Cross-linked amyloid β (Aβ) as a surrogate for amyloid spheroids (ASPD), and analysis of ASPD

Abstract

The present invention relates to a substance that can serve as a surrogate for amyloid spheroids (ASPD) and an analysis method for ASPD. In one embodiment, the present disclosure relates to a substance formed by cross-linking amyloid β (Aβ) using a cross-linking agent having a spacer arm length of at least 100 and no longer than 1000, or a cross-linking agent having 1 or more and no more than 13 oxyethylene (-CH2CH2O-) and / or oxypropylene (-CH2CH2CH2O-) groups as spacer arms. In another embodiment, the present disclosure relates to an analysis method for ASPD using the substance as a standard substance.

Description

Technical Field

[0001] The present disclosure relates to cross-linked forms of amyloid β (Aβ), cross-linked forms of Aβ (cross-linked Aβ) that can serve as a substitute for amyloid spheroids (ASPD), and analysis of ASPD. Background Art

[0002] Amylospheroids (ASPD) are spherical Aβ aggregates with a diameter of approximately 10 to 15 nm, composed of approximately 30 Aβ particles. They are structures believed to play an important role in causing neuronal cell death in Alzheimer's disease.

[0003] ASPD was isolated as an in vitro synthesized Aβ aggregate (i.e., synthetic ASPD) that exhibits strong neurotoxicity (Non-Patent Document 1). Antibodies specific for this synthetic ASPD were produced (Patent Documents 1 and 2), and these antibodies were used to isolate ASPD formed in vivo (i.e., natural ASPD) from the brains of human Alzheimer's disease patients (Non-Patent Document 1).

[0004] Both natural and synthetic ASPD selectively induce cell death in mature neurons. It was discovered that the target of ASPD in this neuronal cell death is the synaptic protein "Na" which plays a vital role in the survival and function of neurons. + , K + -ATPase pump α3 subunit (hereinafter referred to as NAKα3)", it has been shown that the function of NAKα3 is reduced by ASPD binding, leading to excessive neuronal excitation and neuronal cell death (Patent Document 3, Non-Patent Document 2).

[0005] What is most relevant to clinical symptoms in Alzheimer's disease is nerve cell shedding. The natural ASPD amount in the cerebral cortex of the Alzheimer's disease patient who has clearly seen nerve cell shedding increases in correlation with the severity of Alzheimer's disease, and the natural ASPD amount in the cerebellum of the Alzheimer's disease patient who has hardly seen nerve cell shedding is only present in trace amounts (non-patent literature 3). Therefore, it is believed that amyloid spheres play an important role in the irreversible stage of Alzheimer's disease morbidity. In addition, natural ASPD (non-patent literature 3) is also detected in the brain of Lewy body dementia patients, and it is believed that for Lewy body dementia, ASPD also plays an important role in its morbidity.

[0006] Synthetic ASPD, which is considered to be equivalent to natural ASPD, can be produced by slowly stirring a liquid containing Aβ (Non-Patent Document 1, Patent Document 4).

[0007] Furthermore, a cell-secreted ASPD-like structure that is considered to be equivalent to natural ASPD can be secreted from, for example, CHO cells expressing a specific APP protein and can be obtained from the culture supernatant of the CHO cells (Patent Document 5).

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: WO2006 / 016644

[0011] Patent Document 2: WO2009 / 057664

[0012] Patent Document 3: WO2013 / 099806

[0013] Patent Document 4: WO2013 / 094614

[0014] Patent Document 5: WO2017 / 179646

[0015] Non-patent literature

[0016] Non-patent document 1: Hoshi et al., Spherical aggregates of β-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I / glycogen synthase kinase-3β, PNAS May 27, 2003 vol.100no.11 6370-6375

[0017] Non-Patent Literature 2: Ohnishi et al., Na,K-ATPaseα3 is a death target of Alzheimer patient amyloid-βassembly, PNAS August 11, 2015 vol. 112 no. 32 E4465-E4474

[0018] Non-patent document 3: Noguchi et al., Isolation and characterization of patient-derived, toxic, high mass amyloid beta-protein(Abeta) assembly from Alzheimerdisease brains, J Biol Chem. 2009 Nov 20; 284(47): 32895-905 Summary of the Invention

[0019] Problems to be solved by the invention

[0020] As described above, amyloid beta-globules (ASPD) play an important role in Alzheimer's disease and Lewy body dementia. For the prevention, diagnosis, and treatment of diseases in which ASPD is involved, there is an increasing need to analyze ASPD in biological samples of subjects (patients).

[0021] Furthermore, when developing therapeutic drugs targeting ASPD, analysis of ASPD is required for accompanying diagnostics.

