labeled amyloid beta polypeptide in the test cell sample compared to a control cell sample are indicative of Alzheimer's disease.
  • Fig. 1 Flow cytometry of A ⁇ phagocytosis by monocytes: high intracellular uptake by control monocytes.
  • B Monocytes with a high A ⁇ uptake were sorted by FACS Vantage SE sorting flow cytometer into high A ⁇ uptake and low A ⁇ uptake CD14-positive monocytes.
  • C Fluorescence microscopy of high A ⁇ uptake monocytes. Control monocytes show intracellular fluorescence; AD monocytes show surface staining.
  • FITC- A ⁇ microscopic test clustering of control monocytes around aggregated A ⁇ .
  • PBMCs of 12 control subjects and 12 patients were incubated with FITC -A ⁇ (2 ⁇ g/ml) overnight and stained with CD68/ALEXA 555.
  • A) Uptake of A ⁇ by monocytes Photographs of the largest cluster of monocytes with FITC-A ⁇ in each control and patient cytospin preparation (10Ox objective of the Olympus microscope and Hamamatsu camera).
  • Fig. 3 Expression of CD44 on phagocytic cells.
  • PBMCs of a control subject and a patient were incubated with FITC-A ⁇ (2 ⁇ g/ml) overnight and stained with anti- CD44/ ALEXA 555. Note high expression of CD44 on phagocytic cells in control preparation but lack of expression of CD44 on AD cells.
  • Fig. 4 Phagocytosis of FITC-A ⁇ by control and patient monocytes.
  • PBMCs of 30 control subjects and 30 AD patients were incubated with A ⁇ (2 mg/ml) overnight, cytospun on a glass slide, stained with anti-CD68, photographed and uptake of FITC-Ab (IOD) was determined by Image-Pro.
  • IOD uptake of FITC-Ab
  • Fig. 5 Expression of CD44 is increased on monocytes of control subjects.
  • PBMCs of a control subject and a patient were incubated with FITC- ⁇ (2 ⁇ g/ml) overnight, and cytospun, stained with anti-CD44/ ALEXA 555, photographed and IOD of CD44 was determined by Image-Pro. Note high expression of CD44 on phagocytic cells in the control preparation (a, b) but lack of expression of CD44 on AD cells (c,d).
  • Innate immune cells have crucial role in maintaining the pristine milieu of the nervous system; however, their function in the clearance of amyloid- ⁇ (A ⁇ ) is defective in some patients with sporadic Alzheimer disease (AD).
  • AD amyloid- ⁇
  • PBMCs peripheral blood mononuclear cells
  • Flow cytometry showed a significantly higher uptake of FITC -A ⁇ in normal subjects compared to AD patients (PO.020), but some patients' monocytes bound A ⁇ on the surface, as shown by cell sorting and fluorescence microscopy.
  • To distinguish intracellular from surface uptake we performed fluorescence microscopy of PBMCs incubated with FITC-A ⁇ or with unlabeled A ⁇ stained by anti-A ⁇ /fluorescent anti-rabbit IgG and collected by cytocentrifugation.
  • the monocytes of a control subject displayed increased CD44 expression in comparison to the monocytes of a patient.
  • the tests of A ⁇ phagocytosis may serve as immunologic biomarkers for AD. Part 2
  • the transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the present invention relates to methods for the diagnosis of Alzheimer's disease (AD), methods for screening for compounds that treat or prevent AD, and to methods for the treatment of AD which are based on this diagnosis.
  • the present invention provides methods for diagnosing Alzheimer's based on the rate of uptake of A ⁇ in a cell sample. Definitions
  • antibody refers to intact molecules as well as fragments thereof, which binds specifically to an antigenic determinant, and specifically, binds to proteins identical or structurally related to the antigenic determinant which stimulated their production. Thus, antibodies are useful in assays to detect the antigen which stimulated their production.
  • Antibody fragments comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments.
