WO2011036297A2

WO2011036297A2 - Method for the therapy and diagnosis of alzheimer's disease

Info

Publication number: WO2011036297A2
Application number: PCT/EP2010/064289
Authority: WO
Inventors: Helmut E. Meyer
Original assignee: Ruhr-Universität-Bochum
Priority date: 2009-09-27
Filing date: 2010-09-27
Publication date: 2011-03-31
Also published as: WO2011036297A3; EP2504024A2

Abstract

The invention relates to a method for the therapy and diagnosis of Alzheimer's disease, comprising receptor tyrosine kinases and/or membrane-bound cytokine receptors, including involved autoimmune antibodies.

Description

Method for the therapy and diagnosis of Alzheimer's disease

Besehreibung

The invention relates to a method for the therapy and diagnosis of Alzheimer's disease.

In Alzheimer's disease, cholinergic neurons are found in the brain stem (raphe nucleus) and in the cortex of the brain

Neurodegeneration. The first signs of this disease, as has recently been shown, occur in the raphe nucleus and are detectable post mortem (The dorsal raphe nucleus shows phospho-tau neurofibrillary changes before the

Transentorhinal region in Alzheimer's disease. A precocious onset? Grinberg LT, Rüb U, Ferretti RE, Nitrini R, Farfel JM, Polichiso L, Gierga K, Jacob-Filho W, Heinsen H; Brazilian Brain Bank Study Group. Neuropathol Appl Neurobiol. 2009 Aug; 35 (4): 406-16. ). In the cortex, the first signs of disease are first detectable in the entorhinal cortex and, as the disease progresses, they cover additional areas of the cortex (Staging of neurofibrillary pathology in Alzheimer's disease: a study of the BrainNet Europe Consortium.

Arzberger T, Al-Sarraj S, Bodi I, Bogdanovic N, Braak H, Bugiani 0, Del-Tredici K, Ferrer I, Gelpi E, Giaccone G, Graeber MB, Ince P, Kamphorst W, King A, Korkolopoulou P, Kovacs GG, Larionov S, Meyronet D, Monoranu C, Parchi P,

Patsouris E, Roggendorf W, Seilhean D, Tagliavini F,

Stadelmann C, Streichenberger N, Thal DR, Wharton SB,

Kretzschmar H. Brain Pathol. 2008 Oct; 18 (4): 484-96. Epub 2008 Mar 26.) · Characteristic damage in the affected

Nerve tissues are: a) hyperphosphorylation of microtubule-associated tau protein, formation of amyloid plaques, and paired helical filaments. As found in 1992 (The

Alzheimer-like phosphorylation of tau protein reduces

microtubule binding and includes ser-pro and thr-pro-motives. Gustke N, Steiner B, Mandelkow EM, Biernat J, Meyer HE,

Goedert M, Mandelkow E. FEBS Lett. 1992 Jul 28; 307 (2): 199-205; Phosphorylation-dependent epitopes of neurofilament antibodies on dew protein and relationship with Alzheimer's disease.

Lichtenberg Kraag B, Mandelkow EM, Biernat J, Steiner B,

Schröter C, Gustke N, Meyer HE, Mandelkow E. Proc Natl Acad Be U S A. 1992 Jun 15; 89 (12): 5384-8. ), the

Phosphorylation in the case of Alzheimer's typical

Hyperphosphorylation at MAP (mitogen-activated protein) kinase-specific phosphorylation sites (target sequence: serine-proline or threonine-proline), whereby specific serine or threonine residues in the tau protein are phosphorylated.

Antibodies that recognize these corresponding MAP kinase specific phosphorylation sites in the tau protein are described in U.S. Pat histopathological characterization to determine the severity and stage of Alzheimer's disease post mortem (Staging of neurofibrillary pathology in Alzheimer's disease: a study of the BrainNet Europe

Consortium. Alafuzoff I, Arzberger T, Al-Sarraj S, Bodi I, Bogdanovic N, Braak H, Bugiani 0, Del-Tredici K, Ferrer I, Gelpi E, Giaccone G, Graeber MB, Ince P, Kamphorst W, King A, Korkolopoulou P, Kovacs GG, Larionov S, Meyronet D, Monoranu C, Parchi P, Patsouris E, Roggendorf W, Seilhean D, Tagliavini F, Stadelmann C, Streichenberger N, Thal DR, Wharton SB, Kretzschmar H. Brain Pathol. 2008 Oct; 18 (4): 484-96. Epub 2008 Mar 26.).