[0022] When analyzing (including measuring) a substance, a standard substance (standard product) is generally required. Standard substances are used, for example, to create calibration or standard curves. Natural ASPD, synthetic ASPD, and cell-secreted ASPD-like structures can be used as standard substances for ASPD analysis.

[0023] On the other hand, standard substances for diagnostic kits are expected to be stable in production, have excellent storage stability, and be low in production cost. However, natural ASPD, synthetic ASPD, and cell-secreted ASPD-like structures have problems as standard substances for kits used to analyze ASPD, from the perspectives of ease of production, storage stability, and production cost.

[0024] Therefore, in one embodiment, the present disclosure provides a substance that can serve as a substitute for ASPD or a substance that can serve as a standard substance for ASPD in the analysis of ASPD.

[0025] In another aspect, the present disclosure provides a method for analyzing ASPD in a biological sample, such as blood or cerebrospinal fluid.

[0026] Means for solving problems

[0027] In one embodiment, the present disclosure relates to a substance that competes with ASPD-specific antibodies and ASPD, wherein Aβ is cross-linked by a cross-linking agent, wherein the cross-linking agent has a spacer arm length of Above and The following crosslinking agents, or a crosslinking agent having 1 or more and 13 or less oxyethylene groups (-CH2CH2O-) and / or oxypropylene groups (-CH2CH2CH2O-) as a spacer arm.

[0028] In another embodiment, the present disclosure relates to a cross-linked Aβ, wherein Aβ is cross-linked via a spacer arm having a length of Above and The following crosslinking agents are used, or crosslinking agents having 1 or more and 13 or less oxyethylene and / or oxypropylene groups as spacer arms are used to form crosslinked structures.

[0029] In another aspect, the present disclosure relates to an analysis method for ASPD, comprising the following steps:

[0030] (1) a step of contacting a sample derived from a living organism that may contain ASPD with an antibody (primary antibody) that is bound to an insoluble carrier and capable of binding to ASPD, and allowing ASPD to bind to the primary antibody if the sample contains ASPD; and

[0031] (2) a step of reacting an anti-ASPD monoclonal antibody (secondary antibody) with ASPD bound to the primary antibody in step (1);

[0032] The first antibody is the humanized anti-ASPD monoclonal antibody huASD2 or the monoclonal antibody BAN50a, and the second antibody is the anti-ASPD monoclonal antibody mASD3 or a labeled antibody thereof.

[0033] Effects of the Invention

[0034] According to the present disclosure, in one embodiment, it is possible to provide a substance that can serve as a substitute for ASPD or a substance that can serve as a standard substance for ASPD in ASPD analysis.

[0035] Furthermore, according to the present disclosure, in another embodiment, a method for analyzing ASPD in a biological sample, such as blood or cerebrospinal fluid, can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is an example of the results obtained by SDSPAGE of Aβ cross-linked forms 1 to 3.

[0037] Figure 2 This is a schematic diagram illustrating one embodiment of CLEIA for ASPD [anti-ASPD polyclonal antibody / mASD3].

[0038] Figure 3 This is an example of a graph showing the results of a comparison of the reactivity of ASPD in CLEIA using ASPD and Aβ cross-linked forms 1 to 3.

[0039] Figure 4 This is an example of a graph showing the results of a comparison of the reactivity of Aβ cross-linked product 1, ASPD, and Aβ1-16 dimer to commercially available Aβ oligomer ELISA and ASPD in CLEIA.

[0040] Figure 5This is an example of the results obtained by confirming the immunogenicity of the Aβ cross-linked product 1, and shows the results of the reaction between the serum of a rabbit immunized with the Aβ cross-linked product 1 and the immobilized synthetic ASPD.

[0041] Figure 6 This is an example of a graph showing the results of plotting the measured values ​​of ASPD obtained by CLEIA in diluted samples of human CSF to which ASPD was added (dilution ratios were 1-fold, 1 / 2, 1 / 4, and 1 / 8).

[0042] Figure 7 This is an example of a graph showing the results of plotting the measured values ​​of ASPD obtained by CLEIA in diluted samples of human plasma to which ASPD was added (dilution ratios of 1-fold, 1 / 2, 1 / 4, 1 / 8, and 1 / 16).

[0043] Figure 8 This is a schematic diagram illustrating one embodiment of CLEIA for ASPD [huASD2 / mASD3].

[0044] Figure 9 This is an example of a graph showing the results of measuring four independent ASPD-containing samples using CLEIA [huASD2 / mASD3] for ASPD.

[0045] Figure 10 This is a schematic diagram illustrating one embodiment of CLEIA for ASPD [huASD2 / biotinylated mASD3].

[0046] Figure 11 This is an example of a graph showing the results of measuring ASPD-containing samples using CLEIA [huASD2 / biotinylated mASD3] for ASPD.