  • Monoclonal antibodies are derived from a single clone of B lymphocytes (i.e., B cells), and are generally homogeneous in structure and antigen specificity. Polyclonal antibodies originate from many different clones of antibody-producing cells, and thus are heterogeneous in their structure and epitope specificity, but all recognize the same antigen. In some embodiments, monoclonal and polyclonal antibodies are used as crude preparations, while in preferred embodiments, these antibodies are purified. For example, in some embodiments, polyclonal antibodies contained in crude antiserum are used.
  • antibody encompass any immunoglobulin (e.g., IgG, IgM, IgA, IgB, IgD, etc.) obtained from any source (e.g., humans, rodents, non- human primates, lagomorphs, caprines, bovines, equines, ovines, etc.).
  • immunoglobulin e.g., IgG, IgM, IgA, IgB, IgD, etc.
  • auto-antibody or “auto-antibodies” refer to any immunoglobulin that binds specifically to an antigen that is native to the host organism that produced the antibody (i.e., the antibody is directed against 'self antigens).
  • autoimmunity The presence of auto-antibodies is referred to herein as "autoimmunity.”
  • antigen is used in reference to any substance that is capable of being recognized by an antibody. It is intended that this term encompass any antigen and "immunogen” (i.e., a substance which induces the formation of antibodies).
  • antibodies are produced in response to the presence of an antigen or portion of an antigen.
  • antigen and “immunogen” are used to refer to an individual macromolecule or to a homogeneous or heterogeneous population of antigenic macromolecules. It is intended that the terms antigen and immunogen encompass protein molecules or portions of protein molecules, which contains one or more epitopes.
  • epitope refers to that fragment of a molecule that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antigen fragment and “portion of an antigen” and the like refer to a portion of an antigen.
  • Antigen fragments or portions typically range in size, from a small percentage of the entire antigen to a large percentage, but not 100%, of the antigen.
  • antigen fragments and/or portions therefrom comprise an "epitope" recognized by an antibody, and are therefore referred to as “immunoreactive fragments", while in other embodiments these fragments and/or portions do not comprise an epitope recognized by an antibody.
  • antigen fragments andlor portions are not immunogenic, while in other embodiments the antigen fragments and/or portions are immunogenic.
  • peptide As used herein, the terms “peptide,” “polypeptide” and “protein” all refer to a primary sequence of amino acids that are joined by covalent “peptide linkages.” In general, a peptide consists of a few amino acids, typically from 2-50 amino acids, and is shorter than a protein. The term “polypeptide” encompasses peptides and proteins. In some embodiments, the peptide, polypeptide or protein is synthetic, while in other embodiments, the peptide, polypeptide or protein is recombinant or naturally occurring. A synthetic peptide is a peptide which is produced by artificial means in vitro (i.e., was not produced in vivo).
  • sample is used in its broadest sense and encompasses samples or specimens obtained from any source.
  • sample is used to refer to biological samples obtained from animals (including humans), and encompasses fluids, solids, tissues, and gases.
  • biological samples include cerebrospinal fluid (CSF), serous fluid, urine, saliva, blood, and blood products such as plasma, serum and the like.
  • CSF cerebrospinal fluid
  • the sample comprises cells and more preferably, peripheral blood mononuclear cells (PBMC) isolated by centrifugation from a subject's blood.
  • PBMC peripheral blood mononuclear cells
  • a cell sample may be obtained from blood, muscle, connective tissue, brain or nerve tissue, and epithelial tissue.
  • a cell sample is considered essentially free of a specific cell type if less than 1 in 1000 cells of the sample are of that cell type.
  • beta amyloid- ⁇ (or "A ⁇ ") as used herein has the standard meaning understood in the art.
  • the full length beta amyloid precursor protein (APP) occurs in nature in several variants, up to 770 amino acids in length, with other characterized species including variants 695, 639, 574, 547, 484, 352, 327 and 305 amino acids in length.
  • Amyloid- ⁇ polypeptides may be of various lengths, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,
  • a ⁇ polypeptides are at least 80% homologous to the wild-type amyloid- ⁇ protein, or naturally occurring variants, over their length, preferably 85%, 90%, 95%, 99% or 100% homologous.
