JP2008531635A - Screening method, purification method of non-diffusible Abeta oligomer, selective antibody against the non-diffusible Abeta oligomer, and method for producing the antibody - Google Patents

Abstract

  The present invention relates to a non-diffusible spherical Aβ (X-38..43) oligomer (“globulomer”) or a derivative thereof, a method for concentrating the globulomer or derivative, a composition containing the globulomer or derivative, and the globulomer or derivative. Specific antibodies and aptamers, methods for producing such antibodies and aptamers, use of said globulomer or derivative or said antibody or aptamer for diagnostic, therapeutic and other purposes, and said globulomer or derivative or said antibody or It relates to the corresponding use of aptamers.

Description

The present invention provides non-diffusible globular Aβ (X-38..43) oligomers (“globulomers”) or derivatives thereof, methods of concentrating said globulomers or derivatives, compositions comprising said globulomers or derivatives, said globulomers or derivatives. antibodies and aptamers having specificity against, methods for producing such antibodies and aptamers, uses of said globulomers or derivatives or said antibodies or aptamers for diagnostic, therapeutic or other purposes, and of said globulomers or derivatives or said antibodies or aptamers It relates to corresponding methods of use.

Alzheimer's disease (AD) is a common disease characterized by progressive cognitive loss and characteristic neuropathological features including extracellular amyloid deposits, intracellular nerve fiber tangles and neuronal loss in several brain regions. It is a neurodegenerative and dementing disorder (Mattson, M. P. Pathways towards and away from Alzheimer's disease. Nature 430, 631-639 (2004); Hardy, J. & Selkoe, D.J. The amyloid hypothesis of Alzheimer's disease. : progress and problems on the road to therapeutics. Science 297, 353-356 (2002)). The major constituents of amyloid deposits are amyloid β peptides.

One of the hallmarks of pathology in Alzheimer's disease is the excessive formation of amyloid-β (Aβ) 1-42, the major antibody of aggregated and characteristic plaques. Aβ(1-42) is derived from the amyloid precursor protein (APP) and aberrant processing of APP resulting in increased production of Aβ(1-42) has been the subject of intensive research in the past. Hardy, J.A. & Higgins, G,A. Alzheimer's disease: the amyloid cascade hypothesis. Science 256, 184-185 (1992). We hypothesize that increased production causes them to aggregate into large-diameter fibrils (starting from so-called protofibrils) and finally to plaque-like deposits, and that fibrillar Aβ deposits are the reason for the neuropathological symptoms in Alzheimer's disease. was assumed to be Although this hypothesis was the most favored until recently, the correlation between dementia and amyloid plaque burden is rather poor in AD patients (Katzman, R. et al. Clinical, pathological, and neurochemical changes in dementia: a subgroup with preserved mental status and numerous neocortical plaques. Ann Neural 23, 138-144 (1988); Naslund, J. et al. Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. JAMA 283, 1571- 1577 (2000)). Of note, similar findings were obtained in Down syndrome, suggesting the involvement of the same mechanisms.

Initially reported simply as intermediates in the process of fibril and plaque formation, oligomers have recently been discussed as important toxic species with AD pathology. Kuo, Y. M. et al. Water-soluble Abeta (N-40, N-42) oligomers in normal and Alzheimer disease brains. J Biol Chem 271, 4077-4081 (1996) ) correlates better with AD symptoms than with plaque burden (Kuo, Y.M. et al. Water-soluble Abeta (N-40, N-42) oligomers in normal and Alzheimer disease brains. J Biol Chem 271, 4077-4081 (1996); McLean, CA. et al. Soluble pool of Abeta amyloid as a determinant of severity of neurodegen-eration in Alzheimer's disease. Ann Neurol 46, 860-866 (1999)). This led to the revised amyloid cascade hypothesis (Hardy, J. & Selkoe, D.J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353-356 (2002)).

Since the very strong correlation between cortical levels of prefibrillar soluble Aβ and the degree of neuronal loss and severity of cognitive deficit was first observed, Aβ(1-42) globulomers rather than insoluble fibrillar aggregates have been observed. The potential for soluble aggregates to induce early neuronal changes in AD has received much attention (McLean, CA. et al. Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's Ann Neural 46, 860-866 (1999); Lue, L.F. et al. Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer's disease. Am J Pathol. 155, 853-862 (1999); Wang, J., Dickson, D.W., Trojanowski, J.Q. & Lee, V.M. The levels of soluble versus insoluble brain Abeta distinguish Alzheimer's disease from normal and pathologic aging. Exp Neurol 158, 328-337 (1999)).

Recent studies have revealed that soluble and oligomeric forms of Aβ can be generated in vitro (Lambert, M. P. et al. Diffusible, nonfibrillar ligands derived from Abeta 1-42 are potent central nervous system neurotoxins. Proc. Sci U.S.A 95, 6448-6453 (1998); Walsh, D.M. et al. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539 (2002)). Some of these soluble and oligomeric forms of Aβ were found to be toxic to neurons upon specific binding to synaptic spines (Lacor, P. N. et al. Synaptic targeting by Alzheimer's-related amyloid). β oligomers. J Neurosci 24, 10191-10200 (2004)). This specific interaction may be involved in the observed inhibition of hippocampal long-term potentiation (LTP), an experimental paradigm of synaptic plasticity and memory (Lambert, M. P. et al. Diffusible, nonfibrillar ligands derived from Abeta 1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci U. S. A 95, 6448-6453 (1998); Walsh, D. M. et al. vivo. Nature 416, 535-539 (2002)).

Further evidence for the critical role of soluble Aβ forms in AD pathogenesis comes from reports that cognitive deficits develop well before the deposition of insoluble Aβ in mice transgenic for human APP (Buttini et al. , M. et al. Modulation of Alzheimer-like synaptic and cholinergic deficits in transgenic mice by human apolipoprotein E depends on isoform, aging, and overexpression of amyloid beta peptides but not on plaque formation. J Neurosci 22, 10539-10548 (2002) Hsia, A. Y. et al. Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc. Natl. Acad Sci U. S. A 96, 3228-3233 (1999); of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation. J Neurosci 20, 4050-4058 (2000)). Since then, many researchers have used synthetic Aβ preparations to model soluble Aβ-mediated neurotoxicity (Walsh, D. M. & Selkoe, D.J. Deciphering the molecular basis of memory failure in Alzheimer's disease. Neuron 44, 181-193 (2004)).

However, the inherent propensity of Aβ peptides (particularly Aβ(1-42)) to rapidly aggregate in aqueous solution into various polymeric structures such as soluble oligomers, protofibrils and fibrils (Stine, W. B., Jr., Dahlgren, K.N., Krafft, GA & LaDu, M. J. In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J Biol Chem 278, 11612-11622 (2003)) demonstrated that the observed neurotoxic effects were characterized by one specific form of oligomerization. Aβ association could not be attributed. Accordingly, several groups have recently attempted to isolate soluble Aβ(1-42) aggregates and test their neurotoxic activity. Lambert, M. P. et al. Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. 1-42) describe a mixture of aggregates (4 to 18 kDa by SDS-PAGE), which they term "Aβ-derived diffusible ligands" (ADDLs). They were able to show that ADDLs at concentrations as low as 500 nM caused neuronal death in cell cultures and almost completely blocked LTP (Lambert, MP. et al. Diffusible, nonfibrillar ligands derived from Abeta 1 -42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci U. S. A 95, 6448-6453 (1998); Wang, H. W. et al. term depression in rat dentate gyrus. Brain Res 924, 133-140 (2002)).

In another non-synthetic approach, Aβ oligomers were released into the medium by cell lines expressing mutated human APP (Walsh, D.M. et al. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539 (2002); Podlisny, M. B. et al. Aggregation of secreted amyloid beta-protein into sodium dodecyl sulfate-stable oligomers in cell culture. J Biol Chem 270, 9564-9570 (1995)). Aliquots of this conditioned medium containing uncharacterized Aβ(1-42) oligomers blocked LTP in vivo and in vitro at low nanomolar concentrations (Walsh, D.M. et al. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539 (2002); Wang, Q., Walsh, D.M., Rowan, M. J., Selkoe, D.J. & Anwyl, R. Block of long-term potentiation by naturally secreted and synthetic amyloid beta-peptide in hippocampal slices is mediated via activation of the kinases c-Jun N-terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5. J Neurosci 24, 3370-3378 (2004)).

WO 98/33815 and WO 01/10900 (G.A Krafft et al.) describe specific diffuse amyloid beta 1-40- or 1-42-derived dementia as a neurotoxic principle to selectively block long-term potentiation (LTP). Sexual ligands, so-called ADDLs (amyloid beta (Aβ)-derived diffusible ligands) have been described. According to WO98/33815 and WO01/10900 ADDLs are neurotoxic species and the presence of ADDLs in the brain is a causative factor in the development and progression of the major symptoms of Alzheimer's disease (AD) and is therefore a major risk factor. is suggested. The relevance of Aβ oligomers in AD pathology has been highlighted by their detection in the brain of AD patients (Gong, Y. et al. Alzheimer's disease-affected brain: presence of oligomeric Aβ ligands (ADDLs) suggests a Proc. Natl. Acad. Sci U.S.A 100, 10417-10422 (2003)).

Used in Lambert et al., 1998, Walsh et al., 2002, Gong et al., 2003, WO98/33815 and WO01/10900. The term "diffusible" is used to describe the characteristic features of ADDLs (oligomeric Aβ(1-42) or Aβ(1-40)) such that oligo- or poly-Aβ (oligo- or poly-Aβ ( 1-42) or Aβ(1-40) fibrils that diffuse rapidly (A.M. Manelli et al., Journal of Molecular Neuroscience, 2004, Vol. 23, 235-246). Similarly, after intracerebroventricular injection, "diffusible" oligomers were found to spread in fluid and penetrate subsurface layers of the CNS. Despite these reports, experimental approaches to the pathology and function of Aβ oligomers are still difficult because of the current unavailability of well-defined, pure and stable preparations. A pathological structure could not be confirmed.

The neuropathological effects have been attributed to a single distinct oligomeric Aβ species since WO 2004/067561 (Abbott GmbH & Co. KG) published novel and highly stable Aβ(1-42) oligomeric species. It was after describing about.

Because of the homogeneity and characteristic globular spatial structure of this oligomeric Aβ species, it was termed "Aβ(1-42) globulomer". Aβ(1-42) globulomers can be easily and reproducibly formed in vitro from synthetic Aβ peptides. Physicochemical characterization shows that a highly soluble protein of approximately 60 kDa in size, which is a potent antigen in mice and rabbits, results in the development of Aβ(1-42) globulomer-specific antibodies that do not cross-react with amyloid precursor protein. Highly sensitive globular Aβ(1-42) oligomers (“Aβ(1-42) globulomers”) have been demonstrated.

Surprisingly and unexpectedly, said soluble globular Aβ(1-42) oligomers (“globulomers”) described in WO2004/067561 (Abbott GmbH & Co., KG) are specific to neuronal dendrites. but not specifically to glia in hippocampal cell cultures. The binding to tissue is very strong and is readily removed after intracerebroventricular injection in rats. Consequently, within current detection limits, no Aβ(1-42) globulomers were detected in the cerebrospinal fluid of AD patients. Surprisingly and unexpectedly, it was found that the dementing Aβ(1-42) globulomers according to WO2004/067561 (Abbott GmbH & Co. KG) are the toxic principles involved in the development and progression of Alzheimer's disease. /33815 and is characterized by the character of "non-diffusible" rather than "diffusible" as assumed by /33815 and similar publications above. The memory impairment potency of non-diffusible Aβ(1-42) globulomers is demonstrated by complete blockade of long-term potentiation (LTP) in rat hippocampal slices. Therefore, selective neutralization of Aβ(1-42) globulomers is expected to be highly effective in treating AD.

We therefore speculate that this particular non-diffusible globular oligomer is formed by a novel aggregation pathway independent of Aβ fibril formation and represents a novel pathological entity in Alzheimer's disease. ing. Because it is present in the brains of AD patients and APP transgenic mice, and also specifically binds to neurons and blocks LTP, we discovered that this globular Aβ oligomer, characterized by novel structural epitopes believe that it offers unprecedented potential to elucidate the neuropathology associated with Aβ oligomers and to clinically address the deficits in AD patients with specific therapies.

Furthermore, we have shown that by using anti-Aβ globulomer antibodies such as polyclonal antibody 5598 or monoclonal antibody 8F5 in vivo, cross-reactive species are not restricted to the Aβ(1-42) sequence and other Aβ It could be shown that it can also be extended to (x-y) species, eg Aβ(1-40) and Aβ(1-38).

Accordingly, the present invention provides nondiffusible globular Aβ (X-38..43) oligomers (“globulomers”) or derivatives thereof, wherein X is the number 1 . . selected from the group consisting of 24.

As used herein, the abbreviation A. . B refers to the set containing all natural numbers from A to B inclusive, eg "17..20" refers to the group of numbers 17, 18, 19 and 20. A hyphen refers to a contiguous sequence of amino acids, i.e., "X-Y" includes the sequence from amino acid X to amino acid Y, inclusive. Thus "A..B-C..D" includes all possible combinations between members of these two sets, e.g. , 17-41, 17-42, 18-40, 18-41, 18-42, 19-40, 19-41, 19-42, 20-40, 20-41 and 20-42. All numbers refer to the start of the mature peptide and 1 refers to the N-terminal amino acid, unless otherwise indicated. The term "A[beta](X-A..B)" is synonymous with the term "A[beta](X-A/B)" and can be used interchangeably therewith.

According to certain embodiments, the non-diffusible globular oligomers or derivatives thereof are selected from the group consisting of non-diffusible globular Aβ (X-40) oligomers and non-diffusible globular Aβ (X-42) oligomers or derivatives thereof. , and X is Eq. . 24, preferably the number 12 . . 24, more preferably the number 12 . . 20, most preferably the number 17 . . selected from the group consisting of 20;

According to another particular embodiment, the non-diffusible globular oligomers or derivatives thereof include non-diffusible globular Aβ (X-38) oligomers, non-diffusible globular Aβ (X-39) oligomers and non-diffusible globular Aβ ( X-41) selected from the group consisting of oligomers or derivatives thereof;

According to yet another particular embodiment, the non-diffusible globular oligomers or derivatives thereof are selected from the group consisting of non-diffusible globular Aβ (X-43) oligomers or derivatives thereof.

Derivatives of nondiffusible globular Aβ(X-38..43) oligomers especially include crosslinked, preferably chemically crosslinked, more preferably aldehyde crosslinked, most preferably glutardialdehyde crosslinked and nondiffusible spherical Aβ(X-38..43) oligomers.

Still other derivatives of non-diffusible globular Aβ(X-38..43) oligomers may include groups that facilitate detection, preferably such as fluorescein isothiocyanate, phycoerythrin, Aequorea victoria fluorescent protein, gimbo fluorescent protein or combinations thereof fluorophores such as fluorescently active derivatives; chromophores; chemiluminescers such as e.g. luciferase, preferably Photinus filaris luciferase, Vibrio fischeri luciferase or combinations or chemiluminescent derivatives thereof; e.g. peroxidases (e.g. enzymatically active groups such as horseradish peroxidase) or enzymatically active derivatives thereof; electron-dense groups such as heavy metal-containing groups (e.g. gold-containing groups); haptens such as phenol-derived haptens; aptamers to other molecules; chelating groups such as hexahistidinyl a naturally occurring or naturally derived protein structure that mediates another specific protein-protein interaction such as a member of the fos/jun pair; a magnetic group such as a ferromagnetic group; or ¹ H, ¹⁴ C, ³² P, ³⁵ S or and non-diffusible globular Aβ (X-38..43) oligomers labeled by being covalently linked to a radioactive group such as a group containing ¹²⁵ I or combinations thereof. Such labeling groups and methods of attaching them to proteins are known in the art.

In another particular embodiment of the invention, the derivatives of non-diffusible globular Aβ(X-38..43) oligomers are covalently linked or by non-covalent high affinity interactions. , preferably a group promoting in vivo degradation, preferably flagged by being covalently linked to a group promoting deactivation, sequestration, degradation and/or precipitation, more preferably Nondiffusible globular Aβ (X-38..43) oligomers containing at least one type of monomeric subunit flagged with ubiquitin. It is particularly preferred to assemble this flagging oligomer in vivo.

Other derivatives of non-diffusible globular Aβ(X-38..43) oligomers include cross-linked and labeled or cross-linked and flagged non-diffusible globular Aβ(X-38..43) oligomers. .

In a preferred embodiment of the invention the oligomer or derivative thereof is soluble.

Methods for making some Aβ(1-42) and Aβ(X-42) oligomers and their derivatives are described in WO2004/067561. The non-diffusible spherical Aβ(X-38..43) oligomers or derivatives thereof of the present invention can be produced in a similar manner.

Accordingly, the present invention provides a method for the production of non-diffusible globular Aβ (X-38..43) oligomers or in particular cross-linked, labeled and/or flagged derivatives thereof, comprising the preparation of a suitable Aβ protein or derivative thereof with a surfactant and then reducing the surfactant concentration, and obtaining non-diffusible spherical Aβ (X-38..43) oligomers. A detailed description of the method is disclosed in WO2004/067561, the content of which is incorporated herein by reference.

Derivatization and/or cleavage can occur before or after oligomerization. Accordingly, Aβ proteins or derivatives thereof suitable for use in the methods include Aβ (X-38..43) monomers, optionally derivatized, where X preferably has the formula 1..43. . 24, more preferably the number 1 . . 22, most preferably the numbers 1 . . selected from the group consisting of 20;

In another form, and particularly useful for in vitro diagnostic methods, the "non-diffusible" globular Aβ(1-42) oligomers ("globulomers") of the present invention are derived from APP- or amyloid-β expressing recombinant or non-recombinant It can also be extracted from human or animal neural cells such as, but not limited to, cell lines and/or fluids, particularly brain or spinal cord tissue and/or fluids, followed by representative cleaved Aβ (X -38..43) oligomers and/or derivatives thereof, by using antibodies with specificity for "non-diffusible" globular Aβ (1-38..43) oligomers ("globulomers") or derivatives thereof; It can be detected directly in the extract thus obtained or in the treated extract.

In a particularly preferred embodiment of the invention, the resulting non-diffusible globular Aβ(X-38..43) oligomers or derivatives thereof are subjected to one or more additional purification steps, such as precipitation and refolding, by techniques known in the art. Dissolve.

The present invention thus provides a method of concentrating non-diffusible globular Aβ (X-38..43) oligomers or derivatives in a preparation comprising oligomers or derivatives thereof, comprising the steps of capturing the oligomers or derivatives and in enriched form (“active” or “positive” enrichment).

In a preferred embodiment of the invention, the method of concentrating the oligomer or derivative comprises contacting the preparation with an agent that binds to the oligomer or derivative.

In a particularly preferred embodiment of the invention said agent is immobilized.

In a particularly preferred embodiment of the invention said agent is an antibody with specificity for oligomers or derivatives.

In a preferred embodiment of the invention, obtaining the oligomer or derivative in enriched form comprises desorbing the scavenging oligomer or derivative, preferably desorbing the scavenging oligomer or derivative comprises converting the scavenging oligomer or derivative to a high salt It has a step of contacting with a concentration buffer or an acid solution.

The present invention further provides a method of concentrating non-diffusible globular Aβ (X-38..43) oligomers or derivatives thereof in a preparation comprising oligomers or derivatives, wherein at least one species other than the oligomers or derivatives is enriched from the preparation. (“passive” or “negative” enrichment). Preferably, the substance other than the oligomer or derivative is an Aβ monomer or an Aβ fibrillomer, preferably both, and further removing the substance comprises contacting the preparation with an agent that specifically binds to the substance. .

In a particularly preferred embodiment of the invention said agent is immobilized.

In a particularly preferred embodiment of the invention said agent is an antibody that specifically binds to the substance, preferably with high affinity. Antibodies that can be used to remove non-globulomer Aβ species include anti-Aβ(33-42) polyclonal antibodies from Signet Catalog No. 9135 and their monoclonal equivalents, such as mAb12F4 from Signet Catalog No. 9142 or Anti-Aβ(33-42) specific for C-terminal Aβ(1-42) species, such as anti-Aβ(1-42) mAb, C-terminal clone BDI780 (purified, lyophilized; Biodesign Cat. No. Q67780M) ) antibodies.

In a more preferred embodiment of the invention, the step of removing the substance comprises contacting the preparation with an antibody that binds to the substance, and then for the substance bound to the antibody, the affinity for removal of antibody-antigen complexes. Chromatography, preferably Protein A affinity chromatography.

The present invention thus also relates to purified non-diffusible globular Aβ(X-38..43) oligomers or derivatives thereof. According to one embodiment of the invention, the purified non-diffusible globular Aβ(X-38..43) oligomers or derivatives thereof are non-diffusible globular Aβ(X-38..43) oligomers defined above or derivatives thereof. It can be obtained by a method of concentrating the derivative. In another aspect, the purified non-diffusible spherical Aβ(X-38..43) oligomers or derivatives thereof have a purity of greater than 99%, preferably greater than 99.9%, preferably greater than 99.99% by weight. have.

The present invention further provides a composition comprising non-diffusible globular Aβ (X-38..43) oligomers or derivatives thereof obtainable by the method of the invention, preferably non-diffusible globular Aβ (X-38..43). ) oligomers or derivatives thereof, wherein the amount of oligomers or derivatives is at least 99% by weight of the composition, preferably at least 99.9% by weight of the composition, more preferably at least 99.99% by weight of the composition; It is about.

The particularly purified nondiffusible globular Aβ(X-38..43) oligomers or derivatives thereof of the present invention have many uses, some of which are described below.

In one aspect, the invention relates to the use of non-diffusible globular Aβ (X-38..43) oligomers or derivatives thereof in producing antibodies with specificity for the oligomers or derivatives. Accordingly, the present invention provides a method for the production of antibodies having specificity for the non-diffusible spherical Aββ(X-38..43) oligomers defined above or derivatives thereof, comprising:
- providing an antigen comprising an oligomer or derivative;
- exposing an antibody repertoire or potential antibody repertoire to said antigen; and - selecting from said repertoire antibodies that specifically bind to said oligomer or derivative thereof.

It should be understood here that a "potential antibody repertoire" refers to a library, collection, assembly or assembly of amino acid or corresponding nucleic acid sequences, or the ability to produce an antibody repertoire in vivo or in vitro. Refers to the source of such libraries, collections, assemblies or assemblies of amino acid sequences that can be used for. In a preferred embodiment of the invention, the formatogen is the antigen-producing part of an animal's adaptive immune system, particularly a mammalian immune system, which generates antibody diversity by recombinant methods known to those skilled in the art. In another preferred embodiment of the invention, the formatogen is a forming system of random nucleic acid sequences, which can then be used to obtain antibody repertoires in vitro by insertion into suitable antibody scaffolds. can.

In a preferred embodiment of the invention, the antibody repertoire or potential antibody repertoire is exposed in vivo to the antigen by immunizing the organism with the antigen. In another preferred embodiment of the invention, the potential antibody repertoire is a library of suitable nucleic acids, which are screened for antibodies by in vitro affinity screening, e.g., phage display and panning systems, as reported in the art. expose.

In another aspect, the invention also relates to antibodies having specificity for the nondiffusible globular Aβ (X-38..43) oligomers defined above or derivatives thereof.

In a preferred embodiment of the invention, the antibodies are obtainable by a method comprising selecting antibodies from the repertoire or potential repertoire described above.

In a preferred embodiment of the invention, the affinity of the antibody for the oligomer or derivative is at least 2-fold, such as at least 3-fold or at least 5-fold, preferably at least 10-fold, e.g. at least 20-fold, at least 30-fold or at least 50-fold, more preferably at least 100-fold, such as at least 200-fold, at least 300-fold or at least 500-fold, even more preferably at least 1000-fold, such as at least 2000-fold, at least 3000-fold or at least 5000-fold times, more preferably at least 10,000 times, such as at least 20,000 times, at least 30,000 times or at least 50,000 times, most preferably at least 100,000 times.

In a preferred embodiment of the invention, the affinity of the antibody for the oligomer or derivative is at least 2-fold, such as at least 3-fold or at least 5-fold, preferably at least 10-fold, e.g. at least 20-fold, at least 30-fold or at least 50-fold, more preferably at least 100-fold, such as at least 200-fold, at least 300-fold or at least 500-fold, even more preferably at least 1000-fold, such as at least 2000-fold, at least 3000-fold or at least 5000-fold times, more preferably at least 10,000 times, such as at least 20,000 times, at least 30,000 times or at least 50,000 times, most preferably at least 100,000 times.

Conveniently, the antibodies of the invention have low affinity, most preferably an affinity with a KD of 1 x ^10-8 M or less ^, e.g. having an affinity of ⁻⁷ or less, such as having an affinity of KD of 3×10 ⁻⁷ M or less, or having an affinity of KD of 1×10 ⁻⁶ M or less, such as having an affinity of KD of 3×10 ⁻⁵ M or less or binds to one or more preferably both monomers with an affinity with a KD of 1×10 ⁻⁵ M or less.

In a preferred embodiment of the invention, the affinity of the antibody for the oligomer or derivative is at least 2-fold, such as at least 3-fold or at least 5-fold, preferably at least 10-fold, the binding affinity of the antibody to fibrillomer Aβ(1-42). such as at least 20-fold, at least 30-fold or at least 50-fold, more preferably at least 100-fold, such as at least 200-fold, at least 300-fold or at least 500-fold and even more preferably at least 1000-fold, such as at least 2000-fold, at least 3000-fold or at least 5000-fold, more preferably at least 10000-fold, such as at least 20000-fold, at least 30000-fold or at least 50000-fold and most preferably at least 100000-fold.

In a preferred embodiment of the invention, the affinity of the antibody for the oligomer or derivative is at least 2-fold, such as at least 3-fold or at least 5-fold, preferably at least 10-fold, e.g. at least 20-fold, at least 30-fold or at least 50-fold, more preferably at least 100-fold, such as at least 200-fold, at least 300-fold or at least 500-fold and even more preferably at least 1000-fold, such as at least 2000-fold, at least 3000-fold or at least 5000-fold fold, more preferably at least 10,000 fold, such as at least 20,000 fold, at least 30,000 or at least 50,000 fold and most preferably at least 100,000 fold.

Conveniently, the antibodies of the invention have low ^{affinity, most preferably an affinity with a KD of 1 x 10-8 M} or less, e.g. having an affinity of ⁻⁷ or less, such as having an affinity of KD of 3×10 ⁻⁷ M or less, or having an affinity of KD of 1×10 ⁻⁶ M or less, such as having an affinity of KD of 3×10 ⁻⁵ M or less or binds to one or more preferably both fibrils with an affinity with a KD of 1×10 ⁻⁵ M or less.

According to a particularly preferred embodiment, the invention relates to globulomer-specific antibodies. In particular, these include antibodies with relatively lower affinity for both monomeric and fibrilomeric forms of Aβ than for Aβ globulomers.

The term "greater affinity" herein means that the equilibrium between unbound antibody and unbound globulomer on the one hand and the antibody-globulomer complex on the other hand is tilted towards the antibody-globulomer complex. It refers to the degree of action. Similarly, the term "lower affinity" herein means that the equilibrium between unbound antibody and unbound globulomers on the one hand and antibody-globulomer complexes on the other is refers to the degree of interaction that is tilted toward

The present invention further relates to the use of non-diffusible globular A[beta](X-38..43) oligomers or derivatives thereof as defined above in the preparation of aptamers with specificity for the oligomers or derivatives.

The present invention also provides a method for producing an aptamer having specificity for nondiffusible globular Aβ (X-38..43) oligomers or derivatives thereof as defined in the claims, comprising:
- providing a binding target comprising an oligomer or derivative;
- exposing an aptamer repertoire or a potential aptamer repertoire to said binding target; and - selecting from said repertoire aptamers that specifically bind to said oligomer or derivative thereof.

An "aptamer" herein is its target, preferably a peptide, DNA or RNA sequence, more preferably a peptide, DNA or RNA sequence of about 3 to 100 monomers, especially about 5 to 30 monomers, most preferably refers to a small molecule capable of specific non-covalent binding to a peptide of about 5 to 30 amino acids, which at one or both ends is a relatively large molecule, preferably a relatively large molecule that mediates a biochemical function; It may be conjugated to a relatively large molecule, most preferably ubiquitin, which more preferably induces inactivation and/or degradation, or a relatively large molecule, which preferably promotes destruction, more preferably an enzyme or fluorescent protein.