[0047] Figure 12 This is an example of a graph showing the results of detecting ASPD and MAP16 using the CLEIA [huASD2 / biotinylated mASD3] system and the [BAN50 / biotinylated BAN50] system of ASPD.

[0048] Figure 13 This is an example of the results obtained by comparing the results of human plasma samples (dilution ratio 1 / 4) and buffer samples using various [primary antibody / secondary antibody] combinations.

[0049] Figure 14 This is an example of the results obtained by performing CLEIA [huASD2 / biotinylated mASD3] of ASPD on 10 plasma samples from patients diagnosed with Alzheimer's disease (AD) and 10 plasma samples from healthy individuals. DETAILED DESCRIPTION

[0050] In one embodiment, the present disclosure is based on the finding that a cross-linked form of Aβ cross-linked with a specific cross-linking agent exhibits similar reactivity to an ASPD-specific antibody as that of ASPD.

[0051] In another aspect, the present disclosure is based on the finding that ASPD in a biological sample can be efficiently analyzed by using a combination of specific antibodies.

[0052] In the present disclosure, when simply referred to as ASPD, it may include natural ASPD, synthetic ASPD and cell-secreted ASPD-like structures.

[0053] As ASPD-specific antibodies, in one or more embodiments, the polyclonal anti-ASPD antibodies and monoclonal anti-ASPD antibodies disclosed in WO2006 / 016644 and WO2009 / 057664 can be mentioned. In further one or more embodiments, rabbit polyclonal anti-ASPD antibodies (rpASD1, rpASD2, and rpASD3), mouse monoclonal anti-ASPD antibodies (mASD1, mASD2, and mASD3), hamster monoclonal anti-ASPD antibodies (haASD1, haASD2, haASD3, haASD4, and haASD5), and humanized monoclonal anti-ASPD antibody (huASD2) can be mentioned.

[0054] [Amyloid beta]

[0055] Aβ is a protein composed of a sequence corresponding to a portion of the amyloid precursor protein (APP), and is a protein having a peptide sequence that is cleaved from APP by β-selectase and γ-selectase in vivo.

[0056] In the present disclosure, when simply referred to as Aβ, it may refer to Aβ38 (Aβ1-38), Aβ39 (Aβ1-39), Aβ40 (Aβ1-40), Aβ41 (Aβ1-41), Aβ42 (Aβ1-42), Aβ43 (Aβ1-43), or all or part of them.

[0057] In the present disclosure, when simply referred to as Aβ, human Aβ and non-human mammalian Aβ may be included.

[0058] In one or more embodiments, human APP includes splice mutants such as hAPP770, hAPP751, and hAPP695. hAPP770 (NCBI Accession No. NP_000475 (VERSION NP#000475.1GI:4502167) is not limited thereto.

[0059] The amino acid at the N-terminus of human Aβ (the first amino acid of Aβ) corresponds to the 672nd amino acid D (aspartic acid) of hAPP770.

[0060] In the present disclosure, when simply referred to as Aβ, both wild-type and mutant Aβ may be included.

[0061] As mutant Aβ, in one or more embodiments, a mutant Aβ having a sequence of a peptide cut out from mutant APP can be mentioned. As mutant APP, mutations linked to familial Alzheimer's disease can be mentioned, for example, Swedish mutation, Leuven mutation, Icelandic mutation, London mutation, Iranian mutation, Austrian mutation, German mutation, French mutation, Florida mutation, Iberian mutation, Australian mutation, Belgian mutation, Flemish mutation, Icelandic mutation, British mutation, Tottori mutation, Italian mutation, Arctic mutation, Osaka mutation, Iowa mutation, Dutch mutation, etc. As other mutations, mutations to modified amino acids, non-natural amino acids, D-amino acids, etc. can be included. As modified amino acids, in one or more embodiments, amino modification, carboxyl modification, thiol modification, hydroxyl modification, glycosylation modification, PEGylation modification, etc. can be mentioned.

[0062] [Aβ cross-linked body]

[0063] In one embodiment, the present disclosure relates to a cross-linked Aβ formed by cross-linking Aβ via a specific cross-linker.

[0064] In one or more embodiments, the crosslinker used in the Aβ crosslinker of the present disclosure is a crosslinker having a spacer arm length of The following and The following crosslinking agents. The spacer arm length refers to the molecular span of the crosslinking agent (the distance between the bonded molecules). The spacer arm length of the crosslinked product is described in the catalog or product manual of the commercially available crosslinked product and can be appropriately selected and obtained by those skilled in the art.

[0065] In one or more embodiments, the length of the spacer arm is from the viewpoint of making the reactivity with the ASPD-specific antibody close to that of ASPD. Above, preferably Above, preferably More preferably More preferably More preferably Above, more preferably Above, more preferably Above, more preferably Above, more preferably Above, more preferably Above, more preferably above.