  • the A ⁇ polypeptides are 40 or 42 amino acids in length.
  • immunoassay refers to any assay that uses at least one specific antibody for the detection or quantitation of an antigen.
  • Immunoassays include, but are not limited to, Western blots, ELISAs, radio-immunoassays, and immunofluorescence assays.
  • the terms “Western blot,” “Western immunoblot” “immunoblot” and “Western” refer to the immunological analysis of protein(s), polypeptides or peptides that have been immobilized onto a membrane support.
  • the proteins are first resolved by polyacrylamide gel electrophoresis (i.e., SDS-PAGE) to separate the proteins, followed by transfer of the protein from the gel to a solid support, such as nitrocellulose or a nylon membrane.
  • the immobilized proteins are then exposed to an antibody having reactivity towards an antigen of interest.
  • the binding of the antibody i. e., the primary antibody
  • the secondary antibody is typically conjugated to an enzyme which permits visualization of the antigen-antibody complex by the production of a colored reaction product or catalyzes a luminescent enzymatic reaction.
  • radioassay includes any conventional means for determining the level of radioisotope associated with a sample. Such conventional means depend on the radioisotope employed, and may include without limitation for example, use of a gamma counter, scintillation counter, X-ray film exposure, development and densitometry (e.g., Western blotting), etc.
  • a "detection antibody” is an antibody which carries a means for visualization or quantitation, which is typically a conjugated enzyme moiety that typically yields a colored or fluorescent reaction product following the addition of a suitable substrate.
  • Conjugated enzymes commonly used with detection antibodies in the ELISA include horseradish peroxidase, urease, alkaline phosphatase, glucoamylase and ⁇ - galactosidase.
  • the detection antibody is directed against the antibody of interest.
  • the detection antibody is directed against the polypeptide of interest.
  • the detection antibody is prepared with a label such as biotin, a fluorescent marker, or a radioisotope, and is detected and/or quantitated using this label.
  • patient includes human subjects that are diagnosed to suffer from Alzheimer's disease.
  • Alzheimer's disease and “AD” refer to a neurodegenerative disorder and encompass familial Alzheimer's disease and sporadic Alzheimer's disease.
  • familial Alzheimer's disease refers to Alzheimer's disease associated with genetic factors (i.e., inheritance is demonstrated) while “sporadic Alzheimer's disease” refers to Alzheimer's disease that is not associated with prior family history of the disease.
  • Symptoms indicative of Alzheimer's disease in human subjects typically include, but are not limited to, mild to severe dementia, progressive impairment of memory (ranging from mild forgetfulness to disorientation and severe memory loss), poor visual spatial skills, personality changes, poor impulse control, poor judgment, distrust of others, increased stubbornness, restlessness, poor planning ability, poor decision making, and social withdrawal.
  • patients lose the ability to use language and communicate, and require assistance in personal hygiene, eating and dressing, and are eventually bedridden.
  • Hallmark pathologies within brain tissue include extracellular neuritic amyloid plaques, neurofibrillary tangles, neurofibrillary degeneration, granulovascular neuronal degeneration, synaptic loss, and extensive neuronal cell death.
  • the present invention now discloses that the rate of A ⁇ uptake in cells present in a sample of a subject is indicative for Alzheimer's disease.
  • This finding provides tools for diagnosing Alzheimer's disease in a subject, preferably a human subject, and methods for treating the disease, which are based on these diagnostic tools.
  • the present invention provides a method for the diagnosis of Alzheimer's disease, the method comprising: a) providing a sample from a subject; b) contacting the sample with A ⁇ , or a fragment thereof that is capable of being phagocytosed by a control sample, under conditions such that the A ⁇ or fragment thereof may be phagocytosed by a control sample; c) determining the presence of A ⁇ polypetide in said sample; wherein lower levels of A ⁇ polypeptide in said subject's sample relative to a control sample is indicative of Alzheimer's disease.