It should be understood here that a "potential aptamer repertoire" is a library, collection, assembly or assembly or equivalent of amino acid sequences that can be used to form an aptamer repertoire in vivo or in vitro. It refers to a nucleic acid sequence, or the source of a library, collection, assembly or assembly of such amino acid sequences.

The present invention also relates to an aptamer having specificity for nondiffusible globular Aβ(X-38..43) oligomers or derivatives thereof as defined above, which aptamer can be obtained by the method described above.

In a preferred embodiment of the invention, the aptamer comprises a group that facilitates detection, preferably a fluorophore, such as fluorescein isothiocyanate, phycoerythrin, Aequorea victoria fluorescent protein, gimbo fluorescent protein or combinations or fluorescently active derivatives thereof; a chromophore; chemiluminophores such as luciferase, preferably Photinus filaris luciferase, Vibrio fischeri luciferase or combinations or chemiluminescent active derivatives thereof; enzymatic activities such as peroxidase or enzymatically active derivatives thereof heavy metals such as gold; haptens such as phenol-derived haptens; antigenic structures such as peptide sequences predicted to be antigenic; other aptamers; a naturally occurring or naturally derived protein structure that mediates protein-protein interactions; or is covalently linked to a radioactive group such as a group comprising ¹ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I or combinations thereof. It is labeled with

In another preferred embodiment of the invention, by being covalently linked or by non-covalent high affinity interactions, preferably the aptamer is deactivated, sequestered upon binding to its target. , by being covalently linked to a group that promotes degradation and/or precipitation, preferably with a group that promotes in vivo degradation, more preferably with ubiquitin.

The present invention also relates to the use of nondiffusible spherical Aβ(X-38..43) oligomers or derivatives thereof as defined in the claims for the manufacture of pharmaceutical compositions for the treatment or prevention of Alzheimer's disease. It is also about

In a preferred embodiment of the invention the pharmaceutical composition is a vaccine for active immunization.

Accordingly, the present invention provides a method of treating or preventing Alzheimer's disease in a subject in need thereof, comprising treating the subject with delayed non-diffusible globular Aβ (X-38..43) oligomers or It also relates to a method comprising administering the derivative.

In a preferred embodiment of the invention, administration of nondiffusible globular Aβ(X-38..43) oligomers or derivatives thereof is for active immunization of a subject against Alzheimer's disease.

It is particularly preferred that the non-diffusible globular Aβ(X-38..43) oligomers or derivatives thereof of the composition are poorly able to enter the brain of the patient's CNS.

A pharmaceutical composition comprising nondiffusible spherical Aβ (X-38..43) oligomers or derivatives thereof provides a strong immune response against Aβ globulomers, preferably against Aβ globulomers alone, more preferably against Aβ globulomers alone. It is also particularly preferred to be able to elicit an immune response based on non-inflammatory antibodies. Therefore, in a most preferred embodiment of the invention, the pharmaceutical composition comprises an immune adjuvant, preferably an adjuvant that directs the immune response against the non-inflammatory antibody system and signaling molecules such as cytokines. Such adjuvants and signaling molecules are known to those skilled in the art.

It is particularly preferred to administer the pharmaceutical composition for active immunization by a route selected from the group consisting of intravenous, intramuscular and subcutaneous routes. It is also particularly preferred to administer the composition by a method selected from injection, bolus infusion and continuous infusion, each of which can be done once, repeatedly or at regular intervals.

In certain embodiments of the invention, long-term continuous infusion is performed using an implantable device. In another particular embodiment of the invention, the composition is used as an implantable sustained- or sustained-release depot formulation. Suitable formulations and equipment are known to those skilled in the art. The details of the method to be used for a given route will depend on the stage and severity of the disease and the general medical parameters of the subject, and are preferably determined on an individual basis at the discretion of the treating physician or veterinarian.

In a particularly preferred embodiment of the invention, the pharmaceutical composition for active immunization comprises a pharmaceutically acceptable preservative, a pharmaceutically acceptable coloring agent, a pharmaceutically acceptable protective colloid, a pharmaceutically acceptable pH. It comprises one or more substances selected from the group consisting of regulators and pharmaceutically acceptable tonicity regulators. Such substances have been reported in the art.

The antibodies and aptamers of the invention also have many possible uses, some of which are described below. They are presumed to contain or contain amounts of non-diffusible globular Aβ (X-38..43) oligomers or derivatives thereof for screening purposes i.e. detection and optionally such oligomers or derivatives. It is particularly useful for measurements in preparations of known

Thus, the antibodies of the invention are capable of detecting the non-diffusible globular Aβ (X-38..43) oligomers or derivatives thereof to which they bind both in vitro and in vivo. Said antibodies are therefore used to detect said oligomers or derivatives, e.g. can be done.

Accordingly, the present invention provides a method for the detection of non-diffusible globular Aβ (X-38..43) oligomers or derivatives thereof in a preparation, wherein the antibodies of the present invention are directed to the oligomers or derivatives thereof to thereby detect said oligomers or derivatives. It relates to a method comprising conjugating to an oligomer or derivative thereof (and thereby preferably forming a conjugate comprising the antibody and the oligomer or derivative). Said oligomers can be detected, for example, in vitro. For example, the antibody of the invention is added to a preparation, e.g., a sample from a subject or cell culture containing or suspected of containing said oligomer or derivative, to detect said oligomer or derivative in said preparation. can do. Alternatively, oligomers or derivatives can be detected in vivo in an individual.

The present invention also provides an antibody having specificity for nondiffusible globular Aβ (X-38..43) oligomers defined above or derivatives thereof for the manufacture of a pharmaceutical composition for the treatment or prevention of amyloidosis It also concerns the use of

In a preferred embodiment of the invention the pharmaceutical composition is for passive immunization.

Accordingly, the present invention provides a method of treating or preventing amyloidosis in a subject in need thereof, comprising treating the subject with non-diffusible globular Aβ (X-38..43) oligomers as defined in the claims or It also relates to a method comprising administering an antibody having specificity for the derivative.

In a preferred embodiment of the invention, administration of antibodies is for passive immunization.

As used herein, the term "amyloidosis" refers to a number of diseases characterized by abnormal folding, assembly, aggregation and/or accumulation of specific proteins (amyloids, fibrillar proteins and their precursors) in various tissues of the body. pointing to a disability. Alzheimer's disease and Down's syndrome affect nerve tissue, and cerebral amyloid angiopathy (CAA) affects blood vessels. According to a particular embodiment of the invention, the amyloidosis is selected from the group consisting of Alzheimer's disease (AD) and Down's syndrome amyloidosis.

One result obtained by using the antibodies of the present invention is the diagnosis of Alzheimer's disease or an increased risk of developing Alzheimer's disease, within a detectable range of non-diffusible soluble globular Aβ(1-42) and/or Aβ (1-40) Oligomers (“globulomers”) cannot be readily relied upon. Surprisingly and unexpectedly, however, said diagnosis involves determining the presence or absence of autoantibodies that specifically bind to the non-diffusible soluble globular Aβ(1-42) oligomers (“globulomers”) of the invention. recognized that it could be done.

Surprisingly, and consistent with the globulomer hypothesis, globulomer epitopes were found to be endogenous antigens that generate an endogenous immune response. This endogenous immune response is characterized by at least three independent features:

1. Anti-Aβ globulomer autoantibody levels are elevated in Alzheimer's disease. In particular, these autoantibodies are surprisingly elevated in the sera of AD patients. To date, several investigators have reported autoantibodies in sera of AD patients. 3 papers (S. Brettschneider et al., ``Decreased Serum Amyloid 1-42 Autoantibody Levels in Alzheimer's Disease, Determined by a Newly Developed Immuno-Precipitation Assay with Radiolabeled Amyloid 1-42 Peptide, Biol Psychiatry 2005, 57, 813 -816; M. E. Weksler, et al., ″Patients with Alzheimer disease have lower levels of serum antiamyloid peptide antibodies than healthy elderly individuals″ Exp. Gerontology 37, (2002), 943-948; and Y. Du et al., ″ Reduced levels of amyloid β-peptide antibody in Alzheimer disease″, Neurology 57, 801-805 (2001)) reported that anti-Aβ antibody levels were mildly elevated in patients with Alzheimer's disease, but not significantly. It is

2. Anti-Aβ globulomer autoantibodies bind primarily in antigen complexes. The autoantibodies described herein are surprisingly detected in significant amounts even as part of antigen-antibody complexes shown by pretreatment of serum with an acidic buffer according to the method of Example 7. be. This is strong evidence that the immune system is actively trying to clear misfolded globulomer epitopes by complexing to endogenous antibodies.

3. Anti-Aβ globulomer autoantibody levels are significantly higher than those of anti-Aβ monomeric autoantibodies. As described herein in Example 7 and Figure 24, they are about 5- to 10-fold higher compared to monomeric Aβ peptides. In addition to the high proportion of antigen-antibody complexes, this is another parameter indicating that globulomers are being actively cleared by the endogenous immune system.

Accordingly, the present invention provides a composition for the detection of autoantibodies having specificity for the above-defined non-diffusible globular Aβ (X-38..43) oligomers or derivatives thereof in a subject. It also relates to the use of oligomers or derivatives.

In a preferred embodiment of the invention, the subject is suspected of having any form of amyloidosis, such as Alzheimer's disease, and detection of autoantibodies is diagnostic of the presence or absence of any form of amyloidosis, such as Alzheimer's disease, in the subject. It is intended to In a preferred embodiment of the invention the antibody is detected using a labeled derivative of the non-diffusible globular oligomer or cross-linked derivative defined above.

The present invention also relates to the use of oligomers or derivatives in detecting autoantibodies having specificity for nondiffusible globular Aβ (X-38..43) oligomers or derivatives thereof as defined above in a sample. It is also about

In a preferred embodiment of the invention, the sample is from a subject suspected of having amyloidosis, e.g. Alzheimer's disease, and the detection of autoantibodies is for the purpose of diagnosing the presence or absence of amyloidosis, e.g. Alzheimer's disease, in the subject. and In a preferred embodiment of the invention, antibodies are detected using labeled derivatives of the nondiffusible spherical oligomers or crosslinked derivatives described above.

The present invention also provides a method of detecting autoantibodies having specificity for non-diffusible globular Aβ (X-38..43) oligomers or derivatives thereof in a subject, comprising: administering a non-diffusible globular Aβ (X-38..43) oligomer or derivative thereof and detecting complexes formed by the antibody and the oligomer or derivative, wherein the presence of the complex indicates the presence of the autoantibody. It also relates to a method of indicating In a preferred embodiment of the invention, the method involves the use of labeled derivatives of the non-diffusible spherical oligomers described above or crosslinked derivatives thereof.

In a preferred embodiment of the invention, the subject is suspected of having amyloidosis and the detection of autoantibodies is intended to diagnose the presence or absence of the amyloidosis in the subject. In a preferred embodiment of the invention, antibodies are detected using labeled derivatives of the nondiffusible spherical oligomers or crosslinked derivatives described above.

The present invention also provides a method for the detection of autoantibodies having specificity for non-diffusible globular Aβ (X-38..43) oligomers or derivatives thereof in a sample, comprising non-diffusible globular Aβ as defined above (X-38..43) Contacting a sample with an oligomer or derivative thereof and detecting complexes formed by the antibody and the oligomer or derivative, the presence of the complex indicating the presence of the autoantibody. It is also about how to be In a preferred embodiment of the invention, the method involves the use of labeled derivatives of the nondiffusible spherical oligomers described above or crosslinked derivatives thereof.

In a preferred embodiment of the invention, the sample is from a subject suspected of having amyloidosis and the detection of autoantibodies is intended to diagnose the presence or absence of amyloidosis in that subject. In a preferred embodiment of the invention, antibodies are detected using labeled derivatives of the nondiffusible globular oligomers or crosslinked derivatives described above.

It is particularly preferred that the subject suspected of having amyloidosis is a subject who has or is at high risk of developing amyloidosis.

In one embodiment of the invention, the sample is a biological fluid that can be examined by the method. They include plasma, whole blood, dried whole blood, serum, cerebrospinal fluid or aqueous or organic-aqueous extracts of tissues and cells.

According to a particular embodiment of the invention, the step of detecting autoantibodies further comprises pretreatment of the preparation (sample) to effect dissociation of autoantibody/antigen complexes. Thus, methods that include such pretreatment can be used to determine the amount of autoantibodies present in a preparation (sample), and methods that do not include said pretreatment are still capable of binding antigen. The amount of autoantibodies can be measured. Moreover, both methods allow the amount of complexed autoantibodies to be measured indirectly.

Conditions suitable for inducing dissociation of autoantibody/antigen complexes are known to those of skill in the art. For example, the preparation (sample) is prepared (sample) by using a buffer such that the pH of the resulting preparation (sample) is in the range of 1 to 5, preferably in the range of 2 to 4, particularly in the range of 2 to 3. Treatment with acid may be good. Suitable buffers include salts at physiological concentrations, such as NaCl and acetic acid. Methods for separating antibody/antigen complexes are described in WO2005/037209, which is incorporated herein by reference.

That is, dissociating the antibody from the antigen in the antibody/antigen complex comprises contacting the sample containing the antibody/antigen complex with a dissociation buffer; incubating the sample; and optionally concentrating the sample. have.

The Dissociation Buffer can be a PBS buffer with a pH in the range indicated above. For example, PBS buffers containing about 1.5% BSA and 0.2 M glycine acetate (pH 2.5) or 140 mM NaCl and 0.58% acetic acid are suitable.

Incubation for a few minutes, eg 10 to 30 minutes, eg 20 minutes, at a temperature in the range 20 to 40° C. has been found to be sufficient.

Concentration can be carried out in a manner known per se, for example by passing the sample through a Centriprep YM30 (Amincon Inc.).

In one embodiment of the invention, A[beta](X-38..43) globulomers or derivatives thereof are coated on a solid phase. A sample (eg, whole blood, cerebrospinal fluid, serum, etc.) is then contacted with the solid phase. If autoantibodies are present in the sample, such antibodies bind to the Aβ(X-38..43) globulomers or derivatives thereof on the solid phase and are detected by direct or indirect methods. Direct methods involve simply detecting the presence of the complex itself and thus the presence of autoantibodies. In indirect methods, the conjugate is added to the bound autoantibody. The conjugate includes a second antibody that binds to the first bound autoantibody conjugated to a signal-generating compound or label. Upon binding of the second antibody to the bound first autoantibody, the signal generating compound generates a measurable signal. Such signal then indicates the presence of the first autoantibody in the sample.

Examples of solid phases used in diagnostic immunoassays include porous and non-porous materials, latex particles, magnetic particles, microparticles (see US Pat. No. 5,705,330), beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and, if desired, the method of labeling the antigen or antibody present in the conjugate is determined by the performance characteristics of the desired assay format.

As noted above, the conjugate (or indicator reagent) comprises an antibody (or possibly an anti-antibody, depending on the assay) attached to a signal generating compound or "label". The signal generating compound or label is itself detectable or can be reacted with one or more additional compounds to form a detectable product. Examples of signal generating compounds are described above, in particular chromophores, radioisotopes (e.g. ¹²⁵ I, ¹³¹ I, ³² P, ³ H, ³⁵ S and ¹⁴ C), chemiluminescent compounds (e.g. : acridinium), particles (visible or fluorescent), nucleic acids, complexing agents or catalysts such as enzymes (eg alkaline phosphatase, acid phosphatase, horseradish peroxidase, β-galactosidase and ribonuclease). When using an enzyme (eg, alkaline phosphatase or horseradish peroxidase), addition of a chromogenic, fluorogenic or luminescent substrate produces a detectable signal. Other detection systems such as time-resolved fluorescence, internal reflection fluorescence, amplification (eg polymerase chain reaction) and Raman spectrometry are also useful.

Kits are also included within the scope of the invention. More specifically, the invention includes kits for confirming the presence of autoantibodies in a subject. In particular, a kit for confirming the presence of autoantibodies in a sample comprises: a) Aβ (X-38..43) globulomers or derivatives thereof as defined herein; and b) generating a detectable signal. Also included are conjugates comprising an antibody conjugated to a signal-generating compound that is capable of generating a signal. The kit can also include controls or calibrators containing reagents that bind to the antigen.

The invention also includes another type of kit for detecting autoantibodies in test samples. The kit may comprise a) an anti-antibody specific for the autoantibody of interest and b) an A[beta](X-38..43) globulomer or derivative thereof as defined above. Controls or calibrators comprising reagents that bind to A[beta](X-38..43) globulomers or derivatives thereof can also be included. More specifically, the kit can comprise a conjugate comprising a) an anti-antibody that is specific for an autoantibody and b) an Aβ (X-38..43) globulomer or derivative thereof, wherein the conjugate is attached to a signal generating compound capable of generating a detectable signal. Again, the kit can also include controls or calibrators containing reagents that bind to the antigen.

The kit can also contain one container such as a vial, bottle or strip, each container having a preset solid phase, and other containers containing individual conjugates. These kits can also include vials and containers of other reagents needed to perform the assay, such as wash, treatment and indicator reagents.

In a preferred embodiment of the invention, such autoantibodies can also be extracted from commercially available immunoglobulin preparations such as Octagam® (Octapharma Inc., Vienna, Austria), and further used for therapy. It can also be refined.

Surprisingly and unexpectedly, screening of preparations other than the preparations described above revealed that such preparations were highly sensitive to the non-diffusible globular Aβ(X-38..43) oligomers or derivatives of the invention. It has been found that it may contain substances that react with antibodies having specificity. Such substances which have a certain binding affinity for said antibody, but which do not represent non-diffusible globular Aβ (X-38..43) oligomers or derivatives of the invention are hereinafter referred to as globular Aβ Referred to as (X-38..43) oligomeric (“globulomer”) epitopes or globular Aβ (X-38..43) oligomeric (“globulomer”) structural epitopes.

comprising an Aβ (X-38..43) oligomeric epitope in order to have binding affinity for an antibody having specificity for the nondiffusible globular Aβ (X-38..43) oligomer or derivative of the invention; Said substances can be detected in preparations putatively containing such epitopes, their amounts can be determined in said preparations and they can be enriched. Accordingly, the present invention provides such a compound in a preparation suspected or known to contain the Aβ(X-38..43) oligomeric epitope sinAβ(X-38..43) oligomeric epitope. It also relates to screening methods for detecting, quantifying, or enriching for such epitopes. Once detected and concentrated, said substances may have uses similar to those described above for the non-diffusible globular Aβ(X-38..43) oligomers or derivatives of the invention.

In the drawing:
FIG. 1 is a schematic diagram of the Aβ processing pathway. Aβ(1-42) oligomers exist in two distinct forms, fibrillar-type oligomers (“fibriromers”) and globular-type oligomers (“globulomers”). This model shows two mutually exclusive pathways leading from monomers to either fibrils or globulomers. The "decision" for the globulomer pathway over the fibrous pathway is made by a conformational shift at the monomer level that is presumed to be facilitated by fatty acids.

Figure 2 shows Western blots of monomers and globulomers after storage at various temperatures to compare their in vitro stabilities. Row 2: 1 day later, PBS, RT; row 3: 1 day later, PBS, 37°C; row 4: 4 days later, PBS, RT; row 5: 4 days later, PBS, 37°C), B) is Aβ (1-42) Monomer (row 1: standard molecular marker protein; row 2: after 1 day, PBS, −20° C.; row 3: after 1 day, PBS, −0° C.; row 4: after 1 day, PBS, RT; row 5: 1 day later, PBS, 37°C).

FIG. 3 depicts antibody titers of rabbits and mice immunized with Aβ(1-42) globulomers, Aβ(12-42) globulomers and Aβ(20-42) globulomers.

Figure 4 shows the detection results of Aβ(1-42) globulomer epitopes in AD brain and TG2576 brain, A) 3 AD brain samples and 1 brain-net (Brain-Net, Munich). B) are the levels of Aβ(1-42) globulomers, Aβ(1-42) monomers and Aβ(1-40) monomers in AD brain; ) is immunostaining of AD brain plaques with Aβ(1-42) globulomer-specific antibody 8C5; D) is Aβ(1-42) globulomer, Aβ(1-42) monomer in TG2576 and control wild-type mouse brain. and levels of Aβ(1-40) monomers.

Figure 5 shows the detection results of Aβ(1-42) globulomer epitopes in the CNS. capture antibody) and monoclonal non-selective anti-Aβ(1-42) globulomer antibody 6B1 (primary antibody); (1-42) Immunostaining of globulomers; C) Immunostaining of Aβ(1-42) globulomers after Aβ(1-42) globulomer injection in rat hippocampus.

FIG. 6. Reactivity of Aβ(33-42)-directed PAb (Signet 9135) with various Aβ preparations for characterization of Aβ(1-42) globulomer epitopes. . The antibody reacts only with Aβ(1-42) monomer dissolved in ammonia buffer, does not react with Aβ globulomers and Aβ(1-40), and does not react with fibril prone Aβ(1-40) dissolved in ammonia buffer. -42) shows that epitopes on globulomers are inaccessible or altered compared to monomers.

FIG. 7 shows the reactivity of anti-Aβ(20-42) globulomer-derived antibody 5598 in sandwich ELISA. Rabbit Anti-Aβ(1-42) Globulomer Affinity—Purified PAb5598 shows selectivity for Aβ(1-42) globulomers compared to other Aβ forms.

FIG. 8 shows the effect of pretreatment of Aβ(1-42) globulomer and Aβ(1-40) monomer standard preparations with the anti-Aβ globulomer-specific PAb 5598, as measured by the following ELISA,
- Aβ(1-42) globulomer, ±5598 depletion, Aβ globulomer ELISA: >10-fold shift (black to grey);
- Aβ(1-40) monomer, ±5598 deficient, Aβ(30-40) ELISA: no effect (dark green to light green);
- Aβ(1-40) monomer, ±5598 deficient, Aβ globulomer ELISA: 10-fold shift (dark blue to light blue);
We show that Aβ globulomer epitopes are also detectable on Aβ(1-40).

9 is an SDS-PAGE chromatogram of Aβ(20-42) globulomers obtained according to Example 5. FIG.

10 is an SDS-PAGE chromatogram of Aβ(12-42) globulomers obtained according to Example 6. FIG.

FIG. 11 summarizes data on the formation of Aβ(1-42) globulomers. Starting from synthetic Aβ(1-42), 38/48 kDa Aβ(1-42) globulomers, stable and well-defined oligomers with globular structures, can be obtained.

(a) As the first step of 38/48 kDa Aβ(1-42) globulomer formation, synthetic Aβ(1-42) peptide is dissolved in HFIP, then evaporated and resuspended in DMSO. Incubation in PBS with 0.2% SDS yields the intermediate 16/20 kDa Aβ(1-42) globulomer. Further dilution with water and incubation yields the mature 38/48Aβ(1-42)kDa globulomer, which can be dialyzed, precipitated in methanol/acetic acid and buffer exchanged.

(b) Several stages of 38/48 kDa Aβ(1-42) globulomer formation are visualized on a Coomassie-stained 4 20% Tris-glycine SDS-PAGE gel. Rows: (1) intermediate 16/20 kDa Aβ(1-42) globulomer, (2) glutardialdehyde cross-linked intermediate 16/20 kDa Aβ(1-42) globulomer, (3) 38/48 kDa Aβ(1-42) globulomer, ( 4) Glutardialdehyde cross-linked 38/48 kDa Aβ(1-42) globulomer (5) 38/48 kDa Aβ(1-42) globulomer after heat denaturation in SDS sample buffer at 95°C for 5 min, (6) 95°C Glutardialdehyde cross-linking 38/48 kDa Aβ(1-42) globulomers after heat denaturation in SDS sample buffer for 5 min at , (7) Lambert, M. P. et al., Diffusible, non-fibrillary ligands Proc. Natl. Acad. Sci U. S. A 95, 6448-6453 (1998)). Aβ forms are indicated with m=monomer, i=intermediate 16/20 kDa Aβ(1-42) globulomer, g=38/48 kDa Aβ(1-42) globulomer and f=fibrillar Aβ(1-42).

(c) Temporal stability of 38/48 kDa Aβ(1-42) globulomer compared to Aβ(1-42) monomer by incubation in PBS at various temperatures. Rows 1 to 4: 38/48 kDa Aβ(1-42) globulomers after incubation for 1 day at RT and 37°C (1, 2) and 4 days at RT and 37°C (3, 4). Rows 5 to 8: A[beta](1-42) _NH4OH preparations after 1 day at -20<0>C, 0<0>C, RT and 37<0>C.

(d) Size exclusion chromatography on Superose 12 columns: Aβ(1-42) globulomers (top), glutardialdehyde cross-linked Aβ(1-42) globulomers (middle) and 5 min incubation at RT after lysis. Aβ(1-42) _NH4OH preparations (bottom row).

(e) Limited proteolysis of Aβ(1-42) globulomers and the resulting cleavage fragments (indicated by tg) separated by SDS-PAGE and visualized by Coomassie staining. The resulting C-terminal fragment (analyzed by SELDI MS) is shown in brackets. Rows: (1) Undigested Aβ(1-42) globulomer (AA142, 4514 Da) (2) Papain (not detected), (3) Elastase (not detected), (4) Thermolysin (AA4 42, 4200 Da; AA20 42, 2218 Da; AA24 42, 1756 Da), (5) chymotrypsin (AA21 42, 2067 Da), (6) trypsin (AA6 42, 3897 Da; AA17 42, 2578 Da), (7) soluble Aβ _1-19 peptide filtrate isolated after thermolysin digestion. .

(f) Aβ(1-42) globulomers (top) and glutardialdehyde-crosslinked Aβ(1-42) globulomers (bottom) were proteolysed with thermolysin and the resulting peptide fragments analyzed by SELDI-MS.

FIG. 12 shows the reactivity of Aβ(1-42) globulomer-specific antibodies with high affinity and specificity in dot blotting and ELISA.

(a) Comparison of anti-Aβ antibodies for Aβ epitope binding and affinity. The Aβ(1-42) globulomer-specific polyclonal rabbit antibody 5598 and mouse monoclonal antibody 8F5 recognize Aβ intersubunit epitopes, whereas the non-specific anti-Aβ mouse monoclonal antibodies 6G1 and 6E10 recognize Aβ intrasubunit epitopes. Affinities of 8F5 and 6G1 were determined by Scatchard analysis from ELISA.

(b) Specificity of anti-Aβ(1-42) globulomer antibodies for various Aβ forms was determined by dot-blot immunoassay. Aβ(1-42) globulomer-specific antibodies 5598 and 8F5 (bottom) recognize primarily the Aβ(1-42) globulomer form, in contrast to the non-specific antibodies 6G1 and 6E10 (top). or standard preparations of Aβ(1-42) containing aggregated Aβ(1-42).

(c) Quantification of Aβ(1-42) globulomers using 5598 as capture antibody and 6G1 as detection antibody and 6E10 as capture antibody and 9132 (recognizes AA30-40) and 9135 (AA33) as detection antibodies respectively Quantification of Aβ type differentiation in standard preparations by three different sandwich ELISAs, quantification of Aβ(1-40) or Aβ(1-42) levels using Aβ(1-40) or Aβ(1-42) recognition).

(d) A standard of a specific Aβ type is cross-contaminated with other Aβ types. Values are calculated as the ratio between globulomer and non-globulomer forms in % measured by an appropriate ELISA. Previously reported in vitro formed Aβ oligomers (Lambert, ^MP et al. Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci USA 95, 6448- 6453 (1998)) and Aβ oligomers based on cell culture in conditioned medium ( ^** Walsh, DM et al. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539 (2002)) were included in this experiment.

FIG. 13 shows that Aβ(1-42) globulomers are present in the brains of Alzheimer's patients and APP transgenic mice.

(a-f) Thioflavin S(a,d), Aβ(1-42) globulomers in cortical sections of Alzheimer's disease patients (a-c) and cortical cross-sections of 12-month-old Tg2576 mice (d-f). Staining of amyloid plaques with specific antibody 8F5 (b, e), or both (c, f)—scale bar (for a to f): 20 μm.

(g to h) Concentrations of Aβ(1-42) globulomers or monomeric Aβ species in soluble fractions of the cortex of Alzheimer's disease patients (n=3) (g) or 12-month-old Tg2576 mice (n=8) (h). . In the brains of age-matched control humans (g) or C57B16 control mice (n=4) (h), there were no Aβ species concentrations above the limit of detection indicated by horizontal lines (<d.l.).