[0066] As the length of the spacer arm, in one or more embodiments, from the same viewpoint, it is The following are preferably The following are more preferably The following is more preferably The following is more preferably The following is more preferably The following is more preferably The following is more preferably the following.

[0067] In one or more embodiments, the crosslinker used in the Aβ crosslinked product of the present disclosure comprises a crosslinker having one or more and no more than 13 oxyethylene (-CH2CH2O-, EO) and / or oxypropylene (-CH2CH2CH2O-, PO) groups as a spacer arm. A spacer arm refers to a backbone or group between the reactive ends of the crosslinker.

[0068] In one or more embodiments, the total number of EO and PO groups in the spacer arm is 1 or more, preferably 2 or more, more preferably 3 or more, and even more preferably 4 or more, from the viewpoint of making the reactivity with the ASPD-specific antibody close to that of ASPD.

[0069] From the same viewpoint, the total number of EO groups and PO groups in the spacer arm is, in one or more embodiments, 13 or less, preferably 10 or less, more preferably 9 or less, further preferably 8 or less, further preferably 7 or less, and further preferably 6 or less.

[0070] In one or more embodiments, the crosslinking target of the crosslinking agent used in the Aβ crosslinked product of the present disclosure may include an amino group, a thiol group, a carboxyl group, or a non-selective group.

[0071] In one or more embodiments, the reactive end of the cross-linking agent used in the Aβ cross-linked product of the present disclosure is preferably a reactive end that cross-links between amino groups from the viewpoint of making the reactivity with the ASPD-specific antibody close to that of ASPD, and examples thereof include NHS esters and imide esters.

[0072] The crosslinking agent used in the crosslinked Aβ product disclosed herein may or may not have cleavage properties. From the viewpoint of stability, it is preferably not cleavage properties.

[0073] In one or more embodiments, the crosslinking agent used in the Aβ crosslinked product of the present disclosure is preferably ethylene glycol bis(succinimidyl succinate) (EGS, spacer arm length 10000). ), sulfo EGS (spacer arm length is ), bis(succinimidyl)penta(ethylene glycol) (BS(PEG)5, spacer arm length is ), bis(succinimidyl)nona(ethylene glycol) (BS(PEG)9, spacer arm length is ), more preferably BS(PEG)5 and BS(PEG)9, further preferably BS(PEG)5.

[0074] From the viewpoint of making the reactivity with ASPD-specific antibodies close to that of ASPD, Aβ used in the cross-linked Aβ of the present disclosure may be Aβ38, Aβ39, Aβ40, Aβ41, or Aβ42. Aβ40 or Aβ42 is preferred, and Aβ42 is more preferred.

[0075] In one or more embodiments, the molecular weight of the Aβ cross-linked body disclosed herein is greater than 100 kDa or greater than 114 kDa, as measured by 10-20% SDS PAGE analysis.

[0076] In one or more embodiments, the Aβ cross-linked products disclosed herein may have cytotoxicity equivalent to that of ASPD, i.e., selectively inducing cell death in functionally mature neurons, or may be weaker than ASPD, or may not be cytotoxic, or may be more cytotoxic than ASPD. In one or more embodiments, the Aβ cross-linked products disclosed herein may be weaker than ASPD, or may not be cytotoxic.

[0077] In one or more embodiments, the Aβ cross-linked product disclosed herein has a reactivity with ASPD-specific antibodies that is closer to that of ASPD, and more preferably has a reactivity equivalent to that of ASPD, compared to the reactivity with Aβ aggregates other than ASPD.

[0078] In one or more embodiments, the Aβ cross-linked product disclosed herein is a substance that competes with ASPD-specific antibodies and ASPD.

[0079] In one or more embodiments, the Aβ cross-linked product disclosed herein can serve as a substitute for ASPD from the viewpoint of reactivity with ASPD-specific antibodies.

[0080] In one or more embodiments, the Aβ cross-linked product disclosed herein can be used as a standard substance for ASPD analysis or a kit thereof.

[0081] In the present disclosure, analysis of ASPD, in one or more embodiments, may include detection, determination, quantification, and screening utilizing ASPD.

[0082] As an ASPD analysis using the Aβ cross-linked product disclosed herein as a standard substance, in one or more embodiments, examples include an analysis utilizing an immunoassay using an ASPD-specific antibody, more specifically, an enzyme immunoassay using an ASPD-specific antibody, a chemiluminescent enzyme immunoassay, and more specifically, an enzyme-linked immunosorbent assay (ELISA) and a chemiluminescent enzyme immunoassay (CLEIA) using an ASPD-specific antibody.

[0083] In one or more embodiments, the Aβ cross-linked bodies disclosed herein can be used to prepare a standard curve in an ASPD assay. In other one or more embodiments, the Aβ cross-linked bodies disclosed herein can be used as a positive control in an ASPD assay.