  • determination of the presence of A ⁇ in the sample may further comprise: (i) contacting the A ⁇ with a detection antibody such that the detection antibody binds to said A ⁇ ; and (ii) detecting the binding of said detecting antibody to said A ⁇ ; wherein said lower levels of A ⁇ polypeptide in said subject's sample relative to a control sample is indicative of Alzheimer's disease.
  • the diagnostic method of the present invention can be applied to subjects who have been previously diagnosed with Alzheimer's disease, those who are suspected of having Alzheimer's disease, and those at risk of developing Alzheimer's disease.
  • patients diagnosed with dementia in particular, those patients who were previously clinically normal, are suitable subjects.
  • the present invention be limited to use with any particular subject types.
  • the subject is a human subject. According to certain embodiments, the subject is selected from the group consisting of subjects displaying pathology resulting from Alzheimer's disease, subjects suspected of displaying pathology resulting from Alzheimer's disease, and subjects at risk of displaying pathology resulting from Alzheimer's disease.
  • the Alzheimer's disease diagnosed using the method of the present invention is selected from the group consisting of late onset Alzheimer's disease, early onset Alzheimer's disease, familial Alzheimer's disease and sporadic Alzheimer's disease.
  • the present invention provides a method of assessing efficacy of a treatment of Alzheimer's disease in a patient comprising: a) determining a baseline rate of A ⁇ uptake in a first sample obtained from the patient before receiving the treatment; b) determining the rate of A ⁇ uptake in a second sample obtained from said patient after receiving said treatment; wherein an increase in the amount of said A ⁇ uptake in the post-treatment sample is correlated with a positive treatment outcome.
  • the amount of A ⁇ uptake is determined in a sample obtained from a control population and in a sample obtained from a patient suffering from Alzheimer's disease and receiving a treatment, wherein a lack of significant difference between the amount of the A ⁇ uptake measured in a sample obtained after beginning of the treatment compared to the amount of said A ⁇ uptake in a sample obtained from the control population indicates a positive treatment outcome.
  • the amount of A ⁇ uptake for the control population may determined as a standard, wherein the amount of A ⁇ uptake in the patient's post-treatment sample could be compared with the standard in order to evaluate efficacy of treatment.
  • the methods of the present invention are useful for detecting early onset Alzheimer's disease and late onset Alzheimer's disease, as well as for detecting sporadic Alzheimer's disease and familial Alzheimer's disease.
  • the term "early-onset Alzheimer's disease” refers to Alzheimer's disease cases diagnosed as occurring before the age of 65.
  • late-onset Alzheimer's disease refers to Alzheimer's disease cases diagnosed as occurring after the age of 65.
  • the diagnostic method of the present invention is particularly useful for monitoring a course of treatment being administered to a patient.
  • the methods can be used to monitor both therapeutic treatment on symptomatic patients and prophylactic treatment on asymptomatic patients.
  • the diagnostic method of the present invention requires determining a baseline rate of A ⁇ uptake in a sample obtained from a patient before administering a dosage of the agent.
  • the baseline value is then compared with a rate of A ⁇ uptake after the treatment.
  • a significant change i.e., greater than the typical margin of experimental error in repeat measurements of the same sample, expressed as one standard deviation from the mean of such measurements
  • a significant change indicates a positive treatment outcome.
  • patients undergoing an initial course of treatment with an agent are expected to show an increase in the response with successive dosages, which eventually reaches a plateau.
  • Administration of agent is generally continued while the response is increasing.
  • Attainment of the plateau is an indicator that the administered treatment can be discontinued or reduced in dosage or frequency.
  • a control value i.e., a mean and standard deviation
  • rate of A ⁇ uptake is determined for a control population.
  • a lack of significant difference in the antibody concentration in a sample obtained from an AD patient relative to the value obtained in the control population indicates a positive treatment outcome.
  • kits for the detection of the rate of A ⁇ uptake are provided.
  • the kit is a fluorescence microspopy kit.
  • the kit is a flow cytometry kit.
  • the kits are customized for various applications.