Like investigations from the working group of Mandelkow &

Mandelkow (supra) showed, the microtubiii in the

Axons of the affected neurons are unstable due to hyperphosphorylation and can no longer or only to a limited extent perceive their function during vital axonal transport to the terminal synapses and back. In the event of complete loss of function, neurons affected in this way eventually die off and lead to increasing cognitive restrictions.

As described above, the tau protein

Phosphorylations in Alzheimer's disease mediated by the MAP kinase signal transduction pathway. The MAP kinase (s)

is itself initially activated by cell-membrane tyrosine receptor kinases through several intermediate steps. The Activation of these tyrosine receptor kinases occurs physiologically by their specific ligands such as not conclusive eg nerve growth factor (Trk-A =

Growth factor (hormone) receptor), brain-derived neurotrophic factor (TrkB and LNGFR, "low affinity nerve growth factor receptor" or p75) insulin (insulin receptor), insulin-like growth factors, IGF (IGF1R and IGF2R). In case of

membranous cytokine receptors, which include the human growth factor receptor, the activation of the signal cascade is initially via the activation of Janus kinase. The tyrosine receptor kinases (enzyme class EC 2.7.10.1) have in common that they phosphorylate themselves or specific substrate proteins on specific tyrosine residues; these

Finally, tyrosine phosphorylation via GRB2-SOS-ras-raf-MEK leads to the activation of MAP kinase (s). The membranous cytokine receptors are the

Activation first via the Janus kinase, which can lead to activation of the MAP kinase in the further course of the signal cascade.

So far it has been unknown how this process is triggered in the case of Alzheimer's disease and develops into a progressive neurodegenerative disease.

The invention shows for the first time that - analogous to some other autoimmune diseases described, eg Myastenia gravis with autoimmune antibodies to the acetylcholine receptor - in the case of Alzheimer's disease specific autoimmune antibodies are formed, which are against the extracellular

Hormone binding domain of human tyrosine receptor kinases or membrane-bound cytokine receptors are directed. These

Autoimmune antibodies bind to the corresponding ones

Ligand binding sites and solve for the natural

Ligand specific signal. Since autoimmune antibodies are not regulated in their amount or concentration like the natural ligands, they permanently trigger such a signal. Such a permanent, unregulated stimulation leads to the hyperphosphorylation of microtubular tau proteins in the affected axons of the damaged neurons. It can be assumed that the collapse or failure of the counter-regulatory processes ultimately leads to the death of the affected cholinergic neurons.

Autoimmune antibodies in Alzheimer's disease have been reported by Engelhardt et al. in 2000 (supra), which can specifically induce the death of cholinergic neurons in rats. A more detailed functional description or a

however, specific target could not be specified

Engelhardt JI, Le WD, Siklos L, Obal I, Boda K, Appel SH, Arch Neurol 2000 May, 57 (5): 681 -6). The invention is based on the recognition that specific autoimmune antibodies that are involved in the binding and stimulation of specific tyrosine receptor kinases and / or

membrane-bound cytokine receptors, which are causative triggers for Alzheimer's disease.

For the appropriate treatment of Alzheimer's disease, early diagnosis and differentiation are required in conjunction with the need to make clinical decisions.

A disadvantage of known diagnostic methods using the previously known diagnostics, however, is that early and complete detection of high-risk patients fails and therefore a diagnosis or therapy is insufficient or even too late.

It is therefore an object of the invention to develop a method for the therapy and diagnosis of Alzheimer's disease which enables an improved early diagnosis and improved detection of high-risk patients and improves the therapeutic success.

Therefore, an object of the invention is that tyrosine receptor kinases and / or membrane-bound cytokine receptors represent a target for a drug that is able to prevent contact of an autoimmune antibody at the tyrosine receptor kinases or membrane-bound cytokine receptors or the activation of the signal chain from the tyrosine receptor kinases or membrane-bound

Prevent cytokine receptors.