[0084] In one or more embodiments, the Aβ cross-linked product disclosed herein can be used as a standard substance for ASPD analysis in the diagnosis of diseases accompanied by ASPD accumulation. In one or more embodiments, diseases accompanied by ASPD accumulation include Alzheimer's disease and Lewy body dementia.

[0085] In one or more embodiments, the Aβ crosslinked product disclosed herein can be used as a standard substance for an ASPD assay or a kit thereof in a companion diagnostic for a therapeutic drug targeting ASPD. In one or more embodiments, the Aβ crosslinked product disclosed herein can be used as a component of a companion diagnostic for a therapeutic drug targeting ASPD.

[0086] Therefore, in another embodiment, the present disclosure relates to an ASPD analysis kit containing the Aβ cross-linked product disclosed herein as a standard substance. In one or more embodiments, the ASPD analysis kit of this embodiment includes reagents required for an immunoassay using an ASPD-specific antibody. In one or more embodiments, such reagents include an ASPD-specific antibody.

[0087] In one or more embodiments, the ASPD analysis kit of this embodiment is an ASPD analysis kit for diagnosing a disease accompanied by accumulation of ASPD, an ASPD analysis kit for companion diagnosis of a therapeutic drug targeting ASPD, or a companion diagnostic drug for a therapeutic drug targeting ASPD.

[0088] In one or more embodiments, therapeutic drugs targeting ASPD include pharmaceutical compositions for preventing, ameliorating, and / or treating diseases associated with the accumulation of ASPD.

[0089] In one or more embodiments, the Aβ cross-linked product disclosed herein is immunogenic and can induce the production of antibodies against the Aβ cross-linked product disclosed herein.

[0090] In addition, in one or more embodiments, the Aβ cross-linked product disclosed herein is immunogenic and can induce the production of antibodies against ASPD.

[0091] Therefore, in one or more embodiments, the Aβ cross-linked product disclosed herein can serve as a substitute for ASPD from the viewpoint of being able to induce antibodies against ASPD.

[0092] The use of ASPD as an active vaccine against diseases associated with the accumulation of ASPD has been disclosed (e.g. WO 2017 / 179646).

[0093] The Aβ cross-linked product disclosed herein can be a substitute for ASPD, and thus the Aβ cross-linked product disclosed herein can also be used as an active vaccine and can be used in the manufacture of vaccines.

[0094] Therefore, in one embodiment, the present disclosure relates to a pharmaceutical composition comprising the Aβ cross-linked form of the present disclosure as an active ingredient. One or more embodiments of the pharmaceutical composition of the present disclosure include vaccines containing the Aβ cross-linked form of the present disclosure. The pharmaceutical composition and vaccine of the present disclosure may contain a pharmaceutically acceptable excipient and / or adjuvant.

[0095] In one or more embodiments, the pharmaceutical compositions and vaccines disclosed herein can be used to prevent, improve, and / or treat diseases associated with the accumulation of ASPD. In one or more embodiments, the pharmaceutical compositions and vaccines disclosed herein can be used to prevent, improve, and / or treat Alzheimer's disease and / or Lewy body dementia.

[0096] In one or more embodiments, the pharmaceutical compositions and vaccines of the present disclosure can be administered to a subject who may be suffering from a disease associated with the accumulation of ASPD, a subject who may already be suffering from the disease, or a subject who is suffering from the disease. In one or more embodiments, the pharmaceutical compositions of the present disclosure can be administered to a subject who may be suffering from Alzheimer's disease and / or dementia with Lewy bodies, a subject who may already be suffering from the disease, or a subject who is suffering from the disease. Examples of such subjects include mammals, humans, and mammals other than humans.

[0097] One or more embodiments of the pharmaceutical compositions and vaccines disclosed herein for immunizing a subject against ASPD, a disease associated with the accumulation of ASPD, or Alzheimer's disease and / or Lewy body dementia are described. In one or more embodiments, the methods of administering the pharmaceutical compositions and vaccines disclosed herein may include intramuscular, intraperitoneal, intradermal, or subcutaneous injections, or transmucosal administration to the oral cavity, digestive tract, respiratory tract, or urogenital tract. The dosage of the Aβ crosslinked body in the pharmaceutical compositions and vaccines disclosed herein may be an amount that induces an immune defense response without causing significant side effects to the subject. In one or more embodiments, the dosage of the pharmaceutical compositions and vaccines disclosed herein may be an amount containing cell-secreted ASPD-like structures in the range of 0.01 μg to 10 mg, 0.1 μg to 1000 μg, 1 μg to 100 μg, 5 μg to 50 μg, or 5 μg to 25 μg. One or more booster doses may also be administered after the initial administration at sufficient intervals.