  • kits of the present invention include, but are not limited to, materials for sample collection (e.g., spinal and/or vein puncture needles), tubes (e.g., sample collection tubes and reagent tubes), holders, trays, racks, dishes, plates (e.g., 96- well microtiter plates), instructions to the kit user, solutions or other chemical reagents, and samples to be used for standardization, and/or normalization, as well as positive and negative controls.
  • sample collection e.g., spinal and/or vein puncture needles
  • tubes e.g., sample collection tubes and reagent tubes
  • holders e.g., trays, racks, dishes
  • plates e.g., 96- well microtiter plates
  • aspects of the present invention further provide methods for the treatment of Alzheimer's disease comprising modulating the immune response of a patient towards aldolase.
  • a ⁇ , its fragments, analogs and fragment comprising an active epitope for phagocytosis can be synthesized by solid phase peptide synthesis, produced by recombinant expression system, or can be obtained from natural sources. Automatic peptide synthesizers are commercially available from numerous suppliers, for example Applied Biosystems, Foster City, Calif. Recombinant expression can be in bacteria, such as E. coli, yeast, insect cells or mammalian cells. Procedures for recombinant expression are described by Sambrook et al., Molecular Cloning: A Laboratory Manual (CS. H.P. Press, NY 2d ed., 1989). Some forms of A ⁇ are also available commercially.
  • one problem in treating patients suffering from Alzheimer's disease is the inaccuracy or delay in the disease diagnosis, resulting in a sub- optimal treatment regime.
  • embodiments of the present invention provide methods for the diagnosis of Alzheimer's disease and methods for treatment based on the diagnosis outcome. Accordingly, patients amenable to treatment include those diagnosed to have low levels of A ⁇ uptake.
  • Effective treatment regime depends upon many different factors, including means of administration, target site, physiological state of the patient, and other medications administered. Treatment dosages need to be titrated according to the desired outcome (induction or suppression of the immune response) to optimize safety and efficacy.
  • the timing of treatment employment can vary significantly according to the rate of A ⁇ uptake in a patient's sample after the treatment.
  • Agents for modulating A ⁇ uptake can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment.
  • the most typical route of administration is subcutaneous although others can be equally effective.
  • the next most common is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles.
  • Intravenous injections as well as intraperitoneal injections, intraarterial, intracranial, or intradermal injections are also contemplated.
  • Agents for modulating the uptake of A ⁇ may optionally be administered in combination with other agents that are at least partly effective in treatment of Alzheimer's disease.
  • the diagnostic tests described herein can also be used to screen and identify substances useful for the treatment or prevention of Alzheimer's disease. According to this embodiment, substances which reverse or improve the Alzheimer's disease-associated differences described herein (i.e. back to levels found in normal cells) would be identified and selected as substances which are potentially useful for the treatment of Alzheimer's disease.
  • one such method of screening therapeutic substances would involve the steps of contacting sample cells from an AD patient with a substance being screened and an A ⁇ polypeptide, and detecting the level of A ⁇ polypeptide uptake in the sample, wherein an increase in an abnormally low level of A ⁇ uptake associated with Alzheimer's disease cells indicates that the substance is potentially useful for the treatment or prevention of Alzheimer's disease.
  • the sample may be contacted with an A ⁇ polypeptide for various periods of time prior to determining the level of uptake.
  • Incubation with the A ⁇ polypeptide may range from about 0.5 to 24 hours, preferably 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours.
  • a ⁇ polypeptides may be labeled by numerous methods known in the art.
  • the peptides may be labeled with radioactive isotopes, fluorescent dyes or epitopes tags suitable for binding to detection antibodies.
  • the radioisotope may be, for example, 3H, 14C, 32P, 35S, or 1251.
  • Fluorescent dyes may be, for example, Fluorescein (FITC), Phycoerythrin (PE), Cy5PE , Cy7PE, Texas Red (TR), Allophycocyanin (APC), Cy5, Cy7APC, Cascade Blue.