(i) Concentrations of Aβ(1-42) globulomers or monomeric Aβ species in cerebrospinal fluid of patients with mild cognitive impairment (grey bars; n=4) or Alzheimer's disease patients (black bars; n=2). No Aβ globulomers were found in any group above the detection limit of 10 pg/mL (horizontal line).

FIG. 14 shows that Aβ(1-42) globulomer formation can be induced by certain fatty acids. After the general procedure of Aβ(1-42) globulomer formation, SDS in the polymerization step was replaced with various fatty acids at neutral pH. Columns: (1) no inducer control, (2) positive control with 0.2% SDS, (3) 0.5% lauric acid (12:0), (4) 0.5% oleic acid ( 18:1), (5) linolenic acid (18:3), (6) eicosapentanoic acid (20:5), (7) docosatetraenoic acid (22:4), (8) docosahexanoic acid (22) :6), (9) positive control containing 0.2% SDS, (10) 1% arachidonic acid. Yields of Aβ(1-42) globulomers (g, indicated by arrows) differed among the various fatty acids.

FIG. 15 shows that Aβ(1-42) globulomers specifically bind to hippocampal neurons depending on culture age. Cultured hippocampal neurons were incubated with 200 nM Aβ form (monomer concentration=17 nM Aβ(1-42) globulomer based) and anti-Aβ6E10 antibody (Aβ non-specific 6E10 was used to detect Aβ(1-42) monomers and Aβ(1-42) globulomer binding was visualized by immunofluorescence with Aβ(1-42) globulomer binding.

(a) Aβ(1-42) globulomers are expressed on the surface of hippocampal neurons after 15 min of incubation (37° C., 5% CO ₂ ), in contrast to controls without Aβ and Aβ(1-42) monomers. showed a broken bond.

(b) Aβ(1-42) globulomer binding to hippocampal cultures was tested in vitro (DIV) on different days. At DIV8 almost no Aβ(1-42) globulomer binding (approximately 1%) was observed, whereas at DIV11 and DIV15 almost all (approximately 90%) hippocampal neurons bound to the Aβ142 globulomer. The inset in sample DIV15 shows disrupted connections along the dendritic direction and some distance away (arrows).

(c) Hippocampal cultures were double stained with anti-MAP2 as a neuronal marker and anti-GFAP as a glial cell marker. GFAP-positive cells showed no Aβ(1-42) globulomer binding in contrast to MAP2-positive cells. Microscope settings for Aβ signal were kept constant between individual experiments. Scale bar is 40 μm (inset=10 μm).

FIG. 16(I) shows that Aβ(1-42) globulomers have limited tissue penetration due to high binding.

(a) Staining of Aβ(1-42) globulomers with the specific antibody 8F5 (Cy3; red) after intracerebroventricular injection in rat brain. Cell nuclei are counterstained with DAPI (blue). Scale bar: 100 μm.

(b to c) Staining of Aβ(1-42) globulomers (red) 1 day (b) or 7 days (c) after intracortical injection into rat cortex. Cell nuclei are counterstained with DAPI (blue). Scale bar: 200 μm.

FIG. 16(II) shows that Aβ(1-42) globulomers have limited tissue penetration due to high binding.

(a) Staining of Aβ(1-42) globulomers with the specific antibody 8F5 (Cy3) after intracerebroventricular injection in rat brain. Cell nuclei are counterstained with DAPI. Scale bar: 100 μm.

(b) Staining of Aβ(1-42) globulomers 7 days after intracortical injection into rat cortex. Cell nuclei are counterstained with DAPI. Scale bar: 200 μm.

(c) Labeling capacity (mean±s.e.m.) after injection of equivalent amounts of Aβ(1-42) globulomer into the right cortex (filled circles) and Aβ(1-42) monomer into the left cortex (open circles) of the same rat. time lapse. 1 day (n=2), 4 days (n=3) and 7 days (n=2) after injection Aβ(1-42) globulomers were detected with the specific antibody 8F5 and with antibody 6G1. Aβ(1-42) monomer was detected.

FIG. 17. Aβ(1-42) globulomers completely block long-term potentiation in vitro.

(a) LTP induction in brain sections from control (squares; n=9) and 42 nM Aβ(1-42) globulomer-treated (diamonds; n=6) rats. Field EPSP slopes were expressed as percent change from baseline values (mean±s.e.m.).

(b) Representative traces of control (upper row) and Aβ(1-42) globulomer-treated (lower row) sections taken before (1), immediately after (2) and 90 minutes after (3) tonic induction.

(c) Input/output relationships are similar in the control group (squares; n=11) and the Aβ(1-42) globulomer group (diamonds; n=5). Horizontal bar: 2 ms; vertical bar: 1 mV.

FIG. 18 shows a proposed model of the two pathway mechanisms of Aβ-peptide multimerization. The first step is Aβ-monomer formation by β- and γ-secretase cleavage of APP. Two independent pathways of AAβ(1-42) peptide multimerization are believed to occur, possibly depending on intrinsic or environmental factors. Reported fibril pathway (Lomakin, A., Chung, D. S., Benedek, G. B., Kirschner, D.A. und Teplow, D.B., On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantification of rate constants, Proc Natl. Acad. Sci. U.S.A., A93, 1125-1129 (1996)) follows classical polymerization kinetics. Aβ(1-42) monomers polymerize through further association of the monomers into metastable nucleating structures (fibriromers) and stable Aβ fibrils composed of protofibrils and β-sheet secondary structures. The Aβ(1-42) globulomer pathway is characterized by multimerization into structures with different numbers of Aβ-peptides. As a first step, Aβ(1-42) globulomer intermediates are formed, which further self-assemble into stable Aβ(1-42) globulomer structures with different numbers of Aβ-peptides (n=12). . Aβ(1-42) globulomers have no propensity for further polymerization into fibrils. In Aβ(1-42) globulomers, the hydrophobic C-terminus extends inside the globular structure, avoiding exposure to the aqueous phase. The more hydrophobic N-terminus is exposed on the outer surface. Aβ(1-42)2 globulomers are stabilized by β-sheet secondary structures. The high solubility and distinct stable properties of Aβ(1-42) globulomers give rise to this particular globular structure.

FIG. 19 shows circular dichroism spectra of Aβ(1-42) globulomers. Spectra were collected for Aβ(1-42) globulomer at 7.2 mg/mL using freshly buffer exchanged globulomer into PBS according to the reported method (see Supplementary Methods). The photomultiplier tube voltage (HTS) as a function of wavelength is shown in the inset. A strong minimum is observed at 216 nm, indicating a high β-sheet content. No minima are observed at 208 nm or 222 nm, suggesting that there are few alpha helices.

FIG. 20 shows 100 pmol (row A), 10 pmol (row B) and 1 pmol (row C) of Aβ(1-42) globulomers (column 1); Aβ(20-42) globulomers (lane 3); glutaraldehyde cross-linked Aβ(1-42) globulomers (lane 4); MP Lambert et al., J. Neurochem. 79 Aβ(12-42) globulomers (lane 6); Aβ(1-40) monomers (lane 7); 0 A of Aβ(1-42) dissolved in .1% _NH4OH (column 8); Aβ(1-42) fibril preparation (column 9); and PBS diluted APP from Sigma (column 10). FIG. 2 shows dot blots of reactivity with a) human plasma and B) CSF.

FIG. 21. New object recognition task in actively immunized APP/Lo mice. The preference for investigating new subjects over those already known was Aβ(1-42) monomers (n=7), Aβ(20-42) globulomers (n=9) or Aβ(1-42) globulomers (n=9). (n=8)), or in groups of mice injected with vehicle (n=8). Data are mean±s.e.m. After initial immunization, mice were boosted every 3 weeks for 3 months. Immunization with globulomer resulted in a significant increase in the cognitive index, ie preference for new subjects in the study (P<0.01 by ANOVA followed by post-hoc t-test).

FIG. 22 is a bar graph of the cognitive index of mice treated with saline (control), antibody 8F5 and antibody 6G1 (data show the relative Shown is the mean±s.e.m. of 8 animals/group for the amount of time.For each test, 10 minutes of study were allowed).

FIG. 23 shows SELDI mass spectral data from AD brain extracts immunoprecipitated with antibody 8F5. Aβ(1-42) (m/z=4514) as well as Aβ(1-40) species (m/z=4329.9) and Aβ(1-38) species (m/z=4131.6) can also be recognized.

Figure 24. Autoantibody levels from human serum/plasma directed against various amyloid beta antigens.

(a) All reflectance density values are taken from an antigen concentration of 100 pMol.

- Free fraction values are taken from untreated serum/plasma.
- Total fraction values are taken from acid-treated serum/plasma.
- bound fraction value = total fraction value - free fraction value.
(b) Anti-Aβ globulomer autoantibody levels are higher than anti-Aβ monomeric autoantibody levels and are highest in AD patients compared to healthy controls.

Abbreviations throughout the specification, claims and figures: AD, Alzheimer's disease; Aβ, amyloid β peptide; APP, amyloid precursor protein; BSA, bovine serum albumin; CD, circular dichroism spectroscopy; Days in vitro; DMSO, dimethylsulfoxide; GABA, γ-aminobutyric acid; HFIP, 1,1,1,3,3,3-hexafluoro- ₂ -propanol; LTP, _long -term potentiation; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; SELDI-MS, surface-enhanced laser desorption/ionization mass spectrometry; TBS, Tris-buffered saline.

Amyloid β(1-42) (Aβ(1-42)) oligomers have recently been discussed as intermediate toxic species and putative etiologic agents and risk factors in the pathology of Alzheimer's disease (AD) and related conditions such as Down's syndrome. and their involvement with fatty acids, described below, suggests that they may play some drug in the dementia effect of certain metabolic disorders. In particular, the present invention relates to highly stable Aβ(1-42) oligomeric species that are readily manufacturable in vitro and present in the brains of AD patients and Aβ(1-42) overproducing transgenic mice. be. Physico-chemical properties revealed a pure, highly water-soluble globular oligomer with a size of 60 kDa, which we termed "A[beta](1-42) globulomer".

The data presented herein demonstrate that Aβ(1-42) globulomers are persistent structures formed independently of the fibril aggregation pathway. It induces the formation of Aβ(1-42) globulomer epitope-specific antibodies with little cross-reactivity with amyloid precursor protein, Aβ(1-40) and Aβ(1-42) monomers and Aβ fibrils in mice and rabbits. is a potent antigen in Aβ(1-42) globulomers bind specifically to neuronal dendrites, but not to glia in hippocampal cell cultures, and completely block long-term potentiation in rat hippocampal slices. The data provided herein are further representative of the underlying pathogenic structural predisposition believed to be the presence of the Aβ(1-42) globulomer epitope at least in low abundance in the aforementioned oligomeric preparations. formation is an early pathological event in AD. Selective neutralization of Aβ(1-42) globulomer structural epitopes is expected to have high potential in AD therapy.

The ability of Aβ peptides to block LTP has been reported in the art.

Similar blockade of LTP was observed at concentrations as low as about 40 nM of Aβ(1-42) globulomer. However, in contrast to previously reported soluble oligomers, the preparations according to the invention do not contain a mixture of small Aβ(1-42) oligomers, but consist of only one homogeneous and stable chemical species. there were. Therefore, the fact that the effective concentrations required for globulomers are an order of magnitude lower than those reported for previously reported preparations suggests that only a small fraction of the preparations actually exert adverse effects. It may indicate something.

Previously reported Aβ oligomer preparations were characterized primarily by function, ie neurotoxic effects at given concentrations. In addition to these neurotoxic effects, the inherent high stability and well-defined chemical properties and steric arrangement of the Aβ(1-42) globulomers of the present invention contribute to their homogeneity, molecular weight and globularity and structural topology. characterization became possible.

In vitro formation of Aβ(1-42) globulomers begins with the denaturation of synthetic Aβ(1-42) induced by agents that break hydrogen bonds, followed by refolding into a new conformation. Although this conformational transition was nearly complete when analyzed by SDS-PAGE, the Aβ(1-42) globulomers exhibited a characteristic double band with apparent molecular weights of 38/48 kDa. However, cross-linking with glutaraldehyde joins these two bands together, indicating that the Aβ(1-42) globulomer exists in only one conformation. Homogeneity was also shown by size exclusion chromatography, where the Aβ(1-42) globulomer gave rise to one broad peak, which became sharper, ie narrower, after cross-linking. Thus, size exclusion chromatography of cross-linked Aβ(1-42) globulomers under native conditions showed a molecular weight of approximately 60 kDa with minimal variance, which was also confirmed by analytical ultracentrifugation. Calculations suggest that the Aβ(1-42) globulomer consists of about 12 to 14, preferably 12 Aβ subunits, and further experimental data suggest that it is suggested to be formed.

Several experiments have addressed the globularity and structural topology of Aβ(1-42) globulomers. In the case of mature Aβ(1-42) globulomers, fused spherical structures were observed that further condensed upon cross-linking to almost completely spherical structures.

First, size exclusion chromatography of Aβ(1-42) globulomers, especially the cross-linked ones, with narrow elution profiles suggest globularity. Furthermore, the hydrodynamic capacity can be calculated from these data and can be related to the total number of amino acids, providing a measure of the degree of folding (Tcherkasskaya, O. & Uversky, V. N. Denatured collapsed states in protein folding: Proteins 44, 244-254 (2001); Uversky, V.N. Natively unfolded proteins: a point where biology waits for physics. Protein Sci 11, 739-756 (2002)).

Second, digestion of Aβ(1-42) globulomers by various non-specific proteases results in cleavage of approximately 20 amino acids from the N-terminal site, but retains the oligomeric structure of the protease-resistant C-terminal nucleus. . The results also indicated a uniform structural topology in which each of the 12 single Aβ subunits formed co-oriented Aβ(1-42) globulomers.

Third, mass spectrometry confirmed the uniform globularity and structural topology, as cross-linking occurred only between the N-terminus and amino groups of Lys-16, but not Lys-28.

Fourth, and finally, for Aβ(33-42) and Aβ(33-40) used in conventional ELISAs for the measurement of Aβ(1-42) and Aβ(1-40) concentrations Selective antibodies did not react with Aβ(1-42) globulomers, again indicating that the C-terminus is unaffected.

CD spectroscopic measurements suggest that a significant fraction of Aβ(1-42) globulomers, presumably the C-terminus, are present in the β-structure (see FIG. 11), which is a generalization of amyloid proteins in their pathologically polymerized form. (Rochet, J.C. & Lansbury, P.T., Jr. Amyloid fibrillogenesis: themes and variations. Curr Opin Struct. Biol 10, 60-68 (2000)). In the resulting model of Aβ(1-42) globulomers, the hydrophobic C-terminus (Aβ(24-42)) forms the inner core and the hydrophilic N-terminus (Aβ(1-19-23)) forms the outer Surface exposure makes Aβ(1-42) globulomers very water soluble (see FIG. 18).

In summary, the Aβ(1-42) globulomers of the present invention are homogeneous, well-defined and stable chemospecies that are distinguished by their lack of diffusibility and have neuropathological effects on LTP comparable to those previously reported for soluble Aβ oligomers. (Lambert, MP et al. Diffusible, nonfibrillar ligands derived from Abeta 1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci USA 95, 6448-6453 (1998); Walsh, DM et al. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539 (2002); Wang, HW et al. Soluble oligomers of beta amyloid _1-42 inhibit long-term potentiation but not long-term Brain Res 924, 133-140 (2002); Wang, Q., Walsh, DM, Rowan, MJ, Selkoe, DJ & Anwyl, R. Block of long-term potentiation by naturally secreted and synthetic amyloid. beta-peptide in hippocampal slices is mediated via activation of the kinases c-Jun N-terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5. J Neurosci 24, 3370-3378 (2004)).

To understand the concept of diffusible and non-diffusible ligands, the processing of APP-derived Aβ(1-42) monomers formed in situ involves:
a) fibril pathway; b) globulomer pathway.
It is important to understand that there are two independent pathways of

Of these, the fibril pathway has been characterized for over two decades as the quantitatively dominant pathway in Aβ processing in vitro and in vivo. Aβ(1-40) and in particular Aβ(1-42) or related derivatives such as Aβ(1-38), Aβ(1-39), Aβ(1-41) or Aβ(1-43) are monomeric starting from units readily polymerize and grow successively through oligomers (associations of 2 to 24 monomers) and protofibrils (associations of more than 24 monomers) and finally to insoluble fibrils, It has been observed to deposit in vitro or in vivo in brain tissue or blood vessel walls. This fibril formation is so pronounced that early researchers attributed the dementiac effect of Aβ to it. It is important to understand that a special subset of oligomers, different from globulomers, which we propose to call "fibrillomers" (see Figure 1), is intermediate in this case. is.

Herein, the invention describes a new pathway for the stabilization of metastable Aβ formed in situ, which involves changing the structural conformation at the level of the monomer to another three-dimensional There is a step of switching structures, which then leads to a different kind of association which we have given the name 'globulomerization'. Characteristically, the spatial conformation of Aβ(1-42) was altered (Fig. 1) such that its processing gave rise to terminal globular-type oligomeric nuclear structures (“globulomers”), which were replaced by non-terminal fibrillar-type oligomeric nuclear structures. (“Fibriromers”) are completely different. Here, "final" means that unlike fibriromers, globulomers do not further assemble into fibrils.

Because Aβ(1-42) globulomers are stable and not intermediates of macromolecular aggregates, it is proposed that their formation reflects an alternative pathway to fibril aggregate formation. Given that soluble oligomers are toxic to neurons or otherwise detrimental to neuronal function, those along the fibril pathway are 'naturally' lost by further polymerization to mature fibrils. (Caughey, B. & Lansbury, P. T. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu. Rev Neurosci 26, 267-298 (2003)), which is due to sequestration. It is thought to represent nature's attempt at Aβ capture by . In contrast, Aβ(1-42) globulomers are an alternative pathway endpoint as neurotoxic Aβ(1-42) multimers with long-term stability under physiological conditions (see FIG. 18).

Regarding the validity of the Aβ(1-42) globulomer, it needs to be present in the brains of AD patients. Thus, selective monoclonal antibodies have been generated and Aβ(1-42) globulomer epitopes detected along with fibrillar Aβ in brain plaque deposits of both AD patients and mice transgenic for human APP. In parallel with these findings, ADDLs have also been detected in the brains of AD patients (Lacor, PN et al. Synaptic targeting by Alzheimer's-related amyloid beta oligomers. J Neurosci 24, 10191-10200 (2004);
Gong, Y. et al. Alzheimer's disease-affected brain: presence of oligomeric A beta ligands (ADDLs) suggests a molecular basis for reversible memory loss. Proc. Natl. Acad. ). According to the above definition, it should be clear that ADDLs differ essentially from globulomers in that they are described as diffusible. However, as explained above, at least part of these effects may in fact be due to previously undetected amounts of substances present in ADDL preparations that cross-react with globulomer-specific antibodies. have a nature. ADDLs may therefore be said to contain low abundance Aβ(1-42) globulomer epitopes. Using Aβ(1-42) globulomer-specific antibodies, the amount of said epitope can be confirmed to be about 5%. Such amounts may also partially explain the observed neurotoxic effects of the preparations.

Given that Aβ(1-42) globulomers occur naturally in AD brains, it is interesting to see how Aβ(1-42) globulomers, previously shown only in vitro, can arise in vivo. remains unknown. This question has been addressed by substituting natural fatty acids for SDS used in the original Aβ(1-42) globulomer manufacturing process. The presence of some of the fatty acids also resulted in aggregation into Aβ(1-42) globulomers. The same effect is also expected to occur with anionic detergents (eg SDS) or polyanionic agents (eg glycosaminoglycan heparin), either alone or in combination with fatty acids. These chemically distinct agents share the ability to induce amyloid protein aggregation or oligomerization by inducing and stabilizing conformational changes and providing anionically charged surfaces for nucleation, It suggests that anionic surfaces in the form of micelles or vesicles can indeed induce Aβ(1-42) globulomer formation. Anionic membranes or lipid rafts can perform this function in vivo. This may also involve globulomer formation in dementia induced by certain metabolic disorders, particularly by disorders of lipid metabolism, and thus the present invention may also be useful in the diagnosis and treatment of this condition. This suggests that

The mechanism and site of action of Aβ(1-42) globulomers in vivo are currently unknown. Although soluble due to their hydrophilic shell, they are not detectable in cerebrospinal fluid, rather they bind rapidly and tightly to tissues, especially when compared to Aβ(1-42) monomers. This Aβ(1-42) globulomer binding appears to be physiologically relevant as it specifically targeted hippocampal neurons in primary culture, suggesting a specific binding mechanism.

Several findings suggest that the interaction of hydrophilic Aβ(1-42) globulomers with hippocampal neurons is non-specific and based on known insertion-like events of Aβ peptides into lipid bilayers. One possibility is excluded (McLaurin, J. & Chakrabartty, A. Membrane disruption by Alzheimer beta-amyloid peptides mediated through specific binding to either phospholipids or gangliosides. Implications for neurotoxicity. J Biol Chem 271, 26482-26489 (1996) ;V.erdier, Y., Zarandi, M. & Penke, B. Amyloid beta-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer's disease. J Pept Sci 10, 229-248 (2004) )).

Most importantly, monomeric Aβ(1-42), known to be non-toxic and suggested to have anti-dementia effects, shows detectable binding to neuronal structures. (Plant, L.D. et al.: The production of amyloid beta peptide is a critical requirement for the viability of central neurons. J. Neurosci. 23, 5531-5535). Aβ(1-42) globulomer binding, in turn, was strongly dependent on cell culture stage and was restricted to neuronal cells only, leaving glial cells unlabeled. Disrupted binding patterns of Aβ(1-42) globulomers along dendrites have been shown to bind neurons and co-localize with postsynaptic markers such as PSD-95 of ADDLs. pattern (Lacor, P.N. et al. Synaptic targeting by Alzheimer's-related amyloid beta oligomers. J Neurosci 24, 10191-10200 (2004)). Thus, it appears that bound Aβ(1-42) globulomers are specifically localized to postsynaptic processes and that their binding is restricted to dendritic spines. There, it is presumed that it binds to as yet uncharacterized receptors and interferes with electrochemical information processing, but not the essential state-monitoring biochemistry of the cell. This would explain the specific blockade of LTP1 while leaving basic neurophysiology as well as short-term potentiation unchanged. Suppression of LTP (but not of basal membrane physiology) also indicates that Aβ(1-42) globulomers may specifically interfere with learning and memory in vivo, suggesting that Aβ(1 -42) suggests that globulomers are responsible for the memory impairment seen in AD patients.

In the present invention, "neurotoxicity" is understood to refer to a drastic loss of neuronal and/or synaptic function, independent of the effect on individual cell viability.

Although Aβ(1-42) globulomers have been found to be stable under physiological conditions in vitro, degradation under in vivo conditions is similar. Experiments with a non-specific protease revealed that the protruding hydrophilic N-terminus from the globular structure could be cleaved to generate truncated Aβ(1-42) globulomers. Recently, the presence of various N-terminally truncated Aβ species in AD has been reported (Sergeant, N. et al. Truncated beta-amyloid peptide species in pre-clinical Alzheimer's disease as new targets for the vaccination approach. J Neurochem 85 , 1581-1591 (2003)), suggesting that indeed N-terminal truncation is a possible physiological event in the brain.

Blockade of LTP appears to be a common pathophysiological feature of Aβ(1-42) globulomers and reported Aβ oligomer preparations (Lambert, M. P. et al. Diffusible, nonfibrillar ligands derived from Abeta 1-42 are Acad. Sci U. S. A 95, 6448-6453 (1998); Walsh, D. M. et al. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539 (2002); Wang, H.W. et al. Soluble oligomers of beta amyloid 1-42 inhibit long-term potentiation but not long-term depression in rat dentate gyrus. Brain Res 924, 133-140 (2002); Wang, Q., Walsh, D. M., Rowan, MJ. , Selkoe, DJ. & Anwyl, R. Block of long-term potentiation by naturally secreted and synthetic amyloid beta-peptide in hippocampal slices is mediated via activation of the kinases c-Jun N -terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5. J Neurosci 24, 3370-3378 (2004)).

Presumably, several soluble oligomers arise early in AD and target the same neuronal functions. Specifically, other functionally characterized Aβ(1-42) oligomers produced by cell lines expressing mutant human APP (Walsh, D.M. et al. Naturally secreted oligomers of amyloid beta protein potently Inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539 (2002); Wang, Q., Walsh, D.M., Rowan, M.J., Selkoe, D.J. & Anwyl, R. Block of long-term potentiation by naturally secreted and synthetic amyloid beta-peptide in hippocampal slices is mediated via activation of the kinases c-Jun N-terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5. J Neurosci 24, 3370 -3378 (2004)) is rather of the prefibrillar type. We did not find Aβ(1-42) globulomer epitopes among Aβ oligomers released by similar cell lines.

Even so, Aβ(1-42) oligomers obtained from this cell line (Walsh, D.M. et al. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539 ( 2002); Wang, Q., Walsh, D.M., Rowan, MJ., Selkoe, D.J. & Anwyl, R. Block of long-term potentiation by naturally secreted and synthetic amyloid beta-peptide in hippocampal slices is mediated via activation of the kinases c-Jun N-terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5. J Neurosci 24, 3370-3378 (2004)) effectively blocked LTP. Lambert, M. P. et al. Diffusible, nonfibrillar ligands derived from Abeta 1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci U.S.A 95, 6448-6453, 1998); Walsh, D.M. et al Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539 (2002); Wang, H.W. et al. Soluble oligomers of beta amyloid 1-42 inhibit long-term potentiation but not long-term depression in rat dentate gyrus. Brain Res 924, 133 - 140 (2002); Wang, Q., Walsh, D.M., Rowan, M.J. , Selkoe, D.J. & Anwyl, R. Block of long-term potentiation by naturally secreted and synthetic amyloid beta-peptide in hippocampal slices is mediated via activation of the kinases c-Jun N-terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5. J Neurosci 24, 3370-3378 (2004)).

However, residual contamination of Aβ(1-42) globulomer epitopes may be a small but very important part of ADDLs as responsible for the pathological properties of soluble Aβ(1-42) oligomer mixtures containing ADDLs. There is also Independently of this, working with Aβ(1-42) globulomers is highly advantageous in obtaining a distinct and homogeneous population of Aβ(1-42) multimers. This is evident in generating antibodies for scientific or therapeutic purposes.

The invention also relates to monoclonal and polyclonal antibodies with different specificities for various globulomer epitopes.

The first monoclonal mouse antibody, 6G1, has been shown to bind to a representative promiscuous N-terminally located linear Aβ epitope shared by all structural forms of Aβ(1-X) species. . Thus, 6G1, although very potent, has been observed to have an immunotype profile similar to 6E10.

In contrast, two other antibodies, the mouse monoclonal antibody 8F5 and the affinity-purified polyclonal rabbit antibody PAb5598, were selective for Aβ(1-42) globulomers compared to Aβ monomers or fibrils. However, they both map beyond amino acid 20 as they cross-react with truncated Aβ(20-42) oligomers. Since these antibodies access globulomers after native SDS western blotting, but not after denaturing SDS western blotting (data not shown), both antibodies have structural non-linear epitopes in the region of amino acids 20 and 30. It is considered to recognize the unit. Such structural epitopes have recently been shared by diverse amyloid proteins with varying primary sequences, suggesting that the shared common structure induces their neurotoxic function (Kayed, R. et al. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486-489 (2003); Glabe, CG. Conformation-dependent antibodies target diseases of protein misfolding. )).

These antibodies provide a means to look for Aβ(1-42) globulomers in the brains of AD patients and human APP transgenic mice. There were initial concerns about the low selectivity compared to Aβ(1-42) monomer preparations. It was later found that Aβ(1-42) globulomers were depleted in the presence of abundant Aβ monomers both in CSF samples from AD patients and in conditioned media of APP-overexpressing cells, much more than was first confirmed. (1-42) was found to demonstrate high selectivity for globulomers. Indeed, in vitro standard preparations of Aβ(1-42) monomers contain contaminants of Aβ(1-42) globulomer epitopes and the degree of contamination can be determined by Aβ(1-42) globulomer-selective antibodies. I was able to confirm. Thus, the Aβ(1-42) globulomer epitope is a major component in artificial 38/48 kDa globulomer preparations, whereas another reported Aβ oligomer preparation (Lambert, M. P. et al. Diffusible, nonfibrillar ligands derived from Abeta 1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci U. S. A 95, 6448-6453 (1998)).