In the context of this invention, under "tyrosine receptor

Kinases "or" membrane-bound cytokine receptors "are not understood to be complete, as in the sequences SEQ ID No. 1 to SEQ ID No. 179 or its fragments or partial peptides, wherein SEQ ID No. 1 to SEQ ID No. 128 the

"Tyrosine Receptor Kinases" and SEQ ID No. 129 to SEQ ID No. 179 are to be assigned to the "membrane-bound cytokine receptors". Furthermore, the former are in the enzyme class EC 2.7.10.1

disclosed .

Furthermore, the invention also relates to such amino acid sequences (polypeptides, proteins), which have a sequence identity or

Homology of 70% and more, preferably 80% and more, more preferably 90-95% and more with SEQ ID NO. 1 to SEQ ID No. 179 and have the function of a "tyrosine

Receptor kinases "or" membranous cytokine receptors ". Also included are those analogous amino acid sequences that are due to the exchange of one or more multiple amino acid (s) in these sequences, yet have the desired function of a "tyrosine receptor kinases" or "membrane-bound cytokine receptors".

Furthermore, the "tyrosine receptor kinases" or

"Membrane-bound cytokine receptors" are prepared synthetically, in particular in the form of a fusion peptide. Such fusion peptides are known in the art, such as GST, cellulose-binding domain, green fluorescent protein, maltose

binding protein or LacZ, in particular in the form of a tag, which tag may be His-tag, FLAG, c-myc, alkaline phosphatase, EpiTAG, VS tag, T7 tag, Strep tag or the like. Therefore, the invention also relates to such fusion peptides, the at least one sequence of SEQ ID o. 1 to SEQ ID No. 179 or parts or fragments thereof.

Therefore, the invention relates to a target or its use for a medicament for the prophylaxis and treatment of Alzheimer's disease selected from tyrosine receptor kinases and / or membrane-bound cytokine receptors, in particular selected from the sequences SEQ ID No. 1 to SEQ ID No. 179. Such a target binds with specific Alzheimer's disease

Autoantibodies and represents an inhibitor for drugs, as described above, so that autoantibodies can not bind to this target. In a further embodiment, the invention relates to a method for the identification of Alzheimer's disease

specific autoimmune antibodies, wherein a sample or

Substance with tyrosine receptor kinases and / or

membrane-associated cytokine receptors are added and a binding success is detected. By "offsetting" is also meant to bring in contact.

Therefore, the invention also relates to an assay or

Screening system for the identification of Alzheimer's disease-specific autoimmune antibodies, wherein tyrosine receptor

Kinases or membrane-bound cytokine receptors are presented in vitro and tested with a sample or substances.

These substances or samples are preferably made

Body fluids obtained from humans and animals, in particular tissue samples or body fluid, such as blood, plasma, serum, cerebrospinal fluid. Also possible are synthetic, non-synthetic libraries, antibody libraries that may contain any suitable substances or samples.

Therefore, the invention relates to an assay system which

at least one tyrosine receptor kinase or membrane-bound Having cytokine receptor and is mixed with a substance or sample and a binding success is detected.

To prove a binding success, the usual

Detecting agents are used (e.g., radioactive

Labeling, fluorescence and the like relevant known methods).

An assay or screening system may be, for example, the

tyrosine receptor kinases according to the invention or

membrane-bound cytokine receptors, in particular at least one of the sequences SEQ ID NO. 1 to SEQ ID No. 179 or parts or fragments thereof, also in the form of a

Fusion peptides, which are arbitrarily immobilized or fixed on a surface. The surface is particularly suitable nitrocellulose or the like. Furthermore, beads or spheres can be used and these are introduced, for example, in a preparative column.

The method according to the invention can be carried out using such an assay or corresponding device according to the invention.

In a further embodiment, the invention relates

Diagnostics, wherein the tyrosine receptor kinases or membrane-bound cytokine receptors according to the invention, in particular at least one of the sequences SEQ ID NO. 1 to SEQ ID No. 179 or parts or fragments thereof, represent such a diagnostic. With the aid of these diagnostic agents, Alzheimer's disease-specific autoimmune antibodies can be detected, for example, by means of an assay or screening system according to the invention.