[0098] Therefore, in one embodiment, the present disclosure relates to a method for preventing, ameliorating, and / or treating diseases accompanied by accumulation of ASPD (e.g., Alzheimer's disease and / or Lewy body dementia), comprising administering the Aβ cross-linked product, pharmaceutical composition, or vaccine of the present disclosure to a subject.

[0099] In another embodiment, the present disclosure relates to a method for immunizing a subject against ASPD itself, comprising administering the Aβ cross-linked product, or the pharmaceutical composition or vaccine of the present disclosure to the subject.

[0100] In another embodiment, the present disclosure relates to a method for immunizing a subject against a disease associated with accumulation of ASPD, comprising administering the Aβ cross-linked product of the present disclosure, or the pharmaceutical composition or vaccine of the present disclosure to the subject.

[0101] In one embodiment, the present disclosure relates to a method for producing a pharmaceutical composition or vaccine, comprising combining the cross-linked Aβ of the present disclosure with an excipient and / or an adjuvant.

[0102] In another embodiment, the present disclosure relates to a method for producing a pharmaceutical composition or a vaccine, wherein the pharmaceutical composition comprises the cross-linked Aβ of the present disclosure as an active ingredient, or the vaccine comprises the cross-linked Aβ of the present disclosure, the method comprising the step of producing the cross-linked Aβ of the present disclosure by the production method described below.

[0103] [Method for producing cross-linked Aβ]

[0104] In one or more embodiments, the cross-linked Aβ of the present disclosure can be obtained by adding a cross-linking agent to an Aβ aqueous solution prepared by dissolving Aβ in an aqueous solvent, and performing a cross-linking reaction.

[0105] Aβ can be prepared by peptide synthesis or produced by recombinant technology.

[0106] In one or more embodiments, the amount of the cross-linking agent added is 10 times or more, 20 times or more, or 30 times or more relative to the amount of Aβ on a molar basis. In one or more embodiments, the amount of the cross-linking agent added is 200 times or less, 100 times or less, or 75 times or less on a molar basis.

[0107] The cross-linked product after the cross-linking reaction can also be subjected to ultrafiltration. The Aβ cross-linked product of the present disclosure can be obtained in the retained solution or the recovered solution of the filtration residue.

[0108] In one or more embodiments, the molecular weight cutoff (MWCO) of the ultrafiltration filter may be 30 kDa, 50 kDa, or 100 kDa.

[0109] In one or more embodiments, the Aβ cross-linked product disclosed herein can be produced by the methods described in the Examples.

[0110] Therefore, the present disclosure may relate to the following embodiments;

[0111] [A1] An Aβ cross-linked product, wherein Aβ is connected to the cross-linked product by a spacer arm having a length of Above and The following cross-linking agents are used for cross-linking.

[0112] [A2] A cross-linked Aβ, wherein Aβ is cross-linked by a cross-linking agent having 1 or more and 13 or less EO groups and / or PO groups as a spacer.

[0113] [A3] The Aβ cross-linked product according to [A1] or [A2], wherein the Aβ is Aβ38, Aβ39, Aβ40, Aβ41, Aβ42, or Aβ43.

[0114] [A4] The cross-linked Aβ according to any one of [A1] to [A3], wherein the cross-linking agent has a reactive terminal that cross-links amino groups.

[0115] [A5] The cross-linked Aβ according to any one of [A1] to [A4], wherein the molecular weight as analyzed by 10-20% SDS PAGE exceeds 100 kDa.

[0116] [A6] The cross-linked Aβ according to any one of [A1] to [A5], which is a substance that competes with ASPD for an ASPD-specific antibody.

[0117] [A7] The Aβ cross-linked product according to any one of [A1] to [A6], which is a standard substance for ASPD analysis.

[0118] [A8] The Aβ cross-linked product according to any one of [A1] to [A7], which is a standard substance for ASPD analysis in the diagnosis of a disease accompanied by ASPD accumulation.

[0119] [A9] The Aβ cross-linked product according to any one of [A1] to [A8], which is a standard substance for ASPD analysis in companion diagnostics for therapeutic drugs targeting ASPD.

[0120] [A10] The cross-linked Aβ according to any one of [A1] to [A9], which is a component of a companion diagnostic drug for a therapeutic drug targeting ASPD.

[0121] [B1] Use of the cross-linked Aβ according to any one of [A1] to [A10] as a substance that competes with ASPD against an ASPD-specific antibody.

[0122] [B2] Use of the cross-linked Aβ according to any one of [A1] to [A10] as a substitute for ASPD for an ASPD-specific antibody.

[0123] [B3] Use of the cross-linked Aβ according to any one of [A1] to [A10] as a standard substance for ASPD analysis.