  • Epitope tags may include, for example, biotin, FLAG, HIS, c-MYC, HA, VSV-G, HSV, and V5. Flow cytometry
  • Flow cytometry can be used to detect multiple immunofluorescent markers simultaneously in a quantitative manner.
  • the technique of immunofluorescent staining is well known and can be carried out according to any of a variety of protocols, such as those described in Current Protocols in Cytometiy (John Wiley & Sons, NY, NY, Eds. J. Paul Robinson, et a.).
  • a biological sample such as peripheral blood, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, spleen tissue, tumor tissue, and the like, is collected from a subject and cells are isolated therefrom using techniques known in the art.
  • blood is collected from a subject and any PBMCs are isolated.
  • the sample cells are washed with buffered saline and resuspended in buffered saline containing protein for introduction into a flow cytometer.
  • the flow cytometer analyzes the heterogeneous cell population one cell at a time and can classify the cells based on the binding of the immunofluorescent labeled protein or monoclonal antibody and the light scattering properties of each cell (see, for example, Immunol Today 2000; 21 (8):383-90).
  • Fluorescence detection is accomplished using photomultiplier tubes; the number of detectors (channels) determines the number of optical parameters the instrument can simultaneously examine while bandpass filters ensure that only the intended wavelengths are collected.
  • flow cytometry can routinely detect multiple immunofluorescent markers in a quantitative manner and can measure other parameters such as forward light scatter (which is an indication of cell size) and right angle light scatter (which is an indication of cell granularity). Accordingly, a wide variety of cell populations can be differentiated and sorted using immunofluorescence and flow cytometry.
  • a six dimensional data space can be generated wherein specific cell populations found in normal blood or bone marrow are restricted to small portions of the data space.
  • 4 colors of immunofluorescent markers could also be used: Excitation of fluoroflores is not limited to light in the visible spectrum; several dyes, such as the Indo series (for measuring intracellular calcium) and the Hoesch series (for cell-cycle analyses) are excitable in the ultraviolet range.
  • some instruments currently available in the art are configured with ultraviolet-emitting sources, such as the four-laser, 10-color Becton Dickinson LSR II.
  • ultraviolet-emitting sources such as the four-laser, 10-color Becton Dickinson LSR II.
  • a commercially available fluorescence activated cell sorter such as the FACSVantageTM (Becton Dickinson, San Jose, CA), the EPICS® ALTRATM (Beckman Coulter, Fullerton, CA) or the MoFlo® sorter (DakoCytomation, Inc., Carpinteria, CA) cell populations can also be sorted into purified fractions.
  • the abnormal cells are identified by flow cytometry as described herein, the cells are sorted and collected for further confirmatory genetic analysis.
  • a desired number of cells is collected using the parameters of the flow cytometer according to established protocols known to the skilled artisan and as described in the art, for example, in Current Protocols in Cytometry (John Wiley & Sons, NY, NY, Eds. J. Paul Robinson, et a!.).
  • the cells are collected into one or more drops of collection fluid. In one embodiment, single cells are collected in each small drop of collection fluid.
  • the drops containing the desired sorted, purified population of cells (fraction) are deposited into tubes, plates, or onto a solid support. Many distinct populations of cells can be sorted, as defined by immunofluorescent markers as described herein.
  • the sorted cells comprise B cells, T cells, NK cells, plasma cells, stem cells, granulocytes, basophils, or other cells found in the blood.
  • the number of sorted cells to be analyzed can be about 2000, 1500, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200 or fewer cells.
  • Part 3 Monocytes of control subjects show high uptake of FITC-A ⁇ by flow cytometry
  • sorted monocytes were collected by cytocentrifugation on a slide and prepared for immunofluorescence microscopy: whereas control monocytes displayed intracellular fluorescence, AD monocytes displayed surface staining (Fig. 1C). Surface uptake does not necessarily lead to effective phagocytosis. To distinguish surface from intracellular uptake, the method of phagocytosis testing was redirected to cytocentrifugation and fluorescence microscopy.