In a further embodiment, the invention relates to the apheresis of Alzheimer's disease specific

Autoimmune antibodies in the body fluids of a patient by means of the tyrosine receptor kinases or membrane-bound cytokine receptors according to the invention, in particular at least one of the sequences SEQ ID NO. 1 to SEQ ID No. 179 or parts or fragments thereof, also in the form of a fusion peptide, for binding the specific Alzheimer's disease autoimmune antibodies.

Therefore, the invention relates to an apheresis method for

Removal of specific Alzheimer's disease

Autoimmune antibodies, where body fluids of a

Patients with tyrosine receptor kinases or membrane-bound cytokine receptors, in particular at least one of the sequences SEQ ID NO. 1 to SEQ ID No. 179 or a fusion peptide thereof, or parts or fragments thereof, is brought outside the human body or animal into contact and the

Body fluid is returned to the human or animal. Furthermore, the invention relates to a medicament for the prophylaxis or treatment of Alzheimer's disease by means of

Tyrosine receptor kinases or membranous

Cytokine receptors, in particular at least one of the sequences SEQ ID NO. 1 to SEQ ID No. 179 - or in the form of a

Fusionspeptids, or parts and fragments thereof (collectively "active ingredients"), human or animal administered / administered

compete with the native human tyrosine receptor kinases or membrane-bound cytokine receptors for the binding site of the specific Alzheimer's disease autoantibodies and thus at least lead to a reduction or even

Removal of specific Alzheimer's disease

Autoimmune antibodies.

The active compounds according to the invention can be in the form of

pharmaceutical preparations in dosage units

be prepared. This means that the preparation is in the form of individual parts, for example tablets, dragees, capsules, pills, suppositories and ampoules, whose active substance content corresponds to a fraction or a multiple of a single dose. The dosage units may contain, for example, 1, 2, 3 or 4 single doses or 1/2, 1/3 or 1/4 of a single dose. A single dose preferably contains the amount Active substance which is administered in one application and which usually corresponds to one, a half, a third or a quarter of a daily dose.

Among non-toxic, inert pharmaceutically acceptable

Carriers are solid, semi-solid or liquid

To understand diluents, fillers and formulation aids of any kind.

Preferred pharmaceutical formulations are tablets, dragees, capsules, pills, granules, suppositories, solutions, juice, suspensions and emulsions, pastes, ointments, gels,

Creams, lotions, powders and (nose) called sprays. Tablets, dragees, capsules, pills and granules may contain the active ingredient (s) in addition to the usual excipients, such as a) fillers and extenders, e.g. Starches, lactose, cane sugar, glucose, mannitol and silicic acid, b) binders, e.g.

Carboxymethylcellulose, alginates, gelatin,

Polyvinylpyrrolidone, c) humectants, e.g. Glycerine, d) disintegrants, e.g. Agar-agar, calcium carbonate and

Sodium carbonate, e) dissolution inhibitor, e.g. Paraffin and f) absorption enhancer, e.g. quaternary ammonium compounds, g) wetting agents, e.g. Cetyl alcohol, glycerol monostearate, h) adsorbents, e.g. Kaolin and bentonite and i)

Lubricants, for example talc, calcium and magnesium stearate and solid polyethylene glycols or mixtures of the substances listed under a) to i). The tablets, dragees, capsules, pills and granules can be mixed with the usual, optionally opacifying

and may also be composed in such a way that they release the active substance only or preferably delayed in a certain part of the intestinal tract, optionally delayed, as embedding masses e.g. Polymer substances and waxes can be used. The active ingredient (s) may optionally also be incorporated with one or more of the excipients listed above

microencapsulated form.