Antibodies raised against Aβ(1-42) globulomers not only allow detection of this species in a variety of in vitro preparations and in vivo, but also selectively inactivate certain oligomeric species. It also provides means. Besides the possibility of investigating the role of specific neurotoxic proteins in AD development, such specific antibodies recognizing structural epitopes may be useful against other common and relatively less toxic Aβ(1-42) species. It is expected to be superior in therapy because side effects associated with antibody response can be avoided. In this regard, Aβ(1-42) globulomers incorporate superior immune responses resulting in high antibody titers, offering the potential for active and passive immunization against Aβ(1-42) globulomers. That is an interesting point to note.

Accordingly, Aβ(1-42) globulomers and specific antibodies generated against them, including derivatives such as recombinant, humanized and/or chimeric antibodies, are useful for the diagnosis and treatment of Alzheimer's disease and other conditions involving Aβ globulomers. It is expected to be useful in therapy and in the manufacture of materials useful in the diagnosis and/or treatment of those conditions. Clearly, these conditions may include Down's syndrome and dementia caused by metabolic disorders such as certain forms of hyperlipidemia.

As used herein, the term "Aβ(X-Y)" refers to the amino acid sequence from amino acid position X to amino acid position Y of the human amyloid beta protein, including both X and Y, in particular the amino acid sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT (from amino acid position 1 to 43) or the amino acid sequence from amino acid position X to amino acid position Y of a naturally occurring variant thereof, in particular no more than 3 additional amino acid substitutions at position X and position Y at both or neither of which are capable of interfering with globulomer formation. preferably without another amino acid substitution in the portion from amino acid 12 or X (whichever is higher) to amino acid 42 or Y (whichever is lower), more preferably amino acid 20 or X (whichever is most preferably from amino acid 20 or X (whichever is higher) to amino acid 40 or Y (whichever is higher) without additional amino acid substitutions in the region from amino acid 42 or Y (whichever is lower). A2T comprising a sequence without additional amino acid substitutions (where the "additional" amino acid substitutions are derived from the canonical sequence, not found in nature), H6R, D7N, A21G (“Flanders”), E22G (“Arctic”), E22Q (“Holland”), E22K (“Italian”), D23N (“Iowa”), A42T and A42V (numbers indicate the start of the Aβ peptide) are associated with) at least one mutation selected from the group consisting of

More specifically, the term "Aβ(1-42)" herein refers to the amino acid sequence from amino acid position 1 to amino acid position 42 of the human amyloid beta protein, including both 1 and 42, particularly the amino acid sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA or natural variants thereof, in particular A2T, H6R, D7N, A21G (“Flanders”), E22G (“Arctic”), E22Q (“Holland”), E22K (“Italian”), D23N (“Iowa”), with at least one mutation selected from the group consisting of A42T and A42V (numbers refer to the start of the Aβ peptide, including both 1 and 42) or no more than three mutations, none of which interfere with globulomer formation It refers to a sequence that has another amino acid substitution and preferably does not have another amino acid substitution in the region from amino acid 20 to amino acid 42. Similarly, the term "Aβ(1-40)" herein refers to the amino acid sequence from amino acid position 1 to amino acid position 40 of the human amyloid beta protein, including both 1 and 40, particularly the amino acid sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV or its Natural variants, in particular A2T, H6R, D7N, A21G (“Flanders”), E22G (“Arctic”), E22Q (“Holland”), E22K (“Italy”) and D23N (“Iowa”) (number 1) and 40) or no more than 3 alternative amino acid substitutions, none of which interfere with globulomer formation. , preferably refers to a sequence that does not have another amino acid substitution in the portion from amino acid 20 to amino acid 40.

More specifically, the term "Aβ(12-42)" herein refers to the amino acid sequence from amino acid position 12 to amino acid position 42 of the human amyloid beta protein, which includes both 12 and 42, in particular the amino acid sequence VHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA or natural variants thereof, in particular A21G (“Flanders”), E22G (“Arctic”), E22Q (“Holland”), E22K (“Italian”), D23N (“Iowa”), A42T and A42V (values are , 12 and 42) or no more than 3 alternative amino acid substitutions, none of which interfere with globulomer formation. preferably without another amino acid substitution in the region from amino acid 20 to amino acid 42.

More specifically, the term "Aβ(20-42)" herein refers to the amino acid sequence from amino acid position 20 to amino acid position 42 of the human amyloid beta protein, which includes both 20 and 42, in particular the amino acid sequence FAEDVGSNKGAIIGLMVGGVVIA or natural variants thereof, in particular A21G (“Flanders”), E22G (“Arctic”), E22Q (“Holland”), E22K (“Italian”), D23N (“Iowa”), A42T and A42V (values are at least one mutation selected from the group consisting of Aβ peptide initiation, including both 20 and 42) or no more than 3 alternative amino acid substitutions, none of which interfere with globulomer formation. and preferably refers to a sequence without another amino acid substitution.

The term "A[beta](X-Y) globulomer" (A[beta](X-Y) globular oligomer) as used herein refers to the A[beta](X-Y) peptide soluble and spherical oligomer as defined above having homogeneity and distinct physical properties. refers to the non-covalent association of According to one aspect, A[beta](X-Y) globulomers are stable, non-fibrillar, oligomeric assemblies of A[beta](X-Y) peptides obtainable by incubation with an anionic detergent. In contrast to monomers and fibrils, these globulomers have a designated number of subunits (e.g. early assembly type as described in WO2004/067561, n=4 to 6, "oligomer A" and late assembly type, n=12 to 14, "Oligomer B"). Globulomers have a three-dimensional globular-type structure (“molten globules”, see Barghom et al., 2005, J Neurochem, 95, 834-847). They can be further characterized by one or more of the following characteristics.

- The N-terminal amino acid X-23 can be cleaved by promiscuous proteases (such as thermolysin or endoprotease GluC) to generate truncated globulomers.

- C-terminal amino acid 24-Y is inaccessible for promiscuous proteases and antibodies.

- The truncated forms of these globulomers maintain the three-dimensional nuclear structure of the globulomers with better accessibility of the nuclear epitope A[beta](20-Y) in the globulomer conformation.

According to the present invention, in particular with regard to evaluating the binding affinity of the antibodies of the invention, the term "Aβ(X-Y) globulomer" as used herein is referred to in WO 2004/067561 (incorporated herein by reference). It refers to a product that can be obtained by the method of

The method comprises opening a natural, recombinant or synthetic Aβ(X-Y) peptide or derivative thereof; exposing the at least partially opened Aβ(X-Y) peptide or derivative thereof to a detergent; reducing the action of the formulation; and continuing the incubation.

For peptide unfolding, a hydrogen bond scission agent, such as hexafluoroisopropanol (HFIP), can be applied to the protein. If the action temperature is about 20 to 50° C., in particular about 35 to 40° C., an action time of several minutes, for example about 10 to 60 minutes, is sufficient. The evaporated residue, preferably in a concentrated form, is then at least partially dissolved in a suitable organic solvent miscible with aqueous buffers, such as dimethylsulfoxide (DMSO). A suspension of unfolded peptide or derivative thereof is obtained, which can then be used. If desired, the stock suspension can be stored at a reduced temperature, eg, about -20°C, for extended periods of time.

Alternatively, the peptide or derivative thereof can be taken up in a slightly acidic, preferably aqueous solution, eg, about 10 mM HCl in water. After an incubation period, usually several minutes, the insoluble components are removed by centrifugation. 10000 g for several minutes is convenient. These process steps are preferably carried out at room temperature, ie a temperature in the range of 20 to 30°C. The supernatant obtained after centrifugation contains the A[beta](X-Y) peptides or derivatives thereof and can be stored at a low temperature, e.g. about -20[deg.]C, for some time.

Subsequent exposure to a detergent involves oligomerization of the peptide or its derivative to form an intermediate oligomer (referred to as Oligomer A in WO2004/067561). In this regard, the detergent can act on the at least partially unfolded peptide or derivative thereof until sufficient intermediate oligomers are produced.

It is preferable to use an ionic surfactant, particularly an anionic surfactant.

According to certain embodiments, a surfactant of formula (I) below is used.

RX
wherein the group R is unbranched or branched alkyl having 6 to 20, preferably 10 to 14 carbon atoms or unbranched or branched alkenyl having 6 to 20, preferably 10 to 14 carbon atoms and the group X is an acidic group or a salt thereof, and X is preferably:
-COO-M ⁺ , _-SO3M ⁺ , especially _-OSO3M ⁺ , where M ⁺ is a hydrogen cation or an inorganic or organic cation, preferably selected from alkali metal and alkaline earth metal cations and ammonium cations is.

Advantageous are surfactants of formula (I) in which R is unbranched alkyl, among which the alk-1-yl group is particularly to be mentioned. Especially preferred is sodium dodecyl sulfate (SDS). Lauric acid and oleic acid can also be used with advantage. Also particularly advantageous is the sodium salt of the surfactant lauroyl sarcosine (also called sarkosyl NL-30 or Gardol®).

The duration of detergent action depends, inter alia, on whether and to what extent the peptides or derivatives thereof undergoing oligomerization have been unfolded. According to the unfolding step, the working temperature is about 20 to 50° C., especially about 35 to 40° C., when the peptide or derivative thereof is treated with a hydrogen bond scissioning agent, i.e. in particular hexafluoroisopropanol. In some cases action times in the range of several hours, preferably from about 1 to 20 hours, in particular from about 2 to 10 hours, are sufficient. If the starting point is a peptide or derivative thereof with little or no substantial unfolding, a correspondingly long duration of action is good. If the peptide or derivative thereof has been pretreated, for example according to the procedure indicated above as an alternative to HFIP treatment, or if the peptide or derivative thereof undergoes direct oligomerization, the working temperature is about 20 to 50°C. C., in particular about 35 to 40.degree. C., action times in the range of about 5 to 30 hours, in particular about 10 to 20 hours, are sufficient. After incubation, insoluble components are removed, advantageously by centrifugation. A few minutes at 10000g is good.

The surfactant concentration selected will depend on the surfactant used. When using SDS, a concentration in the range of 0.01 to 1% by weight, preferably 0.05 to 0.5% by weight, for example about 0.2% by weight, has been found to be good. If lauric acid or oleic acid is used, slightly higher concentrations, eg in the range of 0.05 to 2% by weight, preferably 0.1 to 0.5% by weight, eg about 0.5% by weight, are good.

Surfactant action should occur at salt concentrations approximately in the physiological range. In particular, therefore, NaCl concentrations in the range from 50 to 500 mM, preferably from 100 to 200 mM, in particular about 140 mM are good.

Subsequent reduction of detergent action and continued incubation is associated with obtaining the Aβ(X-Y) globulomers of the invention by further oligomerization (referred to as Oligomer B in WO2004/067561). . Since compositions obtained from previous steps always contain surfactants and salt concentrations in the physiological range, it is good to reduce surfactant action, preferably even salt concentrations. This can be done by reducing the detergent and salt concentration, for example by conveniently diluting with water or a relatively low salt buffer such as Tris-HCl (pH 7.3). . A dilution factor in the range of about 2 to 10, preferably in the range of about 3 to 8, especially about 4, has been found to be suitable. Reduction of surfactant action can also be achieved by adding substances capable of neutralizing said surfactant action. Examples of these include substances capable of complexing surfactants, such as substances capable of stabilizing cells during purification and extraction procedures, e.g. certain EO/PO block copolymers, especially pyuronic ( and a block copolymer under the trade name Piuronic® F68. Alkoxylation, especially ethoxylated alkylphenols (e.g. Triton X series ethoxylated t-octylphenols, especially Triton®) in a concentration range near or above a certain critical micelle concentration X100, 3-(3-chloramidopropyldimethylammonio)-1-propanesulfonate (CHAPS®) or alkoxylated, especially ethoxylated sorbitan fatty acid esters (e.g. Tween® series, especially can equivalently use Tween® 20).

The solution is then incubated until sufficient A[beta](X-Y) globulomers of the invention are produced. If the working temperature is about 20 to 50° C., especially about 35 to 40° C., working times in the range of several hours, preferably in the range of about 10 to 30 hours, especially in the range of about 15 to 25 hours are sufficient. is. The solution can then be concentrated and any residue removed by centrifugation. Again, 10000 g for several minutes has been found to be good. The supernatant obtained after centrifugation contains the Aβ(X-Y) globulomers of the invention.

The A[beta](X-Y) globulomers of the invention can finally be recovered in a manner known per se, for example by ultrafiltration, dialysis, precipitation or centrifugation.

It is further preferred that electrophoretic separation of Aβ(X-Y) globulomers under denaturing conditions, e.g. by SDS-PAGE, yields double bands (e.g. in the case of Aβ(1-42) the apparent molecular weight is 38 /48 kDa), it is particularly preferred that these two bands converge upon glutardialdehyde treatment of the globulomers prior to separation. Size exclusion chromatography of globulomers also preferably results in a single peak (e.g. corresponding to a molecular weight of about 60 kDa for A[beta](1-42)).

Starting from Aβ(1-42), Aβ(12-42) and Aβ(20-42) peptides, the process proceeds to Aβ(1-42) globulomers, Aβ(12-42) globulomers and Aβ(20 -42) It is particularly suitable for obtaining globulomers.

In certain embodiments of the present invention, Aβ(X-Y) globulomers (where X is selected from the group consisting of the numbers 2..24 and Y is as defined above) are Aβ(1-Y) globulomers to a shorter form, where X is selected from the group consisting of the numbers 2..24, X is preferably 20 or 12, and Y is as defined above. and can be achieved by treatment with an appropriate protease. For example, Aβ(20-42) globulomers can be obtained by subjecting Aβ(1-42) globulomers to thermolysin proteolysis, and Aβ(12-42) globulomers are converted to Aβ(1-42) globulomers. It can be obtained by performing endoproteinase GluC proteolysis. Once the desired degree of proteolysis has been achieved, the protease is inactivated by known methods. The resulting globulomers can then be isolated according to the procedures already described herein and, if necessary, further processed by further work-up and purification steps. A detailed description of said process is disclosed in WO2004/067561, which is incorporated herein by reference.

As used herein, the term "A[beta](X-Y) monomer" refers to an isolated A[beta](X-Y) peptide, preferably A[beta] not associated with other A[beta] peptides in essentially non-covalent interactions. (X-Y) Refers to one form of peptide. In practice, A[beta](X-Y) monomers are usually provided in the form of an aqueous solution. In a particularly preferred embodiment of the invention, the aqueous monomer solution contains 0.05% to 0.2%, more preferably about 0.1% _NH4OH . In another particularly preferred embodiment of the present invention, the aqueous monomer solution contains 0.05% to 0.2%, more preferably about 0.1% NaOH. When used (eg, to measure the binding affinity of an antibody of the invention), it may be advisable to dilute the solution in an appropriate manner. More usually, the solution should be used within 2 hours, especially within 1 hour, especially within 30 minutes, from its preparation.

As used herein, the term "fibril" refers to a non-covalently Refers to a molecular structure comprising an assembly of individual A[beta](X-Y) peptides in association.

In another aspect of the invention, the fibrils comprise self-induced polymer aggregation of suitable Aβ peptides in the absence of detergent, for example in 0.1 M HCl, to more than 24 units, preferably 100 units. Molecular structures obtainable by processes resulting in super-aggregate formation. This process is known in the art. Conveniently, A[beta](X-Y) fibrils are used in the form of an aqueous solution. In a particularly preferred embodiment of the invention, the Aβ peptide is dissolved in 0.1% NH ₄ OH and diluted 1:4 with 20 mM NaH ₂ PO ₄ , 140 mM NaCl (pH 7.4) followed by readjusting the pH. 7.4, the solution was incubated at 37° C. for 20 hours, followed by centrifugation at 10000 g for 10 minutes and resuspension in 20 mM NaH ₂ PO ₄ , 140 mM NaCl (pH 7.4). , an aqueous fibril solution is obtained.

The present invention further relates to the following content.

The results obtained for soluble globular Aβ(1-42) oligomers are consistent with individual soluble globular Aβ(1-40) oligomers as well as individual truncated Aβ(X-38), Aβ(X-39), Aβ(X-40) ), Aβ(X-41), Aβ(X-42) or Aβ(X-43), preferably Aβ(X-42) or Aβ(X-40) oligomers and/or their respective crosslinked oligomers. Relevant and applicable. Thus, according to the present invention, the term "soluble globular Aβ(1-42) oligomers ("globulomers") includes, for example, "soluble globular Aβ(1-40) oligomers" as well as "soluble globular Aβ(X-42) oligomers" and "soluble globular Aβ (X-40) oligomers" and/or crosslinked derivatives thereof ("x is in each case independently the number 2..24, preferably 8..24, more preferably 12 ..24, more preferably 12..20 and most preferably 17..20, the abbreviations having the meanings given above (i.e. 1..24 are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24, etc.).) is also understood to include

Furthermore, the results according to the present invention for non-diffusible water-soluble globular Aβ(1-42) oligomers (“globulomers”) show individual Aβ(X-42) oligomers and/or Aβ(X-40) oligomers (X is In each case independently selected from the group consisting of the numbers 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20. ), as well as crosslinked derivatives thereof, were found to be relevant and applicable.

During further investigation of the above screening methods, the inventors also investigated chemical products described in the literature. Surprisingly, the inventors of the present invention have observed that oligomers according to WO98/33815 (and similar publications) are not homogeneous identifiable substances or homogeneous mixtures of identifiable substances. Unexpectedly, we found that oligomers according to WO 98/33815 (and similar publications) actually contained about 5% of non-diffusible, soluble globular Aβ(1-42) oligomeric epitopes (“globulomer epitopes”). and of about 95% contain a mixture of fibril-prone monomers, such as diffusible oligomers, as protofibril precursors (“fibriromers”) on their way to fibrils.

Even more surprisingly, other known Aβ(1-42) oligomeric preparations also contain non-diffusible soluble globular Aβ(1-42) oligomeric epitopes (“globulomer epitopes”) in amounts of about 5%. is also allowed.

Calculations suggest that Aβ(1-42) globulomers consist of 12 or about 12 Aβ subunits, but with the same characteristic changes in epitope structure but fewer Aβ chains (e.g., 4 subunits only) aggregates are likely and suggestive. Typically this is the case for "Oligomer A" as described in WO2004/067561 (Abbott GmbH & Co. KG) (see Figure 18).

According to one aspect of the invention, non-diffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X- 40), and/or bridged derivatives thereof (where X is independently in each case the numbers 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most qualitative, characterized by determining the presence (=positive diagnosis) or absence (=negative diagnosis) or the amount of autoantibodies with specificity against (preferably selected from the group consisting of 17..20) Methods are provided for diagnosing (=positive or negative) or quantitatively in vitro or in vivo the presence or absence of Alzheimer's disease (AD) or the patient's risk of developing AD in a human or animal patient.

According to a preferred embodiment of the present invention, the above method is characterized in that the sample is selected from the group consisting of cells, cell aggregates, body tissue (cell) samples, body fluid samples and body tissue (cell)/body fluid samples, Each sample is characterized in that it is from an animal body, preferably a human, non-human animal or non-human transgenic animal body.

According to another preferred embodiment of the present invention, the above method is characterized in that the monoclonal or polyclonal antibodies are non-diffusible soluble globular Aβ (X-42) and/or Aβ (X-40) oligomers (“globulomers”) and /or bridged derivatives thereof (where X is independently in each case the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..24). selective for globulomer structure-type-specific epitopes located between about 20 and about 30 amino acid positions of (selected from the group consisting of 20).

The term "about" when used above or elsewhere in the specification and claims means that the exact position is within normal experimental and manufacturing ranges from 0 to 1, 2 or 3 amino acid positions. Used to indicate that something can vary.

The term "about 20 to about 30 amino acid positions" is used in the present invention and herein, without limitation, at least "about 20 to about 30 amino acid positions", "about 21 to about 30 amino acid positions," "about 22 to about 30 amino acid positions," "about 23 to about 30 amino acid positions," and "about 24 to about 30 amino acid positions." be. The term "about 30," independently of the meaning of the term "about 20," is not limited thereto and includes at least 26. . shall be understood to include numbers selected from the group consisting of 34; The term "about 20," independently of the meaning of the term "about 30," is not limited thereto and includes at least 16. . shall be understood to include numbers selected from the group consisting of twenty-four.

According to a preferred embodiment, the above method is characterized in that the sample is selected from the group consisting of cells, cell aggregates, body tissue (cell) samples, body fluid samples and body tissue (cell)/body fluid samples, wherein each said sample is an animal It is characterized by being from a body, preferably a human, non-human animal or non-human transgenic animal body.

According to a preferred embodiment, the above method is characterized in that the monoclonal or polyclonal antibody comprises nondiffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers. (“globulomers”) and/or bridged derivatives thereof (where X is independently in each case the numbers 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and Most preferably selected from the group consisting of 17..20), it is selective for globulomer structure type-specific epitopes located between about 20 and about 30 amino acid positions.

According to a preferred embodiment, the above-described method comprises producing non-diffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42)- and/or Aβ(X-40) oligomers (“globulomers”). is soluble.

According to another aspect of the invention, non-diffusible globular Aβ (X-38/43) oligomers, preferably Aβ (X-42), are isolated from the product by using one or more per se known peptide purification processes. ) and/or Aβ(X-40) oligomers (“globulomers”) (where X is in each case independently the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12 ..20 and most preferably 17..20) and/or crosslinked derivatives thereof are selectively and reversibly captured.

According to yet another aspect of the present invention, at least one non-diffusible spherical Aβ(X-38/43) oligomer, preferably Aβ(X-42) and/or Aβ(X-42), in an artificial or natural preparation. -40) oligomers ("globulomers"), and/or crosslinked derivatives thereof (X is in each case independently the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof, wherein a preparation or a representative portion thereof is subjected to at least one Specific monoclonal or polyclonal specificity for diffusible globular Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers and/or crosslinked derivatives thereof A method is provided comprising contacting with an antibody and then measuring the amount of antibody-globulomer binding product obtained.

According to a preferred embodiment of the invention, said method is a dot blot and ELISA anti-Aβ immunoassay as described below or in WO2004/067561.

According to a preferred embodiment of the invention, the above method is characterized in that the monoclonal or polyclonal antibody is a non-diffusible soluble globular Aβ(X-38/43) oligomer, preferably Aβ(X-42) and/or Aβ(X-42). -40) characterized by being specific for globulomer structure type-specific epitopes located at or between about 20 to about 30 amino acid positions of the oligomer ("globulomer") and/or its crosslinked derivatives .

According to another preferred embodiment of the present invention, the method is characterized in that the non-diffusible globular Aβ (X-38/43) contained in the preparation is purified by using one or more per se known peptide purification method steps. ) oligomers, preferably Aβ(X-42)- and/or Aβ(X-40) oligomers (X is in each case independently the number 1..24, preferably 8..24, more preferably 12..). 24, more preferably selected from the group consisting of 12..20 and most preferably 17..20) and/or its crosslinked derivatives.

According to another preferred embodiment of the present invention, the concentration method described above is ≥50 wt%, preferably ≥60 wt%, more preferably ≥70 wt%, more preferably ≥80 wt%, more preferably % by weight or more, more preferably 95% by weight or more, more preferably 99% by weight or more, more preferably 99.9% by weight or more, more preferably 99.99% by weight or more of non-diffusible spherical Aβ (X-38 /43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (X is in each case independently the number 1..24, preferably 8..24, more preferably 12. .24, more preferably 12..20 and most preferably 17..20) and/or its crosslinked derivatives.

According to another preferred embodiment of the present invention, the enrichment method described above uses one or more per se known peptide purification process steps to extract non-diffusible globular Aβ (X-38/43) from the preparation. oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) (X is in each case independently the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof. and

According to another preferred embodiment of the present invention, the enrichment method described above uses one or more per se known peptide purification process steps to extract non-diffusible globular Aβ (X-38/43) from the preparation. having the step of reversibly or irreversibly removing substances other than oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) and/or crosslinked derivatives thereof. characterized by

According to another preferred embodiment of the present invention, the enrichment method described above comprises nondiffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers. characterized in that the step of measuring the amount of (“globulomer”) and/or crosslinked derivatives thereof is performed only after one or more of the concentration steps have been performed.

According to another preferred embodiment of the present invention, the above method of measurement and/or enrichment is carried out by or Aβ(X-40) oligomers (“globulomers”) are cross-linked using one or more per se known cross-linking steps that can be performed before, during and/or after the enrichment step and/or the measurement step. characterized by

Crosslinking can be achieved by treatment with glutardialdehyde, for example. Suitable cross-linking methods are described in WO2004/067561. A cross-linking step can be performed before, during and/or after the concentration procedure.

According to another preferred embodiment of the invention, the above method of measurement and/or enrichment comprises nondiffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42)- and/or Aβ( X-40) oligomers (“globulomers”) are characterized by being soluble.

According to another preferred embodiment of the present invention, the purified or essentially purified or highly purified non-purified Diffusible globular Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”), where X is in each case independently the number 1..24 , preferably selected from the group consisting of 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof. .

According to another preferred embodiment of the present invention, the purified or essentially purified or highly purified non-purified Diffusible globular Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”), where X is in each case independently the number 1..24 , preferably selected from the group consisting of 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof. .

According to another preferred embodiment of the present invention, the above purified or essentially purified or highly purified non-diffusible globular Aβ(X-38/43) oligomers, preferably Aβ(X- 42) and/or Aβ(X-40) oligomers (“globulomers”) are non-diffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42)- and/or Aβ(X-40) ) oligomers (“globulomers”) are soluble.

According to another preferred embodiment of the present invention, the purified or essentially purified or highly purified non-purified Diffusible globular Aβ(X-38/43) oligomers, preferably Aβ(X-42) or Aβ(X-40) oligomers (“globulomers”) (X is in each case independently the number 1..24, preferably is selected from the group consisting of 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or a crosslinked derivative thereof. .

According to another preferred embodiment of the present invention, the above composition comprises non-diffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42)- and/or Aβ(X-40) Oligomers (“globulomers”) are characterized by being soluble.

According to another aspect of the present invention, purified or essentially purified or highly purified non-diffusible globular Aβ (X -38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) (X is in each case independently the number 1..24, preferably 8..24). 24, more preferably selected from the group consisting of 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof in patients in need of treatment of Alzheimer's disease. Use as a vaccine for active immunization against disease (AD) is provided.

According to another aspect of the present invention, in the manufacture of a specific aptamer against Alzheimer's disease (AD) in a patient in need of treatment, purified or essentially purified or highly purified non-diffusible globular Aβ (X-38/43) oligomers, preferably Aβ (X-42) and/or Aβ (X-40) oligomers (“globulomers”) (X is in each case independently selected from the group consisting of the numbers 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20 provided) and/or the use of crosslinked derivatives thereof.

Purified or essentially purified or highly purified non-diffusible globular Aβ(X-38/43) oligomers identified above, preferably Aβ(X-42) and/or Aβ(X- 40) Methods for making specific aptamers suitable for oligomers (“globulomers”) are known to those skilled in the art. See, for example, published German patent application DE 199 16 417 A1.

The term "active" or "passive" immunization refers to disease treatment of patients in which the onset or development or progression of Alzheimer's disease (AD) is prevented or delayed or is related to the degree of neuronal loss and the severity of cognitive impairment. to halt or slow down the pathogenesis of AD.

According to another aspect of the invention, in the manufacture of a medicament for passive immunization against Alzheimer's disease (AD) in a patient in need of treatment, obtained by or obtainable by the above method , purified or essentially purified or highly purified non-diffusible globular Aβ (X-38/43) oligomers, preferably Aβ (X-42) and/or Aβ (X-40) oligomers ( "globulomers") (X is in each case independently from the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20 (selected from the group consisting of) and/or crosslinked derivatives thereof.

According to another aspect of the invention, nondiffusible spherical Aβ (X-42) and/or Aβ (X-40) oligomers useful in passive immunization against Alzheimer's disease in patients in need of treatment ( "globulomers") and/or crosslinked derivatives thereof, obtained by or by the above methods for producing monoclonal or polyclonal, natural or recombinant human or animal or chimeric antibodies having specificity for Purified or essentially purified or highly purified non-diffusible globular Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X -40) oligomers ("globulomers") (X is in each case independently the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof.