[0124] [B4] Use of the cross-linked Aβ according to any one of [A1] to [A10] for diagnosis of a disease accompanied by accumulation of ASPD.

[0125] [B5] The use according to [B4], wherein the diagnosis is a companion diagnosis for a therapeutic drug targeting ASPD.

[0126] [B6] Use of the cross-linked Aβ according to any one of [A1] to [A10] as a standard substance for ASPD analysis in the diagnosis of a disease accompanied by accumulation of ASPD.

[0127] [B7] The use of the Aβ cross-linked product according to [B6], wherein the disease accompanied by accumulation of ASPD is Alzheimer's disease and / or dementia with Lewy bodies.

[0128] [B8] Use of the cross-linked Aβ according to any one of [A1] to [A10] as a standard substance for ASPD analysis in companion diagnostics for therapeutic drugs targeting ASPD.

[0129] [B9] Use of the cross-linked Aβ according to any one of [A1] to [A10] as a component of a companion diagnostic drug for a therapeutic drug targeting ASPD.

[0130] [C1] An ASPD analysis kit comprising the cross-linked Aβ according to any one of [A1] to [A10] as a standard substance.

[0131] [C2] The ASPD analysis kit according to [C1], further comprising an ASPD-specific antibody.

[0132] [C3] The ASPD analysis kit according to [C1] or [C2], which is used for diagnosing a disease accompanied by accumulation of ASPD.

[0133] [C4] The ASPD analysis kit according to [C3], wherein the disease accompanied by accumulation of ASPD is Alzheimer's disease and / or Lewy body dementia.

[0134] [C5] The ASPD analysis kit according to any one of [C1] to [C4], which is used for companion diagnosis of a therapeutic drug targeting ASPD.

[0135] [C6] A companion diagnostic drug for a therapeutic drug targeting ASPD, comprising the cross-linked Aβ according to any one of [A1] to [A10] as a standard substance.

[0136] [C7] An ASPD analysis method comprising the step of using the cross-linked Aβ according to any one of [A1] to [A10] as a standard substance.

[0137] [C8] The ASPD analysis method according to [C7], comprising performing immunoassay using an ASPD-specific antibody.

[0138] [C9] The ASPD analysis method according to [C7] or [C8], which is used for diagnosing a disease accompanied by accumulation of ASPD.

[0139] [C10] The ASPD analysis method according to [C9], wherein the disease accompanied by accumulation of ASPD is Alzheimer's disease and / or Lewy body dementia.

[0140] [C11] The ASPD analysis method according to any one of [C7] to [C10], which is used for companion diagnosis of a therapeutic drug targeting ASPD.

[0141] [C12] A companion diagnostic method for a therapeutic drug targeting ASPD, comprising the step of performing the ASPD analysis method according to any one of [C7] to [C11].

[0142] [C13] The diagnostic method according to [C12], wherein the therapeutic drug targeting ASPD is a pharmaceutical composition for preventing, ameliorating and / or treating a disease accompanied by accumulation of ASPD.

[0143] [C14] The diagnostic method according to [C13], wherein the disease accompanied by accumulation of ASPD is Alzheimer's disease and / or dementia with Lewy bodies.

[0144] [D1] A pharmaceutical composition comprising the cross-linked Aβ according to any one of [A1] to [A10] as an active ingredient.

[0145] [D2] A vaccine comprising the cross-linked Aβ according to any one of [A1] to [A10].

[0146] [D3] Use of the cross-linked Aβ according to any one of [A1] to [A10] as an active vaccine.

[0147] [D4] Use of the cross-linked Aβ according to any one of [A1] to [A10] in vaccine production.

[0148] [D5] The vaccine according to [D2] or the use according to [D3] or [D4], wherein the vaccine is a vaccine for a disease accompanied by accumulation of ASPD.

[0149] [D6] A method for preventing, ameliorating, and / or treating a disease accompanied by accumulation of ASPD, comprising administering the Aβ cross-linked product according to any one of [A1] to [A10], the pharmaceutical composition according to [D1], or the vaccine according to [D2] to a subject.

[0150] [D7] The method according to [D6], wherein the disease accompanied by accumulation of ASPD is Alzheimer's disease and / or Lewy body dementia.

[0151] [D8] A method for immunizing a subject against ASPD, comprising administering the cross-linked Aβ according to any one of [A1] to [A10], the pharmaceutical composition according to [D1], or the vaccine according to [D2] to the subject.

[0152] [D9] A method for immunizing a subject against a disease associated with accumulation of ASPD, comprising administering the cross-linked Aβ according to any one of [A1] to [A10], the pharmaceutical composition according to [D1], or the vaccine according to [D2] to the subject.

[0153] [D10] The method according to [D9], wherein the disease accompanied by accumulation of ASPD is Alzheimer's disease and / or Lewy body dementia.