The term "Morbus Alzheimer" includes Alzheimer's disease or synonym dementia of the Alzheimer's type, namely a primary degenerative brain disease with progressive dementia usually beginning in the second half of life;

Pathologically / anatomically, a macroscopic cerebral atrophy is observed, especially in the cerebral cortex in the fronto-temporal and parieto-occipital areas. Biochemically u. a. a disorder of the cortical cholinergic system with a

Reduction of choline acetylase (reduced

Acetylcholine synthesis) detectable. Symptoms are initially memory disorders; in the further course there are restlessness,

Orientation disorders, aphasia, agnosia, apraxia,

Irritability, delusion, hallucinations or depression

Foreground. Further, on the above-described Explanations and literature referenced.

The invention also relates to the identification of

Patients at increased risk and / or unfavorable

Prognosis of Alzheimer's disease, especially at

symptomatic and / or asymptomatic patients whose body fluid is assayed for an assay of the invention and corresponding autoimmune antibodies

be identified.

Therefore, the invention also relates to a method for

Diagnosing patients with Alzheimer's disease to make clinical decisions, such as advanced treatment and drug therapy.

In a further preferred embodiment of the

According to the method of the invention, the diagnosis for

Prognosis, for differential diagnostic early detection and detection, for the assessment of severity and for

Therapy-accompanying course assessment of Alzheimer's disease.

In one embodiment of the method according to the invention, the patient to be examined tissue samples or

Body fluid (blood, plasma) taken, and the diagnosis is in vitro / ex vivo, ie outside the human or animal body.

Another task is to provide a

corresponding diagnostic device for carrying out the method according to the invention.

In the context of this invention is under such

diagnostic device, in particular an array or assay (e.g., immunoassay, ELISA, etc.), in particular a protein biochip (US6346413B1, US20050014292) in the broadest sense, an apparatus for performing the invention

Procedure.

The invention additionally relates to a kit for carrying out the methods according to the invention, in particular containing

Detecting reagents and other auxiliaries. Such

Detecting reagents include e.g. Antibodies etc.

The detection and quantification of autoimmune antibodies can also be carried out with the aid of further protein diagnostic methods familiar to the person skilled in the art, in particular using radioactively or fluorescently labeled antibodies

Antibody. These are particularly suitable

bioanalytical methods, such as immunohistochemistry, antibody arrays, Luminex, ELISA, immunofluorescence, Radioimmunoassays and other suitable bioanalytical methods, such as mass spectrometric methods, eg MRM (Multi Reaction Monitoring) or AQUA (absolute

Quantification), with the aid of which the autoimmune antibodies can be quantitatively measured and the respective detection can be carried out.

The following examples serve to illustrate the invention without restricting the invention to these examples.

Example - Preparation of protein arrays (protein chips) for the analysis of blood samples

Design, manufacture and application of a protein array

extracellular domains of tyrosine kinase and cytokine receptors for the identification of autoantibodies in the

CSF and serum from Alzheimer's patients and controls.

Production of proteins (potential autoantigens):

The protein domains are produced as recombinant fusion proteins. The fusion part includes one

Amino acid sequence that is both efficient

Affinity purification as well as the targeted detection of

Fusion protein, e.g. in a Western blot

Analysis. Established candidates for this are the glutathione-S Transferase (GST) or the RGSHis6 tag. Detection takes place via corresponding antibodies available on the market. These are established standard methods of

Molecular biology. The coding sequences of

Protein domains can either be available from the market

Sequences derived or synthesized.

These are then cloned into an expression vector and, if not present, fused therein with the coding DNA sequence of the additional amino acid sequence.

Production of Protein Arrays (Protein Chips):

The proteins expressed in in vitro or in cell culture are purified by affinity chromatography. The

Concentration of the purified proteins is e.g. determined by amino acid analysis and adjusted to the desired value. The proteins are applied by a spotting robot to a suitable support material, for example: nitrocellulose or PVDF membranes or glass or plastic substrates with a modified, protein-binding surface by means of contact or non-contact and immobilized. These products are well established and widely available in the market.

Carrying out the blood analyzes:

The surface of the protein arrays is blocked immediately after production or before use (eg: with a Solution of milk powder or bovine serum albumin). The

excess blocking reagent is washed away. One

defined volume of serum or plasma (e.g., 10 or 100 pL) is diluted with binding buffer in a defined ratio (e.g., 1: 100 or 1: 500) and a blocked protein array is hereby placed in a suitable container (e.g.