  • Monocytes of control subjects show a high intracellular uptake of FITC-A ⁇ by fluorescence microscopy
  • monocytes were incubated with cold A ⁇ and uptake was measured by indirect fluorescence using anti-A ⁇ staining.
  • the test was performed in 18 patients and 18 control subjects; the A ⁇ integrated optical density ("IOD") (calculated as area x density) was measured in the 3 individual monocytes with the largest, second largest, and third largest uptake in each specimen.
  • IOD A ⁇ integrated optical density
  • Significant differences were observed in each category: largest (mean area AD vs. control, 23.8 px vs. 337.4 px), second largest (mean area AD vs. control, 18.2 px vs. 193.5 px), and third largest uptake (mean area AD vs. control, 12.7 px vs. 117.2 px).
  • the analysis showed significantly higher uptake by control monocytes (P ⁇ 0.001).
  • AD biomarkers do not know precisely at which stage of the disease the patients develop these defects of A ⁇ phagocytosis. If, as hypothesized, defective phagocytosis is a link in the immunopathogenesis of AD, then these tests should serve as an early biomarker. We have, however, not excluded the possibility that the defects result from the disease and, therefore represent a late manifestation. Our testing of patients with minor cognitive impairment was likely to shed light on this question. Notwithstanding this question or causality or manifestation, our functional approach to AD biomarkers does not rely on identification of single proteins but takes into consideration the complex process of monocyte/macrophage maturation and phagocytosis.
  • the proteomic technique for detection of biomarkers in CSF has identified 136 CSF proteins unique to AD but encompassing disparate functions, which could be related to various, possibly secondary, mechanisms of the disease, such as inflammation, blood-brain barrier damage and apoptosis. It is desirable to compare the biomarkers reflecting innate immunity or proteomic differences at an early stage of the disease. We have undertaken studies to elucidate the molecular mechanisms responsible for defective phagocytosis. We have recently shown that, after A ⁇ stimulation, RNA for the glycosylating enzyme MGAT3 is not properly transcribed in AD patients' monocytes and macrophages.
  • a ⁇ (1-42) (“cold A ⁇ ") (California Peptide Research, Napa, CA), A ⁇ (1-42) conjugated with fluorescein isothiocyanate (“FITC-A ⁇ ”) (AnaSpec, San Jose, CA); mouse anti-human CD68 (KP-I) (DAKO, Carpinteria, CA); rabbit anti-A ⁇ (specific for 6 amino acid sequence from the C-terminal of human amyloid-beta 1-42) (Chemicon, Temecula, CA); mouse anti-human HLA DR (Becton Dickinson, Pharmingen, San Diego, CA); anti-CD 14- PE (BD Pharmingen, San Diego, CA); tetramethylrhodamine- phalloidin (Sigma, St.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • FITC-A ⁇ 2 ⁇ g/ml
  • the data were gated on monocytes, which were selected as the cells with greater forward scatter and side scatter voltages than lymphocytes.
  • the amplification gain and voltages were adjusted to comprise about 100% unstained cells in LL, about 100% FITC-stained cells in LR, and about 100%
  • PE-stained cells in UL Mean UR fluorescence was analyzed.
  • Control and AD PBMCs were sorted using BD FACSVantageSE sorting flow cytometer according to the FITC-A ⁇ signal intensity.
  • PBMCs were incubated in Iscove's modified Dulbecco's medium (GIBCO) with 10% autologous serum, penicillin (100U/ml)- streptomycin (100 ⁇ g/ml)-fungizone (2.5 ⁇ g/ml), and either cold A ⁇ (2 ⁇ g/ml) in the indirect fluorescence technique, or FITC-A ⁇ (2 ⁇ g/ml) in the FITC-A ⁇ test. After overnight incubation, PBMCs were collected by cytocentrifugation in a double cytology funnel (Fisher Scientific) at 500 RPM for 10 min (Cytospin 2, Thermo Shandon) on a glass slide.