According to another preferred embodiment of the present invention, the use of any of the above comprises nondiffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42)- and/or Aβ(X- 40) Oligomers (“globulomers”) are characterized by being soluble.

According to another aspect of the invention, nondiffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) (X is , in each case independently selected from the group consisting of the numbers 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or a crosslinked derivative thereof. purified or essentially purified or highly purified non-diffusible globular Aβ (X-42) and/or Aβ (X-40) oligomers (“ globulomers") and/or crosslinked derivatives thereof, and for nondiffusible spherical Aβ(X-42) and/or Aβ(X-40) oligomers ("globulomers") and/or crosslinked derivatives thereof. a cell that produces an antibody with specificity for a non-diffusible globular Aβ (X-42) and/or Aβ (X-40) oligomer (“globulomer”) and/or cross-linked derivative. A method of manufacture is provided comprising the steps of isolating from an organism and optionally determining the chemical sequence and/or structure of the obtained antibody.

According to another preferred embodiment of the present invention, the method as described above is characterized in that nondiffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42)- and/or Aβ(X-40) oligomers ("globulomers") are characterized by being soluble.

According to another aspect of the present invention, obtained according to the above method or obtainable according to the above method, for active or passive immunization against Alzheimer's disease (AD) in a patient in need of treatment, Non-diffusible globular Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”), where X is in each case independently the number 1 . 24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof Monoclonal or polyclonal, natural or recombinant, humanized, human, animal or chimeric antibodies with specificity are provided.

According to another preferred embodiment of the invention, said monoclonal or polyclonal, natural or recombinant, humanized, human, animal or chimeric antibody comprises nondiffusible globular Aβ (X-38/43) oligomers, preferably non-diffusible globular Aβ (X-38/43) oligomers. Aβ(X-42)- and/or Aβ(X-40) oligomers (“globulomers”) are characterized by being soluble.

According to another aspect of the present invention, a non-active immunization agent obtained according to the above method or obtainable according to the above method for active or passive immunization against Alzheimer's disease (AD) in a patient in need of treatment. Diffusible globular Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”), where X is in each case independently the number 1..24 , preferably selected from the group consisting of 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof. Compositions are provided comprising monoclonal or polyclonal, natural or recombinant, human, humanized, animal or chimeric antibodies with specificity.

According to another preferred embodiment of the invention, said composition comprises nondiffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42)- and/or Aβ(X-40) oligomers. ("globulomers") are characterized by being soluble.

According to another aspect of the present invention, a method of positively or negatively diagnosing a patient with Alzheimer's disease (AD) or at high risk of developing Alzheimer's disease, comprising: Nondiffusible globular Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”), where X is in each case independently the number 1 . .24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or a crosslinked derivative thereof. A method is provided characterized by determining the presence (positive diagnosis) or absence (negative diagnosis) of antibodies.

According to a preferred embodiment of the present invention, the above method, wherein the sample is selected from the group consisting of cells, cell aggregates, body tissue (cell) samples, body fluid samples and body tissue (cell)/body fluid samples, It is characterized in that the sample is from a human, animal or transgenic animal body.

According to a preferred embodiment of the present invention, the above method comprises nondiffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42)- and/or Aβ(X-40) oligomers (" Globulomers") are characterized by being soluble.

According to another aspect of the invention, nondiffusible globular Aβ (X-38/43) as a diagnostic marker for identifying Alzheimer's disease (AD) or increased risk of developing Alzheimer's disease in a patient in need of treatment. ) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) (X is in each case independently the number 1..24, preferably 8..24, more preferably is selected from the group consisting of 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof. be done.

According to a preferred embodiment of the present invention, humanized to nondiffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) , the use of human, animal or chimeric antibodies to increase the presence of nondiffusible spherical Aβ (X-38/43) oligomers, preferably Aβ (X-42)- and/or Aβ (X-40) oligomers (“globulomers”) It is characterized by being soluble.

According to another aspect of the present invention, in the manufacture of a medicament for the treatment or prevention of Alzheimer's disease or related diseases such as Down's syndrome in a patient in need of such treatment, one or more that reduce fatty acid levels in the brain of a patient. is provided.

According to another aspect of the invention, nondiffusible globular Aβ (X-Y ) oligomers (X is in each case independently the group consisting of the numbers 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20 wherein Y is in each case independently selected from the group consisting of the numbers 38, 39, 41 and 43) and/or bridged derivatives thereof are provided.

With respect to the present invention, including this specification, claims and drawings, the term "Aβ(1-42) oligomers" refers to Aβ(X-42) oligomers, Aβ(X-40) oligomers and/or cross-links thereof. derivatives (X is in each case independently from the group consisting of the numbers 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20 selected).

In particular, the following definitions are applicable throughout the specification, claims and drawings.

According to the present invention, the term "soluble" refers to Aβ(1-Y) globulomers with high or appreciable water solubility (hydrophilicity) of about 15 mg/mL or more, or proteins (e.g. HAS) or cleaved Aβ(X-Y) globulomers (where X is independently a number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20, wherein Y is independently in each case a number 38..43).

According to the present invention, the term "Aβ (X-38/43) oligomers" means "Aβ (X-38) oligomers", "Aβ (X-39) oligomers", "Aβ (X-40) oligomers". , “Aβ (X-41) oligomers”, “Aβ (X-42) oligomers” and “Aβ (X-43) oligomers” and/or bridged derivatives thereof (where X is in each case independently the number 1 . . . ). 24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20). Defined to include Aβ oligomers.

According to the present invention, the term "A[beta](X-38) oligomer" means at least one A[beta](X-38) oligomer and/or bridged derivatives thereof, where X is the number 1..24, preferably 8.0. .24, more preferably 12..24, more preferably 12..20 and most preferably 17..20).

According to the present invention, the term "A[beta](X-39) oligomer" means at least one A[beta](X-39) oligomer and/or bridged derivatives thereof, where X is the number 1..24, preferably 8.0. .24, more preferably 12..24, more preferably 12..20 and most preferably 17..20).

According to the present invention, the term "A[beta](X-40) oligomer" means at least one A[beta](X-40) oligomer and/or crosslinked derivatives thereof, where X is the number 1..24, preferably 8.0. .24, more preferably 12..24, more preferably 12..20 and most preferably 17..20).

According to the present invention, the term "A[beta](X-41) oligomer" means at least one A[beta](X-41) oligomer and/or crosslinked derivatives thereof, where X is the number 1..24, preferably 8.0. .24, more preferably 12..24, more preferably 12..20 and most preferably 17..20).

According to the present invention, the term "A[beta](X-42) oligomer" means at least one A[beta](X-42) oligomer and/or crosslinked derivatives thereof, where X is the number 1..24, preferably 8.0. .24, more preferably 12..24, more preferably 12..20 and most preferably 17..20).

According to the present invention, the term "A[beta](X-43) oligomer" means at least one A[beta](X-43) oligomer and/or crosslinked derivatives thereof, where X is the number 1..24, preferably 8.0. .24, more preferably 12..24, more preferably 12..20 and most preferably 17..20).

According to another aspect of the invention, qualitative (=positive or negative) or quantitative in vitro or in vivo diagnosis in human or non-human animal subjects for Alzheimer's disease (AD) or for the presence or absence of risk of developing AD. wherein at least one non-diffusible globular Aβ (X-38/43) oligomeric structural epitope, preferably at least one non-diffusible globular Aβ ( X-42) and/or Aβ (X-40) oligomeric structural epitopes, and/or bridged derivatives thereof (X is in each case independently the number 1..24, preferably 8..24, more preferably 12 ..24, more preferably selected from the group consisting of 12..20 and most preferably 17..20). ) or measuring the amount.

According to a preferred embodiment of the present invention, the above method is characterized in that the sample is selected from the group consisting of cells, cell aggregates, body tissue (cell) samples, body fluid samples and body tissue (cell)/body fluid samples, Each sample is characterized in that it is from an animal body, preferably a human, non-human or non-human transgenic animal body.

According to a preferred embodiment of the present invention, the method is characterized in that the monoclonal or polyclonal antibody comprises at least non-diffusible globular Aβ(X-38/43) oligomers, preferably non-diffusible globular Aβ(X-42)- and /or Aβ(X-40) oligomers (“globulomers”) and/or crosslinked derivatives thereof (where X is independently in each case the number 1..24, preferably 8..24, more preferably 12..24 , more preferably 12..20 and most preferably 17..20) are selective for globulomer structure type-specific epitopes located between about 20 and about 30 amino acid positions of characterized by

According to a preferred embodiment of the present invention, the above method comprises non-diffusible globular Aβ(X-38/43) oligomers, preferably non-diffusible globular Aβ(X-42)- and/or Aβ(X-40). ) oligomers (“globulomers”) are soluble.

According to another aspect of the invention, at least one non-diffusible spherical Aβ(X-38/43) oligomer, preferably Aβ(X-42) and/or Aβ(X-42), in an artificial or natural preparation. 40) oligomers (“globulomers”), and/or crosslinked derivatives thereof (where X is in each case independently the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12 ..20 and most preferably selected from the group consisting of 17..20) and/or crosslinked derivatives thereof, wherein a preparation or a representative portion thereof is subjected to at least one non-diffusing specificity to a specific globular Aβ(X-38/43) oligomeric structural epitope, preferably at least one Aβ(X-42) and/or Aβ(X-40) oligomeric structural epitope and/or cross-linked derivatives thereof A method is provided comprising contacting with a specific monoclonal or polyclonal antibody having a specific monoclonal or polyclonal antibody and then measuring the amount of antibody-globulomer binding product obtained.

In particular, the monoclonal or polyclonal antibody is a non-diffusible globular Aβ (X-38/43) oligomer, preferably a non-diffusible globular Aβ (X-42) and/or Aβ (X-40) oligomer (“globulomer”). and/or is specific for a globulomer structure type-specific epitope located at or between about 20 to about 30 amino acid positions of and/or a bridged derivative thereof.

According to a preferred embodiment of the present invention, the method described above uses one or more per se known peptide purification process steps to purify the non-diffusible globular Aβ (X-38/43) contained in the preparation. oligomers, preferably nondiffusible globular Aβ(X-42) and/or Aβ(X-40) oligomers (X is in each case independently the number 1..24, preferably 8..24, more preferably 12 ..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof.

According to a preferred embodiment of the present invention, the concentration method described above comprises a More preferably 95% by weight or more, more preferably 99% by weight or more, more preferably 99.9% by weight or more, more preferably 99.99% by weight or more of non-diffusible spherical Aβ (X-38/43 ) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (X is in each case independently the number 1..24, preferably 8..24, more preferably 12..24 , more preferably selected from the group consisting of 12..20 and most preferably 17..20) and/or enriched amounts of crosslinked derivatives thereof.

According to another preferred embodiment of the present invention, the enrichment method described above uses one or more per se known peptide purification process steps to extract non-diffusible globular Aβ (X-38/43) from the preparation. oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) (X is in each case independently the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof. and

According to another preferred embodiment of the present invention, the enrichment method described above uses one or more per se known peptide purification process steps to extract non-diffusible globular Aβ (X-38/43) from the preparation. reversibly or irreversibly remove substances other than oligomers, preferably non-diffusible globular Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) and/or crosslinked derivatives thereof It is characterized by having steps.

According to another preferred embodiment of the present invention, the enrichment method described above comprises nondiffusible spherical Aβ(X-38/43) oligomers, preferably Aβ(X-42) and/or Aβ(X-40) oligomers. characterized in that the step of measuring the amount of (“globulomer”) and/or crosslinked derivatives thereof is performed only after one or more of the concentration steps have been performed.

According to another preferred embodiment of the invention, the above method of measurement and/or enrichment is carried out using the obtained non-diffusible globular Aβ (X-38/43) oligomers, preferably non-diffusible globular Aβ (X-42). )- and/or Aβ(X-40) oligomers (“globulomers”) using one or more per se known cross-linking steps that can be performed before, during and/or after the concentration step and/or the measurement step It is characterized in that it is crosslinked by

According to a preferred embodiment of the present invention, the above method of measurement and/or enrichment comprises non-diffusible globular Aβ(X-38/43) oligomers, preferably non-diffusible globular Aβ(X-42)- and/or Aβ(X-40) oligomers (“globulomers”) are characterized as being soluble.

According to another aspect of the invention, the purified or essentially purified or highly purified non-diffusible as described above, obtained by the above method or obtainable by the above method Globular Aβ(X-38/43) oligomers, preferably nondiffusible globular Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) (where X is independently in each case the number 1 . .24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof. be done.

According to another preferred embodiment of the present invention, the above-mentioned purified or essentially purified or highly purified non-diffusible globular Aβ (X-38/43) oligomers, preferably non-diffusible globular Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) are non-diffusible spherical Aβ(X-38/43) oligomers, preferably non-diffusible spherical Aβ(X-42)- and/or the Aβ(X-40) oligomers (“globulomers”) are soluble.

According to another aspect of the invention, the purified or essentially purified or highly purified non-diffusible as described above, obtained by the above method or obtainable by the above method Globular Aβ(X-38/43) oligomers, preferably nondiffusible globular Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) (where X is independently in each case the number 1 . .24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof A composition is provided.

According to a preferred embodiment of the present invention, the above composition comprises non-diffusible spherical Aβ(X-38/43) oligomers, preferably non-diffusible spherical Aβ(X-42)- and/or Aβ(X- 40) Oligomers (“globulomers”) are characterized by being soluble.

According to another aspect of the present invention, purified or essentially purified or highly purified non-diffusible globular Aβ (X -38/43) oligomers, preferably nondiffusible spherical Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) (X is in each case independently the number 1..24, preferably is selected from the group consisting of 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof in need of treatment. use as a vaccine for active immunization against Alzheimer's disease (AD) in patients with

According to another aspect of the present invention, in the manufacture of a specific aptamer against Alzheimer's disease (AD) in a patient in need of treatment, purified or essentially purified or highly purified non-diffusible globular Aβ (X-38/43) oligomers, preferably non-diffusible globular Aβ (X-42) and/or Aβ (X-40) oligomers ( "globulomers") (X is in each case independently from the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20 (selected from the group consisting of) and/or crosslinked derivatives thereof.

According to another aspect of the present invention, in the manufacture of a medicament for passive immunization against Alzheimer's disease (AD) in a patient in need of treatment, obtained by or obtainable by the above method purified or essentially purified or highly purified non-diffusible globular Aβ(X-38/43) oligomers, preferably non-diffusible globular Aβ(X-42) and/or Aβ(X -40) oligomers ("globulomers") (X is in each case independently the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof.

According to another aspect of the invention, at least one non-diffusible globular Aβ(X-38/43) oligomeric structural epitope, preferably at least one non-diffusible globular Aβ(X-42) and/or Aβ(X-42) -40) in the production of monoclonal or polyclonal, natural or recombinant human or animal or chimeric antibodies with specificity for oligomeric structural epitopes ("globulomers") and/or cross-linked derivatives thereof, obtained by the methods described above or purified or essentially purified or highly purified non-diffusible globular Aβ (X-38/43) oligomers, preferably non-diffusible globular Aβ (X- 42) and/or Aβ(X-40) oligomers (“globulomers”) (where X is in each case independently the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof.

According to a preferred embodiment of the present invention, the above use comprises non-diffusible globular Aβ(X-38/43) oligomers, preferably non-diffusible globular Aβ(X-42)- and/or Aβ(X-40) ) oligomers (“globulomers”) are soluble.

According to another aspect of the invention, at least one non-diffusible globular Aβ(X-38/43) oligomeric structural epitope, preferably at least one non-diffusible globular Aβ(X-42) and/or Aβ(X-42) -40) oligomeric structural epitopes ("globulomers") (X is in each case independently the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most (preferably selected from the group consisting of 17..20) and/or crosslinked derivatives thereof. (i) an organism having an immune system, purified or essentially purified or highly purified non-diffusible globular Aβ obtained by or obtainable by the above method; (X-38/43) oligomers, preferably nondiffusible spherical Aβ(X-42) and/or Aβ(X-40) oligomers (“globulomers”) and/or crosslinked derivatives thereof, and ( ii) at least one non-diffusible globular Aβ (X-38/43) oligomeric structural epitope, preferably at least one non-diffusible globular Aβ (X-42) and/or Aβ (X-40) oligomeric structural epitope (" globulomer") and/or cross-linked derivatives thereof from the organism and (iii) optionally determining the chemical sequence and/or structure of the antibody obtained. or at least one non-diffusible globular Aβ(X-38/43) oligomeric structural epitope, preferably at least one non-diffusible globular Aβ(X-42) and/or Aβ(X-40) oligomeric structure Antibodies with specificity for an epitope (“globulomer”) and/or cross-linked derivatives thereof are combined with at least one non-diffusible globular Aβ (X-38/43) oligomeric structural epitope, preferably at least one non-diffusible Isolate from cells or cell lines that produce antibodies with specificity for globular Aβ(X-42) and/or Aβ(X-40) oligomeric structural epitopes (“globulomers”) and/or crosslinked derivatives thereof A method is provided that features steps.

According to a preferred embodiment of the present invention, the above method comprises non-diffusible globular Aβ(X-38/43) oligomers, preferably non-diffusible globular Aβ(X-42)- and/or Aβ(X-40). ) oligomers (“globulomers”) are soluble.

According to another aspect of the present invention, at least one non-active immunization agent obtained or obtainable according to the above method for active or passive immunization against Alzheimer's disease (AD) in a patient in need of treatment. Diffusible globular Aβ (X-38/43) oligomeric structural epitopes, preferably at least one non-diffusible globular Aβ (X-42) and/or Aβ (X-40) oligomeric structural epitopes (“globulomers”) (X is , in each case independently selected from the group consisting of the numbers 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) Monoclonal or polyclonal, natural or recombinant, humanized, human, animal or chimeric antibodies with specificity for and/or crosslinked derivatives thereof are provided.

According to a preferred embodiment of the invention, said monoclonal or polyclonal, natural or natural or recombinant, humanized, human, animal or chimeric antibody comprises non-diffusible globular Aβ (X-38/43) oligomers, non-diffusible Polyglobular, preferably Aβ(X-42)- and/or Aβ(X-40) oligomers (“globulomers”) are characterized by being soluble.

According to another aspect of the invention, obtained according to or obtainable by the method described above, for active or passive immunization against Alzheimer's disease (AD) in a patient in need of treatment at least one non-diffusible globular Aβ (X-38/43) oligomeric structural epitope, preferably at least one non-diffusible globular Aβ (X-42) and/or Aβ (X-40) oligomeric structural epitope (“globulomer” ) (X is in each case independently from the group consisting of the numbers 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20 selected) and/or crosslinked derivatives thereof.

According to a preferred embodiment of the present invention, the above composition comprises non-diffusible spherical Aβ(X-38/43) oligomers, preferably non-diffusible spherical Aβ(X-42)- and/or Aβ(X- 40) Oligomers (“globulomers”) are characterized by being soluble.

According to another aspect of the invention, a method for positively or negatively diagnosing a human or non-human animal subject for Alzheimer's disease (AD) or for the presence or absence of risk for developing AD, comprising: The sample contains at least one non-diffusible globular Aβ (X-38/43) oligomeric structural epitope, preferably at least one non-diffusible globular Aβ (X-42) and/or Aβ (X-40) oligomeric structural epitope ( "globulomers") (X is in each case independently from the number 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20 (=positive diagnosis) or absence (=negative diagnosis) of autoantibodies having specificity for a cross-linked derivative thereof) and/or crosslinked derivatives thereof. is provided.

According to a preferred embodiment of the present invention, the above method is characterized in that the sample is selected from the group consisting of cells, cell aggregates, body tissue (cell) samples, body fluid samples and body tissue (cell)/body fluid samples, Each sample is characterized as being derived from a human, non-human or transgenic animal body.

According to a preferred embodiment of the present invention, the above method comprises non-diffusible globular Aβ(X-38/43) oligomers, preferably non-diffusible globular Aβ(X-42)- and/or Aβ(X-40). ) oligomers (“globulomers”) are soluble.

According to another aspect of the invention, at least non-diffusible globular Aβ (X-38 /43) oligomeric structural epitopes, preferably at least one nondiffusible globular Aβ(X-42) and/or Aβ(X-40) oligomeric structural epitopes (“globulomers”) (X is in each case independently the number 1 ..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20) and/or crosslinked derivatives thereof. The use of humanized, human, animal or chimeric antibodies with specificity against is provided.

According to a preferred embodiment of the present invention, at least one non-diffusible globular Aβ(X-38/43) oligomeric structural epitope as described above, preferably at least one non-diffusible globular Aβ(X-42) and/or Aβ The use of humanized, human, animal or chimeric antibodies with specificity for (X-40) oligomeric structural epitopes (“globulomers”) is directed to non-diffusible globular Aβ (X-38/43) oligomers, preferably non-diffusible. Diffusible spherical A[beta](X-42)- and/or A[beta](X-40) oligomers ("globulomers") are characterized as being soluble.

According to another aspect of the present invention, in the manufacture of a medicament for the treatment or prevention of Alzheimer's disease or related diseases such as Down's syndrome in a patient in need of such treatment, one or more that reduce fatty acid levels in the brain of a patient. is provided.

According to another aspect of the present invention, non-diffusible globular Aβ _XY oligomers useful as diagnostic markers for identifying Alzheimer's disease (AD) or increased risk of developing Alzheimer's disease in patients in need of treatment. (X is in each case independently selected from the group consisting of the numbers 1..24, preferably 8..24, more preferably 12..24, more preferably 12..20 and most preferably 17..20 and Y is in each case independently selected from the group consisting of the numbers 38..43) and/or bridged derivatives thereof.

In the context of diagnostic applications, the present invention includes methods of diagnosing Alzheimer's disease in patients with suspected Alzheimer's disease. The method includes a) isolating a biological sample from the patient; b) contacting the biological sample with any of the above antibodies for a time and under conditions sufficient to form a globulomer/antibody complex; and c) detecting the presence of globulomer/antibody complexes in the sample, the presence of complexes indicative of a diagnosis of Alzheimer's disease in said patient.

Another method of the invention includes a method of diagnosing Alzheimer's disease in a patient with suspected Alzheimer's disease comprising the steps of a) isolating a biological sample from the patient; contacting the biological sample with the globulomer for a time and under conditions sufficient; c) adding a conjugate to the resulting antibody/globulomer complex for a time and under conditions sufficient for the conjugate to bind to the bound antibody; adding, wherein the conjugate comprises an antibody conjugated to a signal-generating compound capable of generating a detectable signal; and d) detecting the signal generated by the signal-generating compound in the biological sample. Included are methods comprising detecting the presence of possible antibodies, wherein the signal is indicative of a diagnosis of Alzheimer's disease in the patient.

The present invention provides another method of diagnosing Alzheimer's disease in a patient with suspected Alzheimer's disease, comprising the steps of a) isolating a biological sample from the patient; and c) detecting the presence of the antibody/purified globulomer complex in the sample, wherein the presence of the complex is diagnostic of Alzheimer's disease in the patient. A method is included that has the steps shown.

Further, the present invention provides another method of diagnosing Alzheimer's disease in a patient suspected of having Alzheimer's disease, comprising the steps of a) isolating a biological sample from the patient; b) binding the conjugate to the resulting anti-antibody/antibody complex and the conjugate to the bound antibody, for a time and under conditions of adding for as long and under conditions as possible, the conjugate comprising a globulomer bound to a signal generating compound capable of generating a detectable signal; and c) detecting the signal generated by the signal generating compound. and wherein the signal is indicative of a diagnosis of Alzheimer's disease in the patient.

Additionally, the invention includes methods of identifying compounds for the treatment or prevention of Alzheimer's disease. The method includes the steps of a) exposing one or more compounds of interest to the purified globulomer for a time and under conditions sufficient for the one or more compounds to bind to or neutralize the purified globulomer; and b) purification. identifying compounds that bind to or neutralize globulomers, wherein the identified compounds are for use in the treatment or prevention of Alzheimer's disease.

Further included in the invention are methods of designing small molecules useful in the treatment or prevention of Alzheimer's disease in a patient. The method comprises: a) analyzing the three-dimensional structure of a protein, such as a purified globulomer, isolated β-amyloid protein composition or aggregate of isolated β-amyloid protein compositions as described above; and c) designing a small molecule that binds to the identified epitope of step b) or a small molecule that binds to the epitope, wherein the molecule is used for the treatment or prevention of Alzheimer's disease. has a stage that is used for

The invention further includes methods of identifying monoclonal antibodies for use in treating or preventing Alzheimer's disease. The method comprises a) exposing the purified globulomer to a library of monoclonal antibodies for a time and under conditions sufficient for one or more of the monoclonal antibodies to bind to the globulomer and form a globulomer/antibody complex; b) the globulomer / confirming the presence of the antibody complex; and c) determining what the one or more antibodies in the complex are, which antibody or antibodies are used in the treatment or prevention of Alzheimer's disease. have stages.

Additionally, it should be noted that the globulomers of the invention (as well as antibodies directed against them) can be used in a variety of diagnostic assays.

In one embodiment of the invention, globulomers or portions thereof are coated onto a solid phase (or present in a liquid phase). A test sample or biological sample (eg, whole blood, cerebrospinal fluid, serum, etc.) is then contacted with the solid phase. If antibodies are present in the sample, such antibodies will bind to the antigen on the solid phase and be detected by direct or indirect methods. Direct methods involve simply detecting the presence of the complex itself and thus the presence of the antibody. In indirect methods, the conjugate is added to the bound antibody. The conjugate includes a second antibody that binds to the first conjugate, attached to a signal-generating compound or label. Upon binding of the second antibody to the bound first antibody, the signal generating compound generates a measurable signal. Such signal indicates the presence of the first antibody in the test sample.

Examples of solid phases used in diagnostic immunoassays include porous and non-porous materials, latex particles, magnetic particles, microparticles (see US Pat. No. 5,705,330), beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and, if desired, the method of labeling the antigen or antibody present in the conjugate is determined by the performance characteristics of the desired assay format.

As noted above, the conjugate (or indicator reagent) comprises an antibody (or possibly an anti-antibody, depending on the assay) conjugated to a signal-generating compound or label. The signal-generating compound or "label" is itself detectable or can be reacted with one or more additional compounds to form a detectable product. Examples of signal generating compounds include chromophores, radioisotopes (e.g. ¹²⁵ I, ¹³¹ I, ³² P, ³ H, ³⁵ S and ¹⁴ C), chemiluminescent compounds (e.g. acridinium), particles (visible or fluorescent ), nucleic acids, complexing agents or catalysts such as enzymes (eg alkaline phosphatase, acid phosphatase, horseradish peroxidase, β-galactosidase and ribonuclease). When using an enzyme (eg, alkaline phosphatase or horseradish peroxidase), addition of a chromogenic, fluorogenic or luminescent substrate produces a detectable signal. Other detection systems such as time-resolved fluorescence, internal reflection fluorescence, amplification (eg polymerase chain reaction) and Raman spectrometry are also useful.

Examples of biological fluids that can be examined by the above immunoassays include plasma, whole blood, dried whole blood, serum, cerebrospinal fluid or aqueous or organic-aqueous extracts of tissues and cells.

In another embodiment of the invention, a test sample can be exposed to a solid phase (or liquid phase) coated with a specific antibody (e.g., human or humanized monoclonal antibody, polyclonal antibody, etc.) of the invention. If the globulomers described above are present in the sample, they can be bound to the solid phase and then detected by the direct or indirect methods described above. More specifically, indirect methods involve the addition of a conjugate comprising a second antibody (that binds to the bound antigen) conjugated to a label or signal generating compound. When the second antibody binds to the bound antigen, a detectable signal is generated indicating the presence of Alzheimer's proteins, such as globulomers or portions thereof, in the test sample.

The invention also includes a third method of detecting the presence of antibodies in a test sample. This method comprises (a) exposing a test sample, which may contain antibodies, to an antigen-specific anti-antibody (e.g., globulomer or its (b) adding an antigen to the resulting anti-antibody/antigen complex for a time and under conditions sufficient to allow binding of the antigen to the bound antibody, wherein the antigen is defined herein as and (c) adding a conjugate to the resulting anti-antibody/antibody/antigen complex, wherein the conjugate is capable of detecting a detectable signal comprising a composition comprising a monoclonal or polyclonal antibody bound to a generating compound, wherein the monoclonal or polyclonal antibody is directed to the antigen; and (d) detecting the signal generated by the signal generating compound in the test sample. detecting the presence of antibodies that may be present in the Controls or calibrators containing antibodies to anti-antibodies can be used. The antigen mixture can further include P30 if desired.