Tube) over a defined period of time (for example: lh or 12 h). The supernatant is then removed and the

Protein array thoroughly washed with washing buffer and

then with a labeled secondary antibody in a defined concentration (e.g., 100 pg / mL) and in a defined volume (e.g., 5 mL) over a defined concentration

Period (for example: 1 h) incubated. The secondary antibody recognizes and binds human antibodies (e.g., IgG, IgM or IgG and IgM). The label enables its detection, e.g.

by fluorescence (bound fluorescent dye). The supernatant is removed and the protein array washed thoroughly with wash buffer. After that, the remaining ones

detected secondary antibody, e.g. with a

Fluorescence scanner. This results in a quantitative or semiquantitative signal for each sample immobilized on the array (potential autoantigen and controls).

Finding disease-specific markers:

The experiment described is using blood samples Alzheimer's patient cohort (eg 100) as well as a comparable large selection of healthy and diseased controls

carried out. For each potential autoantigen, the recorded signals are normalized and groupwise

compared. For this purpose, special, established statistical methods (for example: M statistics, T-test) and / or

Classification method (e.g. SVM, Support Vector Machine). Alzheimer-specific diagnostic and / or pathologically relevant candidates are proteins whose signals are in the comparative study in the patients concerned

were statistically significantly increased compared to controls.

In this way, the sequences of the invention SEQ ID No. 1-179 can be detected.

Claims

claims

1. Target for a medicament for the prophylaxis and treatment of Alzheimer's disease selected from tyrosine receptor kinases and / or membrane-bound cytokine receptors.

2. Target according to claim 1, characterized in that the target of at least one SEQ ID o. 1 to SEQ ID No. 179 is selected.

3. Assay or screening system containing tyrosine receptor kinases and / or membrane-bound cytokine receptors for the diagnosis of Alzheimer's disease and other adjuvants.

4. assay or screening system according to claim 3, characterized

in that the tyrosine receptor kinases and / or membrane-bound cytokine receptors consist of at least one SEQ ID No. 1 to SEQ ID No. 179 or one

Fusion peptide thereof, or parts or fragments thereof.

5. Assay or screening system for the identification of

Alzheimer's specific autoimmune antibodies, wherein human tyrosine receptor kinases and / or

Membrane-containing cytokine receptors according to claim 3 or 4 are presented in vitro and with a sample or

Substances are tested. Medicaments for the prophylaxis and treatment of Alzheimer's disease selected from tyrosine receptor kinases and / or membrane-bound cytokine receptors.

Medicaments for the prophylaxis and treatment of Alzheimer's disease according to claim 6, characterized in that tyrosine receptor kinases and / or membrane-bound cytokine receptors of at least one SEQ ID o. 1 to 179 or a fusion peptide thereof, or parts or

Fragments thereof is selected.

Diagnostics for the diagnosis of Alzheimer's disease, characterized in that tyrosine receptor kinases and / or membrane-bound cytokine receptors from at least one SEQ ID NO. 1 to 179 or a fusion peptide thereof, or parts or fragments thereof.

Apheresis method for the removal of specific Alzheimer's disease autoimmune antibodies, wherein body fluids of a patient tyrosine receptor kinases and / or membrane-bound cytokine receptors, in particular

at least one of the sequences SEQ ID NO. 1 to SEQ ID No. 179 or a fusion peptide thereof, or parts or fragments thereof, is brought into contact outside the human body or animal.

10. A method for identifying Alzheimer's specific autoimmune antibodies, wherein a sample or substance with tyrosine receptor kinases and / or

membranous cytokine receptors, in particular

at least one of the sequences SEQ ID NO. 1 to SEQ ID No. 179 or a fusion peptide thereof, or parts or fragments thereof, and a binding success is detected.

11. Diagnostic device for performing a

Method according to claim 10, in particular a

Protein biochip, array or assay.

12. Kit containing tyrosine receptor kinases and / or

membranous cytokine receptors, in particular

at least one of the sequences SEQ ID NO. 1 to SEQ ID No. 179 or a fusion peptide thereof, or parts or fragments thereof, for the diagnosis of Alzheimer's disease containing detection reagents and other adjuvants.