  • GEBCO Iscove's modified Dulbecco's medium
  • the cells were stained using anti-A ⁇ /anti-rabbit ALEXA 488 and anti-CD68/anti-mouse ALEXA 594 and DAPI (1:300); in the FITC-A ⁇ test, using anti- CD68/anti-mouse ALEXA 594 and DAPI.
  • the cytospun preparations were examined using the 4Ox objective of the Olympus Bmax fluorescence microscope by scanning a midline vertical strip of the spot with cytospun cells for aggregated monocytes with FITC-A ⁇ . After locating the five largest aggregations (large aggregations were noticeable in control specimens but only small aggregations or single cells with FITC-A ⁇ in AD specimens (see Fig.
  • Reagents and labware provided for an 8 well chamber slide (one embodiment of the disclosed kit)
  • kits are designed to utilize about 5 mL to 10 mL of EDTA- anticoagulated blood and 10 mL of coagulated blood. Of course in other embodiments, other samples may be utilized.
  • PBMC peripheral blood mononuclear cells
  • Stain cells by adding 100 ⁇ L of diluted phalloidin-tetramethylrhodamine for 20 minutes.
  • Figs. 1 and 2 Based on Applicants results shown in Figs. 1 and 2, we expect that the macrophages from Alzheimer patients' will transport significantly less A ⁇ into intracellular locations. In contrast, we expect the macrophages from control subjects' will transport significant amounts of A ⁇ into intracellular locations, such as endosomes and lysosomes. Confocal microscopy can be used to determine the subcellular localization of the transported A ⁇ . Analysis of data We investigated FITC-Ab clearance by monocytes in culture using cytospin centrifugation and fluorescence microscopy. Control monocytes, which were exposed to Ab for 24 to 48h, increased in size, some with a display of dendrites on the surface, and formed multicellular aggregates surrounding globules of Ab.

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Abstract

L'invention concerne un procédé de diagnostic de la maladie d'Alzheimer ou de détection d'une prédisposition à la maladie d'Alzheimer, qui comprend le prélèvement d'un échantillon cellulaire test chez un sujet, l'exposition de l'échantillon cellulaire test à des peptides bêta amyloïdes marqués et la détermination du niveau de peptides bêta amyloïdes marqués dans l'échantillon cellulaire marqué. Selon l'invention, des niveaux de peptides bêta amyloïdes marqués plus faibles dans l'échantillon cellulaire test que dans un échantillon cellulaire témoin indiquent la maladie d'Alzheimer.
PCT/US2007/081989 2007-10-19 2007-10-19 Utilisation de biomarqueurs de la maladie d'alzheimer pour des tests de diagnostic et pour le criblage de médicaments WO2009051599A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2013020724A1 (fr) * 2011-08-05 2013-02-14 Glaxosmithkline Biologicals S.A. Composition contenant un agoniste de tlr et un anticorps spécifique à un antigène et utilisations de celle-ci en tant que vaccin
JP2014529087A (ja) * 2011-10-04 2014-10-30 アフィリス・アクチェンゲゼルシャフトＡｆｆｉｒｉｓ Ａｇ アルツハイマー病の診断方法

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MAGGIO ET AL.: "Reversible in vitro growth of alzheimer disease beta-amyloid plaques by depostion of labeled amyloid peptide.", PNAS., vol. 89, June 1992 (1992-06-01), pages 5462 - 5466 *
VENKITARAMANI ET AL.: "Beta amyloid modulation of synaptic transmission and plasticity.", J. NEUROSCIENCE., vol. 27, no. 44, 31 October 2007 (2007-10-31), pages 11832 - 11837 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013020724A1 (fr) * 2011-08-05 2013-02-14 Glaxosmithkline Biologicals S.A. Composition contenant un agoniste de tlr et un anticorps spécifique à un antigène et utilisations de celle-ci en tant que vaccin
JP2014529087A (ja) * 2011-10-04 2014-10-30 アフィリス・アクチェンゲゼルシャフトＡｆｆｉｒｉｓ Ａｇ アルツハイマー病の診断方法

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