Kits are also included within the scope of the invention. More specifically, the invention includes kits for confirming the presence of antibodies in a patient. In particular, kits for confirming the presence of antibodies in a test sample are capable of producing a) an antigen as defined herein (e.g., a globulomer or portion thereof); and b) a detectable signal. Also included are conjugates comprising an antibody conjugated to a signal-generating compound capable of. The kit can also include controls or calibrators containing reagents that bind to the antigen.

The invention also includes another type of kit for detecting antibodies in test samples. The kit may comprise a) an anti-antibody specific for the antibody of interest and b) an antigen or portion thereof as defined above. Controls or calibrators comprising reagents that bind antigen can also be included. More specifically, the kit can include a conjugate comprising a) an anti-antibody that is specific for the antibody and b) an antigen, wherein the conjugate is capable of generating a detectable signal. bound to a compound. Again, the kit can also include controls or calibrators containing reagents that bind to the antigen.

The kit can also contain one container such as a vial, bottle or strip, each container having a preset solid phase, and other containers containing individual conjugates. These kits can also include vials and containers of other reagents needed to perform the assay, such as wash, treatment and indicator reagents.

The invention is further illustrated by the following examples, which should not be construed as limiting in any respect.

(Example 1)
1 mg of Aβ(1-42) solid powder chemically synthesized by Bachem, Weil am Rhein, Germany, catalog number H-1368, was added to 0.1% aqueous NH ₄ OH (freshly prepared). .5 mL and shaken at room temperature for 30 seconds to give a clear solution with a concentration of 2 mg/mL.

The resulting Aβ(1-42) monomer preparation was diluted with 1.5 mL of 20 mM _NaH2PO4 , 140 mM NaCl, pH _7.4 to a final protein concentration of 0.5 mg/mL. The pH of this Aβ(1-42) fibril-prone monomer preparation was adjusted with 5% hydrochloric acid to pH 7.4, and the preparation was then placed on an ice bath for immediate use.

All steps were performed rapidly to avoid initiation of polymerization of Aβ(1-42) during preparation.

Maximum stability of the monomer was found to be 1 hour at 4°C or 15 minutes at room temperature. Alternatively, samples could be frozen at -80°C immediately and stored at -80°C for up to 1-2 weeks. Even at this extremely low temperature, Aβ protofibril and fibril polymerization processes started and the preparation became unstable when stored for more than 2 weeks.

(Example 2)
Stability of Aβ(X-42) Globulomer and Fibril-Prone Monomer A solution of Aβ(1-42) globulomer was prepared according to the method of Example 5a of WO2004/067561. Briefly, Aβ(1-42) synthetic peptide (catalog number H-1368, Bachem, Switzerland) was added to 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at approximately 6 mg/mL. Suspended and incubated at 37° C. for 1.5 hours with shaking to allow complete dissolution. HFIP was removed by SpeedVac evaporation and Aβ(1-42) was resuspended in dimethylsulfoxide (DMSO) at a concentration of 5 mM and sonicated for 20 seconds. HFIP-pretreated Aβ(1-42) was diluted in phosphate buffered saline (PBS) ( ₂₀ mM _NaH2PO4 , 140 mM NaCl, pH 7.4) to 400 μM and 1/10 volume of 2% SDS (H ₂ O solution) was added (final concentration 0.2% sodium dodecyl sulfate (SDS)). A 6 h incubation at 37° C. yielded the 16/20 kDa Aβ(1-42) globulomer intermediate. Further dilution with 3 volumes of H ₂ O and incubation at 37° C. for 18 hours formed 38/48 kDa Aβ(1-42) globulomers. After centrifugation at 3000×g for 20 minutes, the samples were concentrated by ultrafiltration (30 kDa cutoff), dialyzed against 5 mM NaH ₂ PO ₄ , 35 mM NaCl (pH 7.4) and centrifuged at 10000×g for 10 minutes. , the supernatant containing the 38/48 kDa Aβ(1-42) globulomers was withdrawn. For fatty acid-induced 38/48 kDa Aβ(1-42) globulomers, 1/10 volume of 0.5% fatty acid was added instead of SDS. For cross-linking, 38/48 kDa Aβ(1-42) globulomers were dialyzed against 1 mM glutardialdehyde for 2 h at room temperature, followed by ethanolamine (5 mM) treatment for 30 min at room temperature (RT) prior to concentration and dialysis. bottom. Restricted proteolysis of the 38/48 kDa Aβ(1-42) globulomers was performed using 1/50 (mg/mg) of each protease and 20 h incubation at room temperature to obtain the corresponding truncated forms.

Concentration was evaluated as follows. Based on experimentally determined molecular weights, Aβ(1-42) globulomers were approximated to consist of about 12 monomers. Therefore, the stated concentrations are also approximations. For example, the indicated Aβ(1-42) globulomer concentration of 42 nM is considered equivalent to a 500 nMA β(1-42) globulomer concentration on a monomer basis.

0.5 mg/mL samples in 20 mM NaH ₂ PO ₄ ; 140 mM NaCl; pH 7.4 were incubated at room temperature and 37° C. for 1 day and 4 days, respectively. A 10 μL aliquot of the sample was analyzed by SDS-PAGE.

Aβ(1-42) (Bachem, Peptide Synthesis) was dissolved at 2 mg/mL in 0.1% _NH4OH and diluted 1:4 with water according to the method of Example 1 (0.5 mg/mL). The Aβ(1-42) fibril-prone preparation was incubated at −20° C.; 0° C.; room temperature (corresponding to about 20-22° C.) and 37° C. for 24 hours. Aliquots of 10 μL of each sample were analyzed by SDS-PAGE.

Results are shown in FIG.

Aβ(1-42) globulomers are stable in PBS at both room temperature and 37° C. for at least 4 days, whereas Aβ(1-42) monomers are stable in megadaltons in PBS even after 1 day at 0° C. It was found to show significant polymerization up to 20°C, significant polymerization after 1 day at room temperature, and substantially complete polymerization after 1 day at 37°C.

(Example 3)
Confirmation of the presence of Aβ(1-42) globulomer epitopes in vivo Using the specific antibodies of the invention, Alzheimer's disease patients (see Figures 13a-c) and APP transgenic Tg2576 mice (see Figures 13d-f) ) was able to detect the Aβ(1-42) globulomer epitope in amyloid plaques in the brain of M.

Most of the immunofluorescence was associated with Thioflavin S-positive plaques (see Fig. 13a,d), with Aβ(1-42) globulomer epitopes forming a dense rim around plaques (see Fig. 13c,f). . It was possible to quantify the relative abundance of Aβ(1-42) globulomer epitopes for the PBS soluble fraction of brain extracts. In both Alzheimer's disease patients (see Figure 13g) and Tg2576 mice (see Figure 13h), only a small amount of total Aβ was found to be of Aβ(1-42) globulomer epitope structures. Aβ(1-42) globulomer epitopes were detected not only in plaques, but also in the brains of young 2.5-month-old Tg2576 mice, which also have low total Aβ(1-42) concentrations. This was well before the development of amyloid plaques, which led to a large increase in total Aβ(1-42) levels starting at 11 months of age (see Fig. 13i) and relatively low Aβ(1-42) globulomer epitope formation. 42) have been shown to be frequent even at concentrations. Surprisingly, Aβ(1-42) globulomer epitopes are completely absent in the cerebrospinal fluid of patients with mild cognitive impairment (see Figure 13j, gray bars) and Alzheimer's disease patients (see Figure 13j, black bars). Not detected. However, this negative finding does not support Aβ(1-42) globulomers, as it is a distinct feature of globulomers that they are rapidly and easily bound to neural structures and thus quantitatively cleared from the cerebrospinal fluid. This may be because the epitope concentration is below the detection limit.

The detection was performed using the following materials and methods.

3.1: Extraction and detection of Aβ globulomer epitopes in postmortem AD human brain tissue
3.1. A: Reagent List _- Extraction Buffer: 5 mM _NaH2PO4 ; 35 mM NaCl; pH 7.4.

- Complete Protease Inhibitor Cocktail Tablets; Roche Diagnostics GmbH Catalog No. 697498; stored at 4°C.

- Extraction Buffer + Complete Inhibitor Cocktail: Dissolve 1 tablet of Complete Inhibitor Cocktail in 1 mL of water. Add 1/100 full inhibitor cocktail solution to the extraction buffer.

3.1. B: One-Step Extraction Procedure from Human AD Brains —80° C. Frozen postmortem human AD and age-matched control brain tissue samples were obtained from Brain-Net, Munich. In a 50 mL falcon tube, 9 mL of extraction buffer and complete inhibitor cocktail was added to 1 g of frozen brain tissue material. The samples were homogenized on ice with an Ultra Turrax homogenizer for 2 minutes, the homogenized samples were placed in ultracentrifuge tubes and centrifuged at 150000 g at 8° C. for 1 hour. Supernatants were carefully collected and stored at -80°C in 15 mL Falcon tubes for further ELISA testing as described below.

3.1. C: Sandwich ELISA
3.2. B. as described in

3.2: Extraction and detection of Aβ globulomer epitopes in brain tissue of Aβ overproducing Aβ transgenic mouse Tg2576
3.2. A: Extraction procedure (necessary reagents described in 3.1.A)
Several 3-month-old female Tg2576 mice carrying and overexpressing the human APP gene with the Swedish familial Alzheimer's disease double mutation (K670N;M671L) were purchased from Taconics (USA). , were maintained in our animal care facility until 12 months of age. 100 mg frozen cortices from 12 month old APP-overexpressing TG2576 mice (Taconix) or 3 month old C57 wild-type mice were added to 2.5 mL PBST + 0.5% BSA-buffer + complete inhibitor cocktail (10 mg/0.25 mL wet weight). ). Samples were pre-homogenized in a glass potter and sonicated five times for 1-2 seconds in an ice bath at 0° C. using an ultrasonic cell disruptor. Sample aliquots of 2 mL were incubated overnight at 37° C. for 16 hours in ultracentrifuge tubes and centrifuged at 100000 g at 8° C. for 1 hour. Supernatants were carefully removed and stored at -20°C for ELISA analysis.

3.2. B: Sandwich ELISA
3.2. B. 1: Reagent List1 . F96 Cert. Maxisorp NUNC Immune Plate Catalog No. 439454
2. Capture Antibody Affinity-purified anti-AβpAb rabbit 5598 (purification procedure Labj.: 1286/155); PBS solution; concentration: 0.24 mg/mL; store at -20°C. Coating buffer 100 mM sodium bicarbonate; pH 9.6
4. Blocking reagent for ELISA; Roche Diagnostics GmbH Cat. No. 1112589
5. PBST buffer 20 mM _NaH2PO4 ; 140 mM NaCl; 0.05% Tween ₂₀ ; pH 7.4
6. 6. Protease-free albumin bovine fraction V; Serva catalog number 11926.03; store at 4°C. _PBST + 0.5% BSA buffer: 20 mM _NaH2PO4 ; 140 mM NaCl; 0.05% Tween 20; pH 7.4 + 0.5% BSA
8. 9. Aβ(1-42) oligomer standard stock solution; 5 mM NaH ₂ PO ₄ , 35 mM NaCl (pH 7.4) solution; concentration: 10.77 mg/mL; 10. Anti-Aβ mouse mAb clone 6B1; PBS solution; concentration: 1.2 mg/mL; anti-mouse-POD conjugate; Fa. Jackson Immuno Research Catalog No. 715-035-150
11. Development TMB; Roche Diagnostics Catalog No. 92817060; ₄₂ mM DMSO solution 3% _H2O2 aqueous solution 100 mM sodium acetate, pH 4.9
12. Stop solution: 2M sulfuric acid.

3.2. B. 2: Reagent preparation 1. Capture antibody:
Rabbit polyclonal anti-Aβ(20-42) globulomer antibody 5598 was obtained by immunizing rabbits according to the procedure described in Example 25 of WO2004/067561.

5598 was purified from this rabbit antiserum by affinity chromatography on Sepharose-immobilized Aβ(1-42) globulomers.

- Preparation of Aβ(1-42) globulomer Sepharose:
Production of Aβ(1-42) globulomers: Aβ(1-42) Fa. 9 mg of Bachem was dissolved in 1.5 mL of HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) and incubated at 37° C. for 1.5 hours. The solution was evaporated by speedvac and suspended in 396 μL of DMSO (5 mM Aβ stock solution). Samples were sonicated in an ultrasonic water bath for 20 seconds, shaken for 10 minutes, and stored at -20°C overnight. The sample was diluted with 554 μL of PBS (20 mM NaH ₂ PO ₄ ; 140 mM NaCl; pH 7.4) and 495 μL of 2% SDS aqueous solution was added (SDS content 0.2%).

The mixture was incubated at 37° C. for 7 hours, diluted with 16335 μL H ₂ O and incubated at 37° C. for an additional 16 hours. The Aβ(1-42) globulomer solution was then centrifuged at 3000 g for 20 minutes. The final Aβ(1-42) globulomer supernatant was discarded and used for immobilization.

Preparation of NHS-activated Sepharose: Subsequently, 2.0 g of NHS-activated Sepharose Fast Flow (Amersham Biosciences; catalog number 17-0906-01) was washed twice with the following solutions.

2×25 mL of 1 mM HCl solution in 30% isopropanol (ice cold);
2×25 mL of 1 mM HCl (ice cold);
Finally, 2×25 mL of 50 mM NaHCO ₃ (pH 7.5) (ice cold).

Immobilization: 10 mL of Aβ(1-42) globulomer solution was mixed with 2.0 g of NHS-activated Sepharose and incubated for 2 hours at room temperature. The reaction mixture was centrifuged at 4000g and the supernatant was discarded.

Block: 50 mM NaHCO ₃ ; 250 mM ethanolamine; 0.05% SDS (pH 7.5) 10 mL was added and the resin was incubated for 1 hour at room temperature. The Sepharose material was collected by filtration and washed four _times with 25 mL of 5 mM _NaH2PO4 ; 35 mM NaCl; 0.05% SDS; pH 7.4.

Crosslinking: Sepharose material was resuspended in 10 mL _of 5 mM _NaH2PO4 ; 35 mM NaCl; 0.05% SDS; pH 7.4 and mixed with 1 mL of 40 mM aqueous glutaraldehyde (freshly prepared). After incubation (2 h, _room temperature) and centrifugation (10 min, 4000 g), the supernatant was discarded and Sepharose was added to 5 mM _NaH2PO4 ; 35 mM NaCl; 250 mM ethanolamine; 0.05% SDS; for 1 hour. The mixture was centrifuged at 4000g for 10 minutes and the supernatant was discarded.

The ethanolamine _blocking reaction was repeated and finally the Sepharose material was collected by filtration and washed four times with 25 mL of 5 mM _NaH2PO4 ; 35 mM NaCl; 0.05% SDS; pH 7.4.

₃₅ mM NaCl; 0.05% SDS; 0.02% _NaN3 ; pH _7.4 ; °C (Sepharose 0.5 g/mL) and subjected to further affinity chromatography.

- Affinity Chromatography 0.8 g of Aβ(1-42) globulomer sepharose material was collected by filtration and washed 4 times with 10 mL of TBS buffer (25 mM Tris; 150 mM NaCl; pH 7.5). Rabbit serum 5598 was diluted (1:1) in TBS buffer. 40 mL of this sample was added to 0.8 g of Aβ(1-42) globulomer sepharose and incubated overnight at 8°C. Aβ(1-42) globulomer-Sepharose was packed in a column and washed with 25 mL of TBS buffer. Elution was performed with 0.58% acetic acid in 140 mM NaCl, and the eluted UV active fraction was collected. The eluted fraction (immunopurified Pab5598 stock solution) was immediately adjusted to pH 7.5 by adding 2M Tris buffer, pH 8.5 and stored at -80°C.

- Preparation of antibody solution:
The 5598PAb stock solution was thawed and diluted 1:240 in coating buffer.

2. Blocking reagent:
A blocking stock solution is prepared by dissolving the blocking reagent in 100 mL of water and aliquots of 10 mL are stored at -20°C.

Block by diluting 3 mL of blocking stock solution with 27 mL of water for each plate.

3. Aβ(1-42) Globulomer Standard Diluent:
Add 1 μL A-Aβ(1-42) globulomer standard stock solution to 1076 μL PBST + 0.5% BSA = 10 μg/mL
B- Add 5 μL of 10 μg/mL Aβ(1-42) globulomer standard to 4995 μL of PBST + 0.5% BSA = 10 ng/mL.

4. Primary antibody 6B1:
Mouse monoclonal antibody 6B1 was obtained by standard procedures via immunization with Aβ(1-42) globulomers. The antibody was purified from the hybridoma using standard procedures.

Concentrated anti-Aβ mAb clone 6B1 was diluted in PBST + 0.5% BSA-buffer. The dilution factor was 1/6000=0.2 μg/mL. The antibody was used immediately.

5. Labeling reagent:
The anti-mouse-POD conjugate lyophilisate is reconstituted with 0.5 mL water. Add 500 μL of glycerol and store aliquots of 100 μL at −20° C. for future use. Dilute the concentrated labeling reagent with PBST buffer. The dilution factor is 1/5000. Use immediately.

6. TMB solution:
20 mL of 100 mM sodium acetate (pH 4.9) is mixed with 200 μL of TMB solution and 29.5 μL of 3% peroxide solution. Use immediately.

3.2. B. 3. Sample plate preparation (note that both standards and samples are run in duplicate)

3.2. B. 4: Procedure 1. Add 100 μL of anti-Aβ pAb rabbit 5598 solution per well and incubate at 4° C. overnight.
2. Discard the antibody solution and wash the wells three times with 250 μL of PBST buffer.
3. Add 300 mL of block solution per well and incubate for 2 hours at room temperature.
4. Discard the blocking solution and wash the wells three times with 250 μL of PBST buffer.
5. After preparation of standards and samples, add 100 μL per well of standards and samples to the plate. Incubate for 2 hours at room temperature and overnight at 4°C.
6. Discard standard/sample solution and wash wells 3 times with 250 μL of PBST buffer.
7. Add 299 μL of primary antibody solution per well and incubate at room temperature for 1.5 hours.
8. Discard the antibody solution and wash the wells three times with 250 μL of PBST buffer.
9. Add 200 μL of labeling solution per well and incubate for 1 hour at room temperature.
10. Discard the labeling solution and wash the wells three times with 250 μL of PBST buffer.
11. Add 100 μL of TMB solution to each well and incubate at room temperature (5 to 15 minutes).
12. Observe color development and add 50 μL of stop solution per well.
13. Read at 450 nm.
14. Calculate the results from the standard curve.
15. evaluation:
If the quenching from the unknown sample is not within the linearity range of the calibration curve, repeat the ELISA at the assigned sample dilution.

From these data it can be said that the Aβ(1-42) globulomer epitope is present in vivo in the very early stages of Alzheimer's disease and precedes all clinical manifestations. Therefore, it is presumed that it may be useful in early detection and risk assessment of AD and related conditions.

(Example 4)
Formation and Characterization of Globular Aβ(1-42) Oligomers The first step in the globular Aβ(1-42) oligomerization procedure (see FIG. 11a) is by pretreatment of synthetic Aβ(1-42) peptides with HFIP. removal of existing structural heterogeneities (Stine, WB, Jr., Dahlgren, KN, Krafft, GA & LaDu, MJ In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J Biol Chem 278, 11612 -11622 (2003)). After resuspension in DMSO, incubation in 0.2% SDS initially forms an intermediate oligomeric Aβ form with 16/20 kDa (apparent mass of two bands on SDS-PAGE). do. Upon further dilution and incubation, this intermediate self-associated into the final 38/48 kDa oligomeric Aβ form, which was found to be of a globular structure, hence the 38/48 kDa globular Aβ (1 -42) termed oligomers or simply Aβ(1-42) globulomers (see Figure 11b).

Separation of residual fibril-prone monomers from the globulomer fraction is via affinity chromatography using antibodies specific for fibril-prone Aβ(1-42) monomers bound to immobilized protein A or fibril-prone Aβ(1-42 ) removal of monomers via direct antibody removal (immunoprecipitation or immunoadsorption). These methods are described in more detail below.

Glutardialdehyde cross-linking of both the intermediate 16/20 kDa and final 38/48 kDa Aβ(1-42) globulomers merges the two bands on SDS-PAGE into one, yielding a uniform Aβ(1-42 ) showed a globulomer structure. The Aβ(1-42) peptide interactions that form the Aβ(1-42) globulomer structure are exclusively non-covalent. Heat denaturation reverted Aβ(1-42) globulomers to monomeric Aβ(1-42), but not cross-linked Aβ(1-42) globulomers. Even so, non-covalently linked Aβ(1-42) globulomers showed long-term stability at body temperature (37° C.). The Aβ(1-42) globulomer structure was maintained in vitro for up to 4 days with no apparent degradation or further polymerization into fibrils.

In contrast, standard monomeric preparations of Aβ(1-42) peptide (Bachem, Switzerland) dissolved in 0.1% _NH4OH (Aβ(1-42) _NH4OH preparations) already had 1 day of incubation. Later showed a high degree of aggregation (see Figure 11c).

Aβ forms were analyzed for molecular weight distribution using a Superose 12HR10/30 column (Amersham Biosciences, Germany) and PBS (pH 7.4) as running buffer. A set of standard proteins was used to calibrate the column. Aβ-peptide masses were determined by SELDI-MS using H4 protein chips (Ciphergen Biosystems, USA) and α-cyano-4-hydroxy-cinnamic acid in 50% acetonitrile/0.5% trifluoroacetic acid as substrate. .

Under native conditions (PBS, pH 7.4), size exclusion chromatography of Aβ(1-42) globulomers showed a single but broad peak at approximately 100 kDa (see Figure 11d). Similar to SDS-PAGE, cross-linked Aβ(1-42) globulomers showed a reduced apparent molecular weight with a narrow peak at 60 kDa, and only single spheres in which Aβ(1-42) globulomers aggregated at glutardialdehyde cross-links. suggests the existence of

In contrast, A[beta](1-42) _NH4OH preparations were highly unstable and prone to fibrillation. Already about 5 minutes after resuspension, significant aggregation was detectable (major peak 11 kDa, minor peak 74 kDa) and after 1 hour of incubation no Aβ entered the chromatography column due to apparent fibril aggregation. (data not shown). Proteolysis with promiscuous proteases cleaved the Aβ(1-42) globulomer from the N-terminus forward to approximately amino acids 20, leaving the C-terminal portion and globular structure unaffected (see Figure 11e). SELDI-MS analysis of thermolysin cleavage products showed that cross-linking of Aβ(1-42) globulomers resulted in loss of Aβ(4-42) cleavage products and maintenance of Aβ(20-42) ( 11f). Thus, cross-linking affected only the N-terminus and Lys-16, but not Lys-28, so it must have been hidden within the Aβ(1-42) globulomer.

The following describes a preferred method of cleaning the Aβ(1-42) globulomer solution of residual fibril-prone monomers. Any of these require the following reagents in addition to those listed.

- Protein A Sepharose 4 Fast Flow Amersham Biosciences Catalog No. 17-0974-01
- PBS buffer: 20 mM _NaH2PO4 ; 140 mM NaCl; _0.05 % Pluronic F68; pH 7.4.

4.1: Decontamination of Aβ(1-42) fibril-prone monomers in Aβ(1-42) globulomer preparations via Protein A-bound fibril-prone Aβ(1-42)-specific antibody affinity chromatography ( 1-42) Purification of globulomer 4.1. A: Reagent List -Capturing antibody anti-Aβ(1-42) rabbit pAb; Biotinylated IgG in 50% glycerin in PBS; Concentration: 0.5 mg/mL; Signet Catalog No. 9135; _Aβ (1-42) oligomer standard stock solution; 5 mM _NaH2PO4 solution; 35 mM NaCl; pH 7.4; concentration: 10.77 mg/mL;

4.1. B: Preparation of Reagent 1. Capture Antibody Diluent: Concentrated anti-Aβ(1-42) pAb is diluted 1:5 with PBS to 0.1 mg/mL.

2. Immobilization of Capture Antibody on Protein A Sepharose: Equilibrate 20 μL Protein A Sepharose with 200 μL PBS-buffer, then centrifuge at 3000 g for 5 minutes and discard the supernatant. Repeat procedure 4 times. Add 100 μL of a 1:5 PBS dilution of anti-Aβ(1-42) pAb to the Protein A Sepharose pellet and shake for 1 hour at room temperature. Centrifuge at 3000 g for 5 minutes. The supernatant is discarded and the Protein A Sepharose is washed 3 times with 200 μL of PBS and then centrifuged at 3000 g for 5 minutes each time. Discard the supernatant.

3. A _1-42 Oligomer Standard Diluent: Add 50 μL Aβ(1-42) Oligomer Standard Stock Solution to 450 μL PBS = 1.08 mg/mL.

4.1. C: Procedure Add 500 μL of Aβ(1-42) oligomer standard dilution to 20 μL of Protein A Sepharose-immobilized anti-Aβ(1-42) pAb resin. The samples are shaken for 2 hours at room temperature and 16 hours at 4° C., then centrifuged at 3000 g for 5 minutes.

Aβ fibril-prone contaminants (fibriromers) react with the affinity substrate and the supernatant contains purified Aβ globulomers with globulomer epitope purity>>95%.

The purity of this sample was further improved by repeating the procedure twice, resulting in >99% globulomer epitope purity in the supernatant.

4.2: Purification of Aβ globulomers by decontaminating Aβ(1-42) fibril-tendency monomers in crude Aβ(1-42) preparations via direct removal of fibril-tendency Aβ(1-42) .2. A: Reagent List - F96 Sert Maxisorp NUNC Immunoplate Catalog No. 439454
- capture antibody anti-Aβ(1-42) rabbit pAb; biotinylated IgG solution in PBS containing 50% glycerol; concentration: 0.5 mg/mL; signet catalog number 9135; sodium hydrogen; pH 9.6
- Blocking reagent for ELISA; Roche Diagnostics Catalog No. 1112589
_- PBST buffer: 20 mM _NaH2PO4 ; 140 mM NaCl; 0.05% Tween 20; pH 7.4
- Aβ (1-42) oligomer standard stock solution; 5 mM _NaH2PO4 , ₃₅ mM NaCl (pH 7.4) solution; concentration: 10.77 mg/mL; stored at -20°C

4.2. B: Preparation of Reagent 1. Capture Antibody: Dilute concentrated anti-Aβ(1-42) pAb 1:100 in coating buffer.
2. Blocking reagent: Dissolve blocking reagent in 100 mL water to prepare blocking stock solution and store 10 mL aliquots at -20°C. Block by diluting 3 mL of blocking stock solution with 27 mL of water for each plate.

3. Aβ(1-42) Globulomer Standard Diluent: Add 1 μL Aβ(1-42) Globulomer Standard Stock Solution to 1076 μL PBST=10 μg/mL Aβ(1-42) Globulomer Standard Solution.

4.2. C: Procedure Add 100 μL of anti-Aβ(1-42) mAb Clone Signet 9135 solution per well and incubate at 4° C. overnight. Discard the antibody solution and wash the wells three times with 250 μL of PBST buffer. Add 300 μL of blocking solution per well and incubate for 2 hours at room temperature. Discard the blocking solution and wash the wells three times with 250 μL of PBST buffer. After standard preparation, add 100 μL of Aβ(1-42) globulomer standard solution to the plate for each well. Incubate for 2 hours at room temperature and overnight at 4°C. Aβ(1-42) globulomer standard solutions are collected in 15 mL Falcon tubes and have >95% purity.

The globular epitope purity can be further increased by repeating the above immunoaffinity procedure in microtiter plates. After this procedure, the Aβ(1-42) globulomer solution is >>99% pure with respect to Aβ fibril-prone structures (monomers and fibriromers).

4.3: Aβ Globulomer Epitopes by Decontamination of Aβ Fibril-Prone Monomers in Aβ(1-42) ADDL Preparations via Protein A-Binding Fibril-Prone Aβ(1-42)-Specific Antibody Affinity Chromatography 4.3. A: Reagent List - Capture antibody anti-Aβ(1-42) rabbit pAb; biotinylated IgG solution in 50% glycerin in PBS; concentration: 0.5 mg/mL; 1-42) ADDL standard stock solution; solution in F12 medium; concentration 0.12 mg/mL; store at -80°C.

4.3. B: Preparation of Reagent 1. Equilibration of Protein A-Sepharose: 20 μL Protein A Sepharose is equilibrated with 200 μL PBS buffer and then 200 μL 0.58% acetic acid and further washed in the column with 200 μL PBS buffer. Place the equilibrated protein A-sepharose in the tube and add an equal volume of PBS-buffer to the substrate.

2. Aβ(1-42) ADDL standard diluent: Add 42 μL Aβ(1-42) ADDL standard stock solution to 58 μL PBS = 0.05 mg/mL.

4.3. C: Procedure Anti-Aβ(1-42) rabbit pAb; add 20 μL of biotinylated IgG solution to 100 μL of Aβ(1-42) ADDL solution. Shake the samples for 2 hours at room temperature. Add 10 μL of equilibrated Protein A Sepharose suspension in PBS buffer to the sample, shake for 1 hour at room temperature and then centrifuge at 3000 g for 5 minutes.

Aβ fibril-prone contaminants (fibriromers) react with the affinity substrate and the supernatant exhibits globulomer epitope purity >>55%.

The purity of this sample was further improved by repeating the procedure twice, resulting in >95% globulomer epitope purity in the supernatant.

4.4: Enrichment of Aβ globulomer epitopes by decontamination of fibril-prone Aβ in Aβ(1-42) ADDL preparations via Protein A-bound fibril-prone Aβ(1-42)-specific antibody affinity chromatography. 4. A: Reagent List -Capture Antibody: Anti-Aβ(1-42) mAb C-terminal clone BDI780; purified, lyophilized; Biodesign Catalog No. Q67780M; solution in F12 medium; concentration: 0.12 mg/mL; stored at -80°C.

4.4. B: Preparation of Reagent 1. Equilibration of Protein A-Sepharose: 20 μL Protein A Sepharose is equilibrated with 200 μL PBS buffer and then 200 μL 0.58% acetic acid and further washed in the column with 200 μL PBS buffer. Place the equilibrated protein A-sepharose in the tube and add an equal volume of PBS-buffer to the substrate.

2. Reconstitution of Capture Antibody: Reconstitute 100 μg of antibody with 1 mL of water to 0.1 mg/mL.

3. Aβ(1-42) ADDL standard diluent: Add 42 μL Aβ(1-42) ADDL standard stock solution to 58 μL PBS = 0.05 mg/mL.

4.4. C: Procedure Anti-Aβ(1-42) rabbit pAb; add 100 μL of biotinylated IgG solution to 100 μL of Aβ(1-42) ADDL solution. Shake the samples for 2 hours at room temperature. Add 10 μL of equilibrated Protein A Sepharose suspension in PBS buffer to the sample, shake for 1 hour at room temperature and then centrifuge at 3000 g for 5 minutes. Aβ fibril-prone contaminants (fibriromers) react with the affinity substrate and the supernatant exhibits globulomer epitope purity >>50%.

The purity of this sample was further improved by repeating the procedure twice, resulting in >90% globulomer epitope purity in the supernatant.

4.5: Enrichment of Aβ Globulomer Epitopes by Decontamination of Aβ Fibrilomers in Aβ(1-42) _NH4OH Preparations via Protein A-Binding Fibril-Prone Aβ(1-42)-Specific Antibody Affinity Chromatography .5. A: Reagent List - Capture antibody anti-Aβ(1-42) rabbit pAb; biotinylated IgG solution in 50% glycerin in PBS; concentration: 0.5 mg/mL; 1-42) _NH4OH standard stock solution; solution in 0.1% _NH4OH ; concentration: 2 mg/mL; store at -80<0>C.

4.5. B: Preparation of Reagent 1. Equilibration of Protein A-Sepharose: 20 μL Protein A Sepharose is equilibrated with 200 μL PBS buffer and then 200 μL 0.58% acetic acid and further washed in the column with 200 μL PBS buffer. Place the equilibrated protein A-sepharose in the tube and add an equal volume of PBS-buffer to the substrate.

2. Aβ(1-42) _NH4OH standard diluent: Add 2.5 μL Aβ(1-42) ADDL standard stock solution to 97.5 μL PBS = 0.05 mg/mL.

4.5. C: Procedure: 4.3. Same as the method under C.

(Example 5)
Formation of Aβ(20-42) Globulomers Starting from Peptide Aβ(20-42) a) Preparation of Globulomer Stock Solution /452-5055/33908) was dissolved in 25 μL of 1,1,1,3,3,3-hexafluoro-2-propanol and incubated in an Eppendorf tube at 37° C. for 1 hour, then at 10000 g for 10 minutes. Centrifuged. The supernatant was removed to give an Aβ(20-42) stock solution of 20 mg/mL (9 mM), which could be stored at -80°C.

b) Preparation of final stable Aβ(20-42) globulomers 4 μL of Aβ(20-42) stock solution from Example 5a) was added to 196 μL of 2 mM sodium phosphate, 14 mM NaCl (pH 7.4) and 1% dodecyl sulfate. After mixing with 20 μL of sodium (SDS) solution, incubation was carried out at 6° C. for 20 hours and finally centrifuged at 10000 g for 10 minutes. Supernatants were collected as approximately 180 μM Aβ(20-42) globulomer preparations, which could be stored at -20°C. Samples were analyzed by subsequent SDS-PAGE (Figure 9) and the product appeared at 32 kDa. It can be further purified by the purification method described in Example 4.

(Example 6)
Formation of Aβ(12-42) Globulomers Starting from Peptide Aβ(12-42) a) Preparation of Globulomer Stock Solution /452-5055/33907A) 0.5 mg, and the procedure described in Example 5a) was carried out to give an Aβ(12-42) stock solution of 20 mg/mL (6.2 mM), which at −80° C. was able to save.

b) Preparation of final stable Aβ(12-42) globulomers 4 μL of the Aβ(12-42) stock solution from Example 6a) are treated according to the method of Example 5b) to yield approximately 120 μM Aβ(12-42) ) A globulomer preparation was obtained, which could be stored at -20°C.

Samples were analyzed by subsequent SDS-PAGE (Figure 10) and the product appeared almost quantitative at 12 kDa. Further purification by the purification method described in Example 4 will yield ultrapure Aβ(12-42) globulomers.

(Example 7)
Detection of Anti-Globulomer Autoantibodies in Human Body Fluids by Plasma or CSF Dot Blots
Processing of Plasma or Serum Samples a) Titers of free fractions of autoantibodies were measured directly from serum or plasma.

b) Total autoantibody titers were determined by pretreatment of plasma/serum samples as described below. 100 μL of plasma/serum was mixed with 10 mL of 140 mM NaCl+0.58% acetic acid, incubated for 20 minutes, and then concentrated to 1 mL with Centriprep YM30 (Amicon Inc.).

Anti-Aβ autoantibodies were then assessed on plasma/serum samples. For this purpose serial dilutions of individual A[beta](1-42) forms ranging from 100 pmol/[mu]L to 0.01 pmol/[mu]L in PBS supplemented with 0.2 mg/mL BSA were prepared. 1 μL of each sample was blotted onto a nitrocellulose membrane. Corresponding mouse plasma samples were used for detection (1:400 dilution). Immunostaining was performed using alkaline phosphatase-conjugated anti-mouse-IgG and the staining reagent NBT/BCIP.

Aβ-standards for dot blots1 . Aβ(1-42) Globulomers The production of Aβ(1-42) oligomers (hereinafter referred to as Aβ(1-42) globulomers) is described in WO2004/067561.

2. Aβ(1-42) Monomer 3 mg of human Aβ(1-42) (Bachem catalog number H-1368) was dissolved in 0.5 mL of HFIP (6 mg/mL suspension) in a 1.7 mL Eppendorf tube and cleared. Shake at 37° C. for 1.5 hours until a solution is obtained (Eppendorf Thermomixer, 1400 rpm). Samples were dried in a speedvac concentrator (1.5 hours), resuspended in 13.2 μL DMSO, shaken for 10 seconds, then sonicated (20 seconds) and shaken for 10 minutes (e.g. thermomixer, 1400 rpm). 140 mM NaCl; 0.1% Pluronic F68; pH 7.4 6 mL was added and stirred at room temperature for ₁ hour _. Samples were centrifuged at 3000 g for 20 minutes. The supernatant was discarded and the precipitate dissolved in _0.6 mL of 20 mM _NaH2PO4 ; 140 mM NaCl; 1% Pluronic F68, pH 7.4. 3.4 mL of H ₂ O was added, stirred at room temperature for 1 hour, and then centrifuged at 3000 g for 20 minutes. Eight aliquots of 0.5 mL each of supernatant were stored at -20°C for further use.

3. Aβ(12-42) Globulomers The preparation of Aβ(12-42) oligomers (hereinafter referred to as Aβ(12-42) globulomers) is described in WO2004/067561.

4. Aβ(12-42) Monomer A 5 mg/mL solution (Anaspec) in 0.1% NaOH was prepared and stored at -20°C.

5. Aβ(17-42) Globulomer Production of Cleaved Aβ(17-42) Globulomers from Aβ(1-42) Oligomers by Cleavage with Trypsin For the preparation of (1-42)-globulomer, described in WO2004/067561) 0.1 mL was added to 2.4 mL of buffer (20 _{mM NaH2PO4} , 140 mM NaCl, pH 7.4) and 0.2 mg/ml. Mix mL trypsin solution (Roche) with 125 μL of 20 mM NaH ₂ PO ₄ , 140 mM NaCl (pH 7.4) solution. The reaction mixture is stirred at room temperature for 20 hours. Then 25 μL of an aqueous solution of 100 mM diisopropylfluorophosphate are added and the mixture is further adjusted with 80 μL of 1% strength SDS solution to an SDS content of 0.03%. The reaction mixture is concentrated to approximately 0.25 mL using a 10 kDa centriprep tube. The concentrate is mixed with 0.625 mL buffer (20 mM NaH ₂ PO ₄ , 140 mM NaCl, pH 7.4) and concentrated again to 0.25 mL.

6. Aβ(17-42) Monomer A 5 mg/mL solution (Anaspec) in 0.1% NaOH was prepared and stored at -20°C.

7. Aβ(20-42) Globulomers The preparation of Aβ(20-42) oligomers (hereinafter referred to as Aβ(20-42) globulomers) is described in WO2004/067561.

8. Aβ(20-42) Monomer A 5 mg/mL solution (Anaspec) in 0.1% NaOH was prepared and stored at -20°C.

9. Aβ(1-40) Monomer 1 mg of human Aβ(1-40), (Bachem catalog number H-1194) was suspended in 0.25 mL of HFIP (4 mg/mL suspension) in an Eppendorf tube. The tube was shaken at 37° C. for 1.5 hours (eg, 1400 rpm in an Eppendorf thermomixer) to obtain a clear solution and then dried in a speedvac concentrator (1.5 hours). Samples were redissolved in 46 μL of DMSO (21.7 mg/mL solution=5 mM), shaken for 10 seconds, then sonicated for 20 seconds. After 10 minutes of shaking (eg 1400 rpm in an Eppendorf thermomixer) the samples were stored at -20°C for further use.

10. Aβ(1-42) fibrils 1 mg of human Aβ(1-42) (Bachem catalog number H-1368) was dissolved in 500 μL of 0.1% NH ₄ OH aqueous solution (Eppendorf tube), and the sample was stirred at room temperature for 1 minute. . 100 μL of this freshly prepared Aβ(1-42) solution was neutralized with 300 μL of 20 mM NaH ₂ PO ₄ ; 140 mM NaCl (pH 7.4). The pH was adjusted to pH 7.4 with 1% HCl. Samples were incubated at 37° C. for 24 hours and centrifuged (10000 g for 10 minutes). The supernatant was discarded and the fibril pellet was resuspended in 400 μL of 20 mM NaH ₂ PO ₄ ; 140 mM NaCl (pH 7.4) by vortexing for 1 minute.

Materials for dot blot
Serial dilution of Aβ-antigen in Aβ-standard ₂₀ mM _NaH2PO4 , 140 mM NaCl, pH 7.4 + 0.2 mg/mL BSA 1) 100 pmol/μL
2) 10 pmol/μL
3) 1 pmol/μL
4) 0.1 pmol/μL
5) 0.01 pmol/μL.

Nitrocellulose trans-blot transfer medium, pure nitrocellulose membrane (0.45 μm); Fa. BIO-RAD
Anti-mouse AP
AQ330 (Fa. Chemicon)
Detection reagent NBT/BCIP tablets (Roche)
bovine serum albumin (BSA)
A-7888 (SIGMA)
Blocking reagent 5% low-fat milk in TBS
buffer
TBS
25 mM Tris/HCl-buffer pH 7.5
+150mM NaCl
TTBS
25 mM Tris/HCl-buffer pH 7.5
+150mM NaCl
+0.05% Tween 20
PBS + 0.2 mg/mL BSA
20 mM _{NaH2PO4 buffer} pH 7.4
+140 mM NaCl
+0.2 mg/mL BSA
Antibody solution I
Mouse plasma samples from active immunization studies with Aβ(20-42) globulomers (diluted 1:400 in 20 mL of 1% low-fat milk in TBS)
Antibody solution II
1:5000 dilution anti-mouse-AP in TBS solution of 1% low fat milk.

Dot Blot Procedure 1) 1 μL of each of the different Aβ-standards (in 5 serial dilutions) were dotted onto a nitrocellulose membrane approximately 1 cm apart from each other.

2) Air-dry the Aβ-standard dots on the nitrocellulose membrane for at least 10 minutes at room temperature (RT) (=dot blot).

3) Blocking Incubate the dot blot with 30 mL of 5% low-fat milk in TBS for 1.5 hours at room temperature.

4) Washing Discard the blocking solution and incubate the dot blot with 20 mL TTBS for 10 minutes at room temperature with shaking.

5) Antibody solution I
Discard the wash buffer and incubate the dot blot with Antibody Solution I overnight at room temperature.

6) Washing Discard Antibody Solution I and incubate the dot blot with 20 mL TTBS for 10 minutes at room temperature with shaking. The wash solution is discarded and the dot blot is incubated with 20 mL of TTBS for 10 minutes at room temperature with shaking. Discard the wash solution and incubate the dot blot with 20 mL of TBS for 10 minutes at room temperature with shaking.

7) Antibody solution II
The wash buffer is discarded and the dot blot is incubated with Antibody Solution II for 1 hour at room temperature.

8) Washing Discard the antibody solution II and incubate the dot blot with 20 mL of TTBS for 10 minutes at room temperature with shaking. The wash solution is discarded and the dot blot is incubated with 20 mL of TTBS for 10 minutes at room temperature with shaking. Discard the wash solution and incubate the dot blot with 20 mL of TBS for 10 minutes at room temperature with shaking.

9) Development Discard the wash solution. Dissolve 1 tablet of NBT/BCIP in 20 mL of H ₂ O and incubate the dot blot with this solution for 5 minutes. Development is stopped by washing thoroughly with _H2O .

10) Densitometric analysis Quantitative evaluation was performed using a densitometric analysis of intensity (BioRad densitometer and software package Quantity one (BioRad)). For the evaluation in FIG. 24, 100 pMol refractive density values were taken as readings.

The results are shown in Figures 24a and b.

The plasma of human Alzheimer's disease patients contains autoantibodies reactive to Aβ(12-42) globulomers and Aβ(20-42) globulomers, and the CSF of human Alzheimer's disease patients to Aβ(12-42) globulomers and Aβ (20-42) The conclusion is that it contains autoantibodies that are reactive against globulomers.

These results demonstrate the feasibility of examining the presence of globulomers (which by themselves are difficult to detect due to their high affinity for the target) in body fluids by assessing the presence of antibodies to the globulomers. showing. It further enhances the status of globulomers as pathophysiological entities.

(Example 8)
active immunization
Description Memory in novel object recognition tasks by active immunization of APP transgenic mice harboring and overexpressing human APP with the London mutation under the control of the Thy1 promoter (APP/Lo; Moechars et al., 1999). disability improved. APP/Lo mice immunized with Aβ(1-42) globulomers or Aβ(1-42) globulomers had a higher search index for new subjects compared to known subjects the mice had encountered 3 hours earlier. but not in mice immunized with Aβ(1-42) monomer (FIG. 21). Mice's curiosity, measured as subject investigation time at the first encounter, did not vary between groups. Thus, active immunization with Aβ(1-42) and Aβ(20-42) globulomers selectively improved short-term memory in subjects.

Methods —Active Immunization of APP Transgenic Mice APP[V717I]C57BI×FVB female mice (n=36; 6 weeks old) were used in this study (Moechars et al., 1999). All mice were housed and bred in an animal care facility (the Catholic University Leuven, Campus Gasthuisberg) in accordance with the Belgian Ethical Code for the Care, Breeding and Handling of Laboratory Animals.

Animals were genotyped by polymerase chain reaction (PCR) at 3 weeks of age and double checked by a second round of PCR at study initiation. All mice were blind-randomized, age-matched, and randomly assigned to treatments. Animals were re-caged by treatment group the day before study initiation to acclimate to the new cage conditions. Animals were allowed free access to filtered, sterile (UV light) drinking water and standard mouse chow (Muracon-G, Trouw Nutrition, Gent). Food was stored under dry and cold conditions in a well-ventilated storage room. The amount of water and food was checked once daily and replenished if necessary, and standard food was replaced twice weekly. Mice were housed in standard metal cages type RVST2 (area 540 cm ² ) with a hard floor and a layer of straw shavings under a reversal day-night rhythm of 14 hours light/10 hours dark with light onset at 7 pm. bottom.

Mice were given one of the following solutions: phosphate buffered saline (PBS; pH 7.4), 100 μg of Aβ(1-42) (Bachem, H1368) in 0.25% NH ₄ OH, Aβ(1-42) 42) 100 μg globulomer or 30 μg Aβ(1-42) globulomer were administered intraperitoneally (200 μL per injection; n=7-9 animals per group). The initial injection contained 50% (by volume) complete Freund's adjuvant (Difco Laboratories, Detroit, Michigan, USA, reference number 263810) and the booster injection contained 50% (by volume) incomplete Freund's adjuvant (Difco Laboratories, Detroit, Michigan, USA, Docket No. 263910). Compound dosing occurred every 3 weeks for 3 months (weeks 0/3/6/9/12).

- Novel Object Recognition Task in Immunized Mice After the last injection, all mice were subjected to a novel object recognition task. The protocol used followed the method reported by Dewachter et al. (Dewachter I. et al., Journal of Neuroscience, 2002, 22(9): 3445-3453). Mice were habituated for 1 hour to an open Plexiglas box (52 x 52 x 40 cm) with black vertical walls and a transparent floor that was dimly lit by a lamp placed on the underside of the box. The next day, animals were placed in the same box and subjected to a 10 minute acquisition test. During this test, mice were placed individually in an open field in the presence of object A (a blue ball or red cube, of similar size ±4 cm), and the duration (time _AA ) and frequency (frequency _AA ) of exploring object A were measured. (where the animal's nose turns toward the object at a distance of <1 cm and the mouse actively sniffs in the direction of the object) was recorded by a computerized system (Ethovision, Noldus information Technology, Wageningen, the Netherlands). During a 10-minute retention test (second test) performed 3 hours later, a new subject (subject B, red cube or blue ball) was placed with the open-field-familiar subject (subject A) (each frequency _A and frequency _B and time _A and time _B ).

Non-spatial memory was measured using the Recognition Index (RI), defined as the ratio of the duration of exploring a novel object to the duration of exploring both objects [time _B /(time _A +time _B )×100]. Curiosity was measured using the duration and frequency with which object A was explored during the acquisition test (time _AA and frequency _AA ).

- Results During the active immunization process, the weight gain between different treatment groups of mice was the same, indicating no side effects of the immunization procedure. All groups exhibited the same curiosity during the first encounter with the subject. That is, there was no statistically significant difference in time _AA and frequency _AA . At the second encounter, mice immunized with Aβ(1-42) and Aβ(20-42) globulomers explored the unknown subject significantly longer (i.e., 50% (= chance level for no memory) P<0.01; RI) greater than Student's t-test), but not in mice treated with Aβ(1-42) monomer or vehicle. Furthermore, the RIs of mice immunized with Aβ(1-42) and Aβ(20-42) globulomers were significantly higher than those of mice treated with Aβ(1-42) monomers or vehicle (P<0 .01; ANOVA followed by post-hoc Holm-Sidak t-test).

(Example 9)
passive immunization
Description Memory in a novel object recognition task by passive immunization of APP transgenic mice harboring and overexpressing human APP with the London mutation under the control of the Thy1 promoter (APP/Lo; Moechars et al., 1999). disability improved. We observed that mouse monoclonal antibodies 6G1 and 8F5 compared to PBS-treated mice resulted in a higher survey index for new subjects compared to known subjects that the mice had encountered 3 hours earlier. (Fig. 22). Mice's curiosity, measured as subject investigation time at the first encounter, did not vary between groups. Thus, passive immunization with mouse monoclonal antibodies 6G1 and 8F5 selectively improved short-term memory in subjects.

Methods Mice and breeding conditions were the same as the protocol used in Example 8. Mice were treated with 250 μg of monoclonal antibodies 6B1 and 8F5 directed against Aβ globulomers in phosphate buffered saline (PBS) (250 μL) or PBS alone (n=8 for all groups) once weekly for 2 weeks. Administered intraperitoneally (2 injections in total). One day after the last injection, the object recognition task described in Example 8 was performed.

Results All groups exhibited the same curiosity during the first encounter with the object. That is, there was no statistically significant difference in frequency _AA . At the second encounter, mice immunized with mouse monoclonal antibodies 6G1 and 8F5 explored unknown subjects significantly longer (i.e., 50% (= chance level for no memory; P at least <0.05; RI) over Student's t-test) was absent in mice injected with PBS alone.

(Example 10)
Induction of Aβ(1-42) globulomer formation by fatty acids Various fatty acids were investigated to see if they could be used instead of SDS as inducers of in vitro Aβ(1-42) globulomer formation.

Results are shown in FIG. As can be seen, lauric acid, oleic acid and arachidonic acid also induced characteristic 38/48 kDa Aβ(1-42) globulomers, whereas eicosapentanoic acid gave rise to partially migrated oligomers, linolenic acid, docosahexane Acid and docosatetraenoic acid failed to produce a characteristic 38/48 kDa Aβ double band within detection limits.

These data indicate not only as yet uncharacterized factors that cause overproduction of Aβ by causing miscleavage of APP, but also possibly induce conformational changes in Aβ or scaffolds where globulomer formation can occur. It has been suggested that the direct catalytic influence of lipids by providing an anionic surface in the form of micelles or vesicles acting as lipids also promotes globulomer formation in vivo. This is of particular interest as it may indicate a role for globulomers in the dementiac effects of certain disorders of lipid metabolism as well as potentially beneficial effects of agents or compositions that affect brain lipid metabolism on AD and related conditions.

(Example 11)
In Vivo Binding of Aβ(1-42) Globulomer Intracranial injections in rats were performed to investigate the feasibility of in vivo administration of Aβ(1-42) globulomer. For electrophysiological and histological studies, male Sprague-Dawley rats were obtained from Janvier (France) at approximately 8 weeks of age. All animals were maintained under standard conditions (12 h day/night cycle, 22° C., 50% humidity), had free access to food and tap water, and were fed at least one hour after arrival at our animal colony. Recovered after a week.

Rats (260 to 430 g) were anesthetized with pentobarbital (60 mg/kg ip; Narcoren) and fixed in a stereotaxic apparatus according to the literature (Paxinos and Watson ³⁶ ). For intraventricular administration, 18 rats were injected with Aβ(1-42) globulomer into the right ventricle using a combination (mm to bregma) of 1.0 tail, 1.5 lateral and 3.9 ventral. A stereotaxic microinjection of 0.8 nmol was performed. A total volume of 4.3 μL was injected during 5 minutes by means of a 10 μL Hamilton syringe attached to a motorized micropump (WPI, Germany) and the cannula was left in place for an additional 2 minutes. For intracortical injection, 8 rats were dosed with Aβ(1-42) globulomer (in 0.15 nmol) was performed. The infusate was administered via a 10 μL Hamilton syringe for 5 minutes and the cannula was left in place for an additional 5 minutes.

Animals were allowed to recover and then sacrificed 1, 4 and 7 days later to examine penetration of Aβ(1-42) globulomers from the ventricles/injection site into surrounding tissues by immunohistochemical and biochemical dot-blot analyses. . Therefore, rats were deeply anesthetized with Narcoren. To analyze whether Aβ(1-42) globulomers are cleared from the cerebrospinal fluid (CSF), in some rats the atlas occipital periosteum was exposed and the cisterna magna was treated with a 1 mL syringe and a 0.3 mm cannula. A few μL of CSF were withdrawn after blocking with . The rats were then transcardially perfused with 10 mM PBS (pH 7.2) for 5 min followed by 4% paraformaldehyde (PFA) in PBS. Brains were excised, postfixed in 4% PFA in PBS containing 30% sucrose overnight, transferred to 30% sucrose in PBS and cryoprotected.

For staining of Aβ(1-42) globulomers, 40 μm sections were cut with a cryomicrotome and incubated free-floating in 5% donkey serum (Serotec) in TBST for 20 minutes. Sections were then transferred to primary monoclonal antibody 8F5, which is specific for Aβ(1-42) globulomers, at a dilution of 1:1000 in TBST for 24 hours. Fluorescent labeling was performed for 1 hour using a Cy3-labeled donkey anti-mouse antibody (Jackson Laboratories). Between steps and after completion, sections were washed extensively with TBST three times for 5 minutes before being mounted on glass slides, air dried and coverslipped with Vectashield maximum hardness mounting medium containing DAPI (Vector Laboratories). The cell nucleus was observed with the naked eye. All incubations were performed at room temperature. Some sections were also stained with 0.05% Thioflavin S (Sigma, Germany) in 50% ethanol for 10 min, followed by two 5 min rinses in 100% ethanol before embedding in Vectashield. bottom. Labeling analysis was performed under a confocal fluorescence microscope (Zeiss LSM 510 Meta, Germany).

A third ventricular injection resulted in rapid and persistent binding of Aβ globulomers to cells of the ventricular wall (see FIG. 16(II)a), but they were completely eliminated from the cerebrospinal fluid (see FIG. 16(II)a). CSF; data not shown). Injected Aβ(1-42) globulomers were not masked by other proteins in CSF, as administration of various concentrations to rat CSF in vitro resulted in dose-dependent recovery. Bound Aβ globulomers remained in the ependyma with no detectable diffusion into underlying brain tissue and no apparent cytotoxicity as cell density appeared unchanged towards the ventricular wall. Small tissue penetration was also shown by intracerebral injection, where Aβ globulomer staining was restricted near the injection site and did not decline for several days (see Figure 16(II)b-c). Biochemical analysis confirmed that the majority of injected Aβ(1-42) globulomers were still present in the hippocampus for at least 7 days (data not shown). In contrast, injected Aβ(1-42) monomers stained a relatively large area, with decreased labeling after 7 days (FIG. 16(II)c), indicating greater tissue penetration than Aβ(1-42) globulomers. was suggested to be good.

(Example 12)
Specific Binding of Aβ(1-42) Globulomers to Hippocampal Neurons We investigated the physiological relevance of Aβ(1-42) globulomers by studying their interactions with primary hippocampal neurons.

Hippocampal cells from rat brain (QBM Cell Science, Canada) were incubated with poly-L-lysine in Neurobasal medium (Invitrogen, Germany) supplemented with B27 at 37° C., 5% CO _{2 .} Cultures were made in 24-well plates on coated coverslips (BD Biosciences, Germany). Mitotic inhibitors (uridine 35 μg/mL, 5-fluoro-2′-deoxyuridine 15 μg/mL) were added to some cultures at DIV 6 to prevent overgrowth with glia. Hippocampal cells were used at DIV 13-14 unless otherwise stated. When Aβ was added, 750 μL of fresh 37° C. warm medium containing Aβ was replaced for a total of 1 mL. Incubation was at 37° C., 5% CO ₂ for 15 minutes. Aβ-monomer (0.5 mg/mL) stock solution was HFIP solution and Aβ(1-42) globulomer was in 35 mM NaCl, 5 mM NaH ₂ PO ₄ , pH 7.4 solution.

Cells were rinsed three times with 1 mL medium and fixed with 3.7% buffered formaldehyde (Accustain, Sigma, Germany). Non-specific binding sites were blocked by incubation with 10% normal goat serum in PBS (pH 7.4) for 90 minutes at room temperature, to which 0.1% Triton X-100 was added for intracellular counterstaining. rice field. Next, primary antibodies (anti-Aβ6E10 (Signet Laboratories, USA), 1:2000; anti-GFAP (Dako Cytomation, Germany), 1:2500; anti-MAP2 (Chemicon International, Germany), 1:1000) and secondary antibodies ( Alexa 488, 1:500; Alexa 635, 1:500; Molecular Probes, Germany) were incubated in 10% normal goat serum in PBS (pH 7.4) for 2 hours at room temperature. After each antibody incubation, cells were washed three times with PBS and finally placed in ProLong antifade reagent (Molecular Probes, Germany).

Aβ(1-42) globulomers stimulated hippocampal neurons at a concentration of 17 nM (equivalent to a 200 nM monomeric concentration) in contrast to controls without Aβ(1-42) and controls with 200 nM monomeric Aβ(1-42). showed strong binding to (see Figure 15a). The monomeric structure of Aβ(1-42) was secured by the HFIP stock solution. A possible negative effect of HFIP on the ability of hippocampal neurons to bind Aβ(1-42) was prevented by incubating an equivalent volume of HFIP with Aβ(1-42) globulomers, and Aβ(1-42) globulomers There was no difference compared with the single case. Hippocampal neurons bound Aβ(1-42) globulomers at levels as low as 2 nM (data not shown). Binding of Aβ(1-42) globulomers to neurites is strongly disrupted, suggesting specific binding to synaptic terminals or dendritic spines (Fig. 15b, see inset). A strong dependence of Aβ(1-42) globulomer binding on the age of cultured neurons was observed. Little hippocampal binding was observed at DIV8 (approximately 1%), whereas Aβ(1-42) globulomer binding was prominent at DIV11 and DIV15 (approximately 90%) (see Figure 15b). Its high specificity of Aβ(1-42) globulomer binding was further demonstrated by its restricted binding to neurons, whereas glia in this hippocampal preparation did not bind Aβ(1-42) globulomers ( See Figure 15c). Moreover, various neuronal cell lines were negative for Aβ(1-42) globulomer binding (data not shown).

(Example 13)
Complete Inhibition of Long-Term Potentiation by Aβ(1-42) Globulomer To confirm the effect of Aβ(1-42) globulomer on synaptic function, we performed Extracellular potentials were recorded from the CA1 region of perfused brain slices.

Sections were obtained from male Sprague-Dawley rats (7-8 weeks old). The excised brain hemispheres were shaved and sectioned using a vibratome. Sections (400 μM) were transferred directly into the interface recording chamber and oxygenated artificial CSF (aCSF: 118 mM NaCl, 1.24 mM KH ₂ PO ₄ , 10 mM glucose, 2 mM MgSO ₄ , 2.5 mM CaCl) at a flow rate of 1.8 mL/min. ₂ , 5 mM KCl, 25.6 mM NaHCO ₃ ) were continuously perfused at 33°C. Freshly obtained sections were allowed to equilibrate for at least 1 hour before recording. Field potentials were obtained from the stratum radiatum of CA1 using CSF-filled glass microelectrodes (0.8 to 1.2 MΩ). Schaffer collaterals were stimulated by bipolar tungsten electrodes (0.5 MΩ, Science Products GmbH, Germany). Constant voltage biphasic pulses (0.1 ms/phase; range 1 to 5 V) were applied once per minute at an intensity giving 30% of the fEPSP maximum. LTP was induced by two trains of 100 Hz (1 sec/train with 10 sec intertrain interval) using the same intensity and pulse length. The Aβ group was perfused with 42 nM Aβ(1-42) globulomer throughout the experiment beginning 80 to 120 minutes prior to tetanus induction. Input/output relationships were measured prior to baseline recordings (64 to 94 minutes after Aβ(1-42) globulomer wash-in). Signals were digitized by Power CED1401 and analyzed using the software Signal 2.14 (Cambridge Electronic Design Ltd., Cambridge). For statistical comparisons, data were analyzed by two-way repeated measures ANOVA using SAS.

Field EPSPs obtained from the CA1 dendritic layer of Aβ(1-42) globulomer-treated sections appeared normal in size and shape (see FIG. 17b), indicating that excitatory synaptic transmission was unaffected. was showing. Field EPSPs also showed no lack of synaptic inhibition (ie, multiple assembly spikes), indicating that GABAergic transmission was unaffected. Furthermore, comparison of mean input/output relationships in Aβ(1-42) globulomer-treated and untreated sections (see FIG. 17c) shows that basic synaptic function is not impaired by Aβ(1-42) globulomer. rice field. However, when examining high-frequency stimulation-induced LTP, Aβ(1-42) globulomer-treated (42 nM) sections showed complete block (see Figure 17a). Potentiation decayed to baseline levels within 30 minutes of tetanic stimulation. Short-term potentiation could be easily induced in the Aβ(1-42) globulomer group, but potentiation was significantly impaired as early as 5 min after tonic induction (p<0.05). In contrast, control sections showed stable LTP for at least 1 hour, with an enhancement of at least 30% over the entire recording time. In some experiments, we measured the Aβ(1-42) globulomer concentration at the outflow of the recording chamber and were able to detect 50 to 70% Aβ(1-42) globulomers, which is a significant amount (total Aβ(1-42) globulomers of Aβ(1-42) reached the slice during the experiment. Thus, less than 42 nM Aβ(1-42) globulomers were sufficient to specifically inhibit LTP while leaving basal synaptic transmission unaffected.

(Example 14)
Antibodies to Aβ(1-42) Anti-Aβ(20-42) globulomer polyclonal antibody 5598 was generated from immunized rabbits and affinity purified against immobilized Aβ(1-42) globulomers. The methods used are industry-proven. Briefly, Aβ(1-42) synthetic peptide (catalog number H-1368, Bachem, Switzerland) was added to 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at approximately 6 mg/mL. Suspended and completely lysed by incubating at 37° C. with shaking for 1.5 hours. HFIP was completely removed by speedvac evaporation and Aβ(1-42) was resuspended in dimethylsulfoxide (DMSO) at a concentration of 5 mM and sonicated for 20 seconds. HFIP-pretreated Aβ(1-42) was diluted to 400 _μM in phosphate buffered saline (PBS) (20 mM _NaH2PO4 , 140 mM NaCl, pH 7.4) and 1/10 volume of 2% SDS ( _H2O ). solution) was added (final concentration 0.2% sodium dodecyl sulfate (SDS)). A 6 h incubation at 37° C. yielded the 16/20 kDa Aβ(1-42) globulomer intermediate. Further dilution with 3 volumes of H ₂ O and incubation at 37° C. for 18 hours formed 38/48 kDa Aβ(1-42) globulomers. After centrifugation at 3000 xg for 20 minutes, samples were concentrated by ultrafiltration (30 _kDa cutoff), dialyzed against 5 mM _NaH2PO4 , 35 mM NaCl, pH 7.4, and centrifuged at 10000 xg for 10 minutes. , the supernatant containing the 38/48 kDa Aβ(1-42) globulomers was withdrawn. For fatty acid-induced 38/48 kDa Aβ(1-42) globulomers, 1/10 volume of 0.5% fatty acid was added instead of SDS. For cross-linking, 38/48 kDa Aβ(1-42) globulomers were treated prior to concentration and dialysis with 1 mM glutardialdehyde for 2 h at RT, and then ethanolamine for 30 min at room temperature (RT). 5 mM) treatment was performed. Restricted proteolysis of the 38/48 kDa Aβ(1-42) globulomers was performed using 1/50 (mg/mg) of each protease and incubation at room temperature for 20 hours.

Immunization of mice yielded high titers (1:10,000 to 100,000) and potent Aβ(1-42) globulomer-specific (8F5) and non-specific monoclonal antibodies (6G1). In contrast to the non-specific 6G1 and reference antibody 6E10, the specific antibody 8F5 detected Aβ(1-42) globulomers only by native PAGE-Western blot, but not by SDS-PAGE Western blot analysis. (data not shown) indicated binding to a more complex detergent-cleavable intersubunit epitope in the nuclear Aβ(1-42) globulomer structure (see Figure 12a). Dot blot analysis on various Aβ(1-42) and Aβ(1-40) standard preparations showed that Aβ(1-42) globulomers and non-globulomer Aβ forms (in standard Aβ(1-40/42) monomer preparations Significant differences in recognition were shown, with aggregation of Aβ(1-42)) for the specific 8F5 and 5598, but not for the isoform non-specific antibodies 6G1 and 6E10 (see Figure 12b). The globulomer specificity of 8F5 and 5598, but not 6G1 and 6E10, was confirmed by quantification of Aβ(1-42) globulomer, Aβ(1-42) monomer, Aβ(1-40) and sAPPα binding in a sandwich ELISA ( See Figure 12c). However, since our specific antibodies did not detect the Aβ(1-42) globulomer epitope at levels as low as <0.5% compared to Aβ monomers in CSF samples, we It is assumed that antibody selectivity was underestimated due to slight cross-contamination with Aβ(1-42) globulomer epitopes in their synthetic standard preparation (see data in Figure 13). The extent of cross-contamination in synthetic Aβ preparations was calculated from ELISA value ratios (see Figure 12d). Aβ(1-42) _NH4OH preparations and Lambert, MP et al. Diffusible, non-fibrillar ligands derived from Abeta 1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. A 95, 6448-6453 (1998)), approximately 5% Aβ(1-42) globulomer epitopes and 95% Aβ(1-42) monomeric epitopes were measured in Aβ oligomers prepared according to the present invention. A[beta](1-42) globulomer preparation of was of 95% purity with only 5% cross-contamination signal for monomeric A[beta](1-42). In conditioned medium from CHO cells expressing APP with the V717F mutation (Walsh, DM et al. Naturally secreted oligomers of amyloid beta protein pqtently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535 -539 (2002)), Aβ(1-42) globulomer epitope levels were below the detection limit.

(Example 15)
Endogenous amyloid-β (1-38), (1-40) and (1-42) levels in AD brain tissue extracts after immunoprecipitation with globulomer-selective anti-Aβ mAb 8F5 in human AD brain (Brain-Net -net), sample from Munich) was suspended in 1.39 mL of extraction buffer at 0°C. The sample was homogenized in a glass crucible by pressing the pestle 20 times. The homogenized tissue was centrifuged at 100000 g for 1 hour. Supernatants were taken as human AD brain extracts.

Materials Basic buffer:
50 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.01% Tergitol NP-40, 0.1% SDS pH 7.6 (adjusted with 10M HCl)
Extraction buffer:
100 mL buffer
+ 1 tablet of Complete Protease Inhibitor Cocktail (Fa. Roche No. 1697498) dissolved in 1 mL H ₂ O + 200 μL of 250 mM PMSF (in methanol) prepared immediately prior to use.

Immobilization of anti-Aβ mAbs to CNBr-activated Sepharose 4B mAb 8F5:
0.4 g of CNBr-activated Sepharose 4B (Amersham Inc. No. 17-0430-01) was added to 10 mL of 1 mM aqueous HCl and incubated for 30 minutes at room temperature. CNBr-activated Sepharose 4B was washed three times with 10 mL of 1 mM HCl and finally twice with 10 mL of 100 mM NaHCO ₃ ; 500 mM NaCl; pH 8.3. For each immobilized antibody, 100 μL of CNBr-activated Sepharose 4B substrate was added to 950 μL of 0.5 mg/mL anti-Aβ mAb 8F5 in 100 mM NaHCO ₃ ; 500 mM NaCl; pH 8.3. After shaking for 2 hours at room temperature, the samples were centrifuged at 10000 g for 5 minutes. 500 μL of 100 mM ethanolamine; 100 mM NaHCO ₃ ; 500 mM NaCl; pH 8.3 buffer was then added to the beads and the samples were shaken for 1 hour at room temperature.

Anti _- A[beta] mAb 8F5-Sepharose samples were centrifuged at 10000 for 5 minutes and washed 5 times with 20 mM _NaH2PO4 ; 140 mM NaCl; pH 7.4500 [mu]L. Samples were stabilized by adding sodium azide to a final concentration of 0.02% before freezing for storage.

Immunoprecipitated mAb 8F5-Sepharose:
150 μL of human AD brain extract was diluted with 150 μL of 20 mM NaH ₂ PO ₄ ; 140 mM NaCl; 0.05% Tween 20; pH 7.4. These samples were added to 2 μL of anti-Aβmab8F5-Sepharose substrate and stirred for 1.5 hours at room temperature. Samples were centrifuged at 10000 g for 5 minutes. The supernatant was discarded and the anti-Aβ mAb 8F5-Sepharose was washed twice with 50 μL PBS, vortexed for 1 minute and centrifuged (10000 g for 5 minutes). The supernatant was discarded and the Sepharose beads were suspended in 50 μL of 2 mM NaH ₂ PO ₄ ; 14 mM NaCl; pH 7.4, then stirred for 1 minute at room temperature and centrifuged at 10000 g for 5 minutes. In the next step, the anti-A[beta] mAb-Sepharose beads were treated with 10 [mu]L of 50% _CH3CN ; 0.2% TFA in water. After shaking for 10 minutes at room temperature, the samples were centrifuged at 10000 g for 5 minutes. The supernatant was collected.

Measurement of Aβ(1-xx) using SELDI-MS:
2 μL of the anti-Aβ mAb 8F5-Sepharose IP eluate was plated on the H4 protein chip array configuration A-HFa. Dots were formed on spots of Fa. Ciphergen part number C553-0028. The chips were air dried. Then 2 μL of 3.33 mg/mL CHCA in 50% CH ₃ CN; 0.5% TFA in water was dotted onto the spot. The chips were air-dried again and analyzed on a SELDI mass spectrometer (Sifelgen).

conditions:
Strength: 200
Sensitivity: 6
Mass range: 800 to 10000 Da
Calibration: 7 Fa. All-in-one peptide standard mixture of Schifelgen part number C100-0005.

Schematic diagram of Aβ processing pathways. FIG. 2 shows Western blots of monomers and globulomers after storage at various temperatures to compare in vitro stability. FIG. 1 depicts antibody titers of rabbits and mice immunized with Aβ(1-42), Aβ(12-42) and Aβ(20-42) globulomers. FIG. 3 shows the detection results of Aβ(1-42) globulomer epitopes in AD brain and TG2576 brain. FIG. 3 shows the detection results of Aβ(1-42) globulomer epitopes in the CNS. FIG. 3 shows the reactivity of Aβ(33-42)-directed PAb (Signet 9135) with various Aβ preparations for characterization of Aβ(1-42) globulomer epitopes. FIG. 4 shows reactivity of anti-Aβ(20-42) globulomer-derived antibody 5598 in sandwich ELISA. FIG. 2 shows the effect of pretreatment of Aβ(1-42) globulomer and Aβ(1-40) monomer standard preparations with the anti-Aβ globulomer-specific PAb 5598, as measured by the ELISA below. FIG. 5 shows an SDS-PAGE chromatogram of Aβ(20-42) globulomers obtained according to Example 5; FIG. 3 shows an SDS-PAGE chromatogram of Aβ(12-42) globulomers obtained according to Example 6; FIG. 2 summarizes data on the formation of Aβ(1-42) globulomers. FIG. 2 shows the reactivity of Aβ(1-42) globulomer-specific antibodies with high affinity and specificity in dot blot and ELISA. FIG. 1 shows that Aβ(1-42) globulomers are present in the brains of Alzheimer's patients and APP transgenic mice. FIG. 1 shows that Aβ(1-42) globulomer formation can be induced by certain fatty acids. FIG. 4 shows that Aβ(1-42) globulomers specifically bind to hippocampal neurons depending on culture age. FIG. 16(I) shows that Aβ(1-42) globulomers have limited tissue penetration due to high binding. FIG. 16(II) shows that Aβ(1-42) globulomers have limited tissue penetration due to high binding. FIG. 2 shows that Aβ(1-42) globulomers completely block long-term potentiation in vitro. FIG. 2 shows a proposed model of the two-pathway mechanism of Aβ-peptide multimerization. The first step is Aβ-monomer formation by β- and γ-secretase cleavage of APP. Circular dichroism spectra of Aβ(1-42) globulomers. FIG. 100 pmol (row A), 10 pmol (row B) and 1 pmol (row C) of Aβ(1-42) globulomer (column 1); Aβ(1-42) monomer in 0.1% Pluronic F58 (column 2); Aβ(20-42) globulomers (lane 3); glutaraldehyde crosslinked Aβ(1-42) globulomers (lane 4); Lambert et al., J. Neurochem. 79, 595-605 (2001)) at 4°C or room temperature or 37°C (lane 5); Aβ(12-42) globulomers (lane 6); Aβ(1-40) monomers (lane 7); Aβ(1-42) dissolved in ₄ OH (column 8); Aβ(1-42) fibril preparation (column 9); and PBS diluted APP from Sigma (column 10) A) human plasma and B) Dot blot of reactivity with CSF. FIG. 13 shows a novel object recognition task in actively immunized APP/Lo mice. Bar graph of recognition index of mice treated with saline (control), antibody 8F5 and antibody 6G1. SELDI mass spectral data from AD brain extracts immunoprecipitated with antibody 8F5. FIG. 1 shows autoantibody levels from human serum/plasma directed against various amyloid beta antigens.

Claims (72)

  X is the number 1. . A non-diffusible spherical Aβ (X-38..43) oligomer selected from the group consisting of 24 or a derivative thereof.   The oligomer or derivative according to claim 1, wherein the oligomer is a non-diffusible spherical Aβ (X-40) oligomer.   The oligomer or derivative according to claim 1, wherein the oligomer is a non-diffusible spherical Aβ (X-42) oligomer.   X is the number 8. . 4. The oligomer or derivative according to any one of claims 1 to 3 selected from the group consisting of 24.   X is Equation 12. . 4. The oligomer or derivative according to any one of claims 1 to 3 selected from the group consisting of 24.   X is Equation 12. . 4. The oligomer or derivative according to any one of claims 1 to 3 selected from the group consisting of 20.   The oligomer or derivative according to any one of claims 1 to 6, which is soluble.   The oligomer or derivative according to any one of claims 1 to 7, wherein the derivative is an oligomer covalently labeled with a group facilitating detection.   Groups that facilitate detection include fluorophores, chromophores, chemiluminescent groups, enzyme active groups, high electron density groups, haptens, strong antigenic structures, aptamers, chelating groups, specific protein-protein interaction mediators, magnetism 9. The oligomer or derivative of claim 8 selected from the group consisting of groups and radioactive groups.   10. The oligomer or derivative according to any one of claims 1 to 9, wherein the derivative is an oligomer flagged with a group that promotes in vivo degradation.   The oligomer or derivative according to claim 10, wherein the group that promotes in vivo degradation is ubiquitin.   12. A purified oligomer or derivative according to any one of claims 1 to 11.   Method for concentrating non-diffusible spherical Aβ (X-38..43) oligomers or derivatives thereof in a preparation comprising oligomers or derivatives, the step of capturing the oligomers or derivatives and obtaining the oligomers or derivatives in concentrated form Having a method.   14. The method of claim 13, wherein capturing the oligomer or derivative comprises contacting the preparation with an agent that binds to the oligomer or derivative.   15. The method of claim 14, wherein the drug is immobilized.   16. The method of claim 14 or 15, wherein the agent is an antibody having specificity for an oligomer or derivative.   17. The method of any one of claims 13 to 16, wherein obtaining the oligomer or derivative in concentrated form comprises desorbing the captured oligomer or derivative.   18. The method of claim 17, wherein desorbing the capture oligomer or derivative comprises contacting the capture oligomer or derivative with a high salt buffer or acidic solution.   19. A method according to any one of claims 13 to 18, further comprising the step of determining the amount of non-diffusible spherical A [beta] (X-38..43) oligomer or derivative in the preparation.   A method of concentrating a non-diffusible spherical Aβ (X-38..43) oligomer or derivative thereof in a preparation comprising the oligomer or derivative, the method comprising removing at least one substance other than the oligomer or derivative from the preparation Having a method.   21. The method of claim 20, wherein the substance other than the oligomer or derivative is an Aβ monomer or Aβ fibrillomer.   23. The method of claim 21 or 22, wherein removing the substance comprises contacting the preparation with an agent that specifically binds to the substance.   23. The method of claim 22, wherein the drug is immobilized.   24. The method of claim 22 or 23, wherein the agent is an antibody that specifically binds to a substance.   25. A method according to any of claims 20 to 24, wherein the step of removing the substance comprises contacting the preparation with an antibody that binds to the substance and then performing protein A affinity chromatography on the substance bound to the antibody.   A method further comprising measuring the amount of non-diffusible spherical Aβ (X-38..43) oligomer or derivative thereof in the preparation.   27. A composition comprising a non-diffusible spherical Aβ (X-38..43) oligomer or derivative thereof obtainable by the method of any one of claims 13 to 26.   The composition comprising the non-diffusible spherical Aβ (X-38..43) oligomer or derivative thereof according to any one of claims 1 to 12, wherein the amount of oligomer or derivative is at least 99% by weight of the composition.   29. The composition of claim 28, wherein the amount of oligomer or derivative is at least 99.9% by weight of the composition.   29. The composition of claim 28, wherein the amount of oligomer or derivative is at least 99.99% by weight of the composition.   31. A composition according to any one of claims 27 to 30 wherein the composition is a vaccine.   Use of a non-diffusible spherical Aβ (X-38..43) oligomer or a derivative thereof as defined in any one of claims 1 to 12 for the production of an antibody having specificity for the oligomer or derivative. .   A method for producing an antibody having specificity for a non-diffusible globular Aβ (X-38..43) oligomer or derivative thereof as defined in any one of claims 1 to 12, comprising an oligomer or derivative Providing an antibody repertoire or potential antibody repertoire to the antigen; and selecting an antibody from the repertoire that specifically binds to the oligomer or derivative thereof.   34. The method of claim 33, wherein the repertoire is exposed to the antigen in vivo by immunizing the organism with the antigen.   An antibody having specificity for a non-diffusible globular Aβ (X-38..43) oligomer or derivative thereof as defined in any one of claims 1-12.   36. The antibody of claim 35 obtainable by the method of claim 33 or 34.   37. The antibody of claim 35 or 36, wherein the affinity for the oligomer or derivative of the antibody is at least 10 times greater than for monomeric A [beta] (1-42).   38. The antibody of any one of claims 35 to 37, wherein the affinity for the oligomer or derivative of the antibody is at least 10 times greater than for monomeric Aβ (1-40).   39. The antibody of any one of claims 35 to 38, wherein the affinity for the oligomer or derivative of the antibody is at least 10 times greater than for fibrillar Aβ (1-42).   40. The antibody of any one of claims 35 to 39, wherein the affinity for the oligomer or derivative of the antibody is at least 10 times greater than for fibrillar Aβ (1-40).   41. The antibody of any one of claims 35 to 40, wherein the affinity for the oligomer or derivative of the antibody is at least 10 times greater than for protofibrillar Aβ (1-42).   42. The antibody of any one of claims 35 to 41, wherein the affinity for the oligomer or derivative of the antibody is at least 10 times greater than for protofibrillar Aβ (1-40).   43. The antibody of any one of claims 35 to 42, wherein the antibody binds to an epitope located between about 20 to about 30 amino acid positions of the oligomer or derivative.   Use of a non-diffusible spherical Aβ (X-38..43) oligomer or derivative thereof as defined in any one of claims 1 to 12 for the manufacture of an aptamer having specificity for an oligomer or derivative. .   A method for producing an aptamer having specificity for a non-diffusible spherical Aβ (X-38..43) oligomer or derivative thereof as defined in any one of claims 1 to 12, comprising the oligomer or derivative Providing a binding target comprising: exposing an aptamer repertoire or potential aptamer repertoire to the binding target; and selecting from the repertoire an aptamer that specifically binds to the oligomer or derivative thereof.   45. An aptamer having specificity for a non-diffusible spherical Aβ (X-38..43) oligomer or derivative thereof as defined in any one of claims 1 to 12 obtainable by the method of claim 45 .   47. The aptamer of claim 46, wherein the aptamer is labeled and / or flagged.   Use of a non-diffusible globular Aβ (X-38..43) oligomer or a derivative thereof as defined in any one of claims 1 to 12 for the manufacture of a pharmaceutical composition for the treatment or prevention of amyloidosis .   49. Use according to claim 48, wherein the pharmaceutical composition is a vaccine for active immunization.   50. Use according to claim 48 or 49, wherein the oligomer cannot enter the CNS.   51. Use according to any of claims 48 to 50, wherein the composition is capable of eliciting a strong antibody-mediated non-inflammatory response against globulomer.   52. Use according to any of claims 48 to 51, wherein the pharmaceutical composition is administered via a route selected from intravenous, muscle and subcutaneous.   A method for the treatment or prevention of amyloidosis in a subject in need of treatment or prevention, wherein the subject is non-diffusible globular Aβ (X-38.43) as defined in any one of claims 1 to 12 Administering the oligomer or derivative thereof.   54. The method of claim 53, wherein the step of administering a non-diffusible globular A [beta] (X-38..43) oligomer or derivative thereof is for active immunization.   44. Use of an antibody as defined in any one of claims 35 to 43 for the manufacture of a pharmaceutical composition for treating or preventing amyloidosis.   56. Use according to claim 55, wherein the pharmaceutical composition is for passive immunization.   44. A method of treating or preventing amyloidosis in a subject in need of treatment or prevention, comprising administering to the subject an antibody as defined in any one of claims 35 to 43.   58. The method of claim 57, wherein the step of administering the antibody is for active immunization.   44. Use of an antibody as defined in any one of claims 35 to 43 for detecting an oligomer or derivative in a preparation.   44. Use of an antibody as defined in any one of claims 35 to 43 for determining the amount of oligomers or derivatives in a preparation.   44. A method for detecting a non-diffusible globular A [beta] (X-38..43) oligomer or derivative thereof in a preparation, wherein the preparation is contacted with an antibody as defined in any one of claims 35 to 43. And a step of detecting a complex formed by the oligomer or derivative and the antibody, wherein the presence of the complex indicates the presence of the oligomer or derivative.   62. The method of claim 61, wherein the method further comprises measuring the amount of oligomers or derivatives present in the preparation.   Detecting autoantibodies having specificity for an oligomer or derivative in a subject of a non-diffusible globular Aβ (X-38..43) oligomer or derivative thereof as defined in any one of claims 1 to 12 Use for manufacturing a composition for.   64. The use of claim 63, wherein the subject is suspected of having amyloidosis and the detection of autoantibodies is for diagnosing Alzheimer's disease in the subject.   Detection of autoantibodies having specificity for an oligomer or derivative in a sample of a non-diffusible spherical Aβ (X-38..43) oligomer or derivative thereof as defined in any one of claims 1 to 12 Use to do.   66. The use of claim 65, wherein the sample is from a subject suspected of having amyloidosis, and the detection of autoantibodies is for diagnosis of amyloidosis in the subject.   A method for detecting an autoantibody having specificity for a non-diffusible globular Aβ (X-38..43) oligomer or derivative thereof in a subject, the method comprising any one of claims 1 to 12 for the subject. Administering a non-diffusible globular Aβ (X-38..43) oligomer or derivative thereof as defined in 1 and detecting a complex formed by the antibody and oligomer or derivative, wherein the presence of the complex A method that indicates the presence of autoantibodies.   68. The method of claim 67, wherein the subject is suspected of having amyloidosis and the detection of autoantibodies is for diagnosis of amyloidosis in the subject.   A method for detecting an autoantibody having specificity for a non-diffusible globular Aβ (X-38..43) oligomer or derivative thereof in a sample, the sample according to any one of claims 1 to 12. Contacting the defined non-diffusible spherical Aβ (X-38..43) oligomer or derivative thereof and detecting the complex formed by the antibody and oligomer or derivative, wherein the presence of the complex is self A method that indicates the presence of an antibody.   70. The method of claim 69, wherein the sample is from a subject suspected of having amyloidosis and the detection of autoantibodies is for diagnosis of amyloidosis in the subject.   71. The method of claim 69 or 70, wherein the subject suspected of having amyloidosis is a subject having amyloidosis or an increased risk of developing amyloidosis.   72. The method or use of any one of claims 48 to 71, wherein the amyloidosis is selected from the group consisting of Alzheimer's disease and Down syndrome amyloidosis.

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