[0154] [D11] A method for producing a pharmaceutical composition or vaccine, comprising the step of combining the cross-linked Aβ described in any one of [A1] to [A10] with a pharmaceutically acceptable excipient.

[0155] [ASPD analysis method]

[0156] In one embodiment, the present disclosure relates to an analysis method for ASPD (hereinafter also referred to as “the analysis method for ASPD of the present disclosure”) comprising the following steps (1) and (2).

[0157] (1) A step of contacting a sample derived from a living organism that may contain ASPD with an antibody (primary antibody) that is bound to an insoluble carrier and capable of binding to ASPD, and allowing ASPD to bind to the primary antibody if the sample contains ASPD.

[0158] (2) A step of reacting an anti-ASPD monoclonal antibody (secondary antibody) with ASPD bound to the primary antibody in step (1).

[0159] Here, the first antibody is the humanized anti-ASPD monoclonal antibody huASD2 or the monoclonal antibody BAN50a.

[0160] The second antibody is the mouse monoclonal anti-ASPD antibody mASD3 or its labeled antibody.

[0161] The ASPD analysis method disclosed herein is a method utilizing the principle of immunoassay. In one or more embodiments, the method is an enzyme immunoassay such as ELISA or a chemiluminescent enzyme immunoassay such as CLEIA.

[0162] In one or more embodiments, the first antibody bound to the insoluble carrier is preferably huASD2 or BAN50a from the viewpoint of improving detection sensitivity when blood, plasma components, cerebrospinal fluid, or dilutions thereof are used as samples.

[0163] huASD2 is described in WO2009 / 057664 and is a humanized monoclonal anti-ASPD antibody having a heavy chain variable region with the amino acid sequence described in SEQ ID NO: 1 and a light chain variable region with the amino acid sequence described in SEQ ID NO: 2.

[0164] Serial number 1: EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWVRQAPGKGLEWVGGIEIKSYFYATYYFGSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTNREVGGLDNWGQGTLVTVSS

[0165] Serial number 2: QSVLTQPSSLSASPGASASLTCTLRSGISVGGKNIYWYQQKPGSPPQYLLKYSSYSNKQLGPGVPSRFSGSKDASANAGILLISGLQSEDEADYYCSIHESNAYVFGGGTKLTVLGR

[0166] huASD2 can be obtained, for example, by co-transfecting ASD2RHApG1D200 (Japanese National Biotechnology Depository No.: FERM BP-11040) and ASD2RLApLN100 (Japanese National Biotechnology Depository No.: FERM BP-11041) into known animal cells and expressing them.

[0167] In addition, in one or more embodiments, huASD2 in the present disclosure can be replaced with an anti-ASPD antibody having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 4.

[0168] Serial number 3: EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWVRQAPGKGLEWVXGIEIKSYFYATYYFGSVKGRFTISRDDSKNTXYLQMNSLKTEDTAVYYCTXNREVGGLDNWGQGTLVTVSS

[0169] Serial number 4: QXVLTQPXSLSASPGASASLTCTLRSGISVGGKNIYWYQQKPGSPPQXXLXYSSYSNKQLGPGVPSRFSGSKDXSANAXILLISGLQSEDEADYYCSIHESNAYVFGGGTKLTVLG

[0170] (In sequence number 3, X at position 49 is G or A, X at position 81 is L or V, and X at position 100 is T or R. In sequence number 4, X at position 2 is S or A, X at position 8 is S or A, X at position 48 is Y or F, X at position 49 is L or F, X at position 51 is K, F, or R, X at position 74 is A or T, and X at position 79 is G or A.)

[0171] In the present disclosure, BAN50a is a known monoclonal antibody described in International Publication No. WO94-17197 that specifically recognizes the N-terminal region of Aβ. BAN50a can be commercially available or produced by known methods using hybridoma BAN50 (Deposit No. FERM BP4163) from the Japanese National Biotechnology Collection.

[0172] In one or more embodiments, the second antibody may be a labeled mASD3 antibody from the viewpoint of improving detection sensitivity. In one or more embodiments, the labeling may be biotinylation.

[0173] In one or more embodiments, the insoluble carrier in the ASPD analysis method of the present disclosure may be a solid phase such as beads or a multi-well plate.

[0174] Examples of biologically derived samples that may contain ASPD include blood, plasma, cerebrospinal fluid, and dilutions thereof in one or more embodiments.

[0175] In one or more embodiments, the ASPD analysis method of the present disclosure may include a step (0) of preparing a sample derived from a living organism prior to step (1). In one or more embodiments, the sample preparation step (0) includes diluting a biological sample such as blood, plasma, or cerebrospinal fluid into an appropriate solvent or buffer. In one or more embodiments, the solvent or buffer in the sample preparation step (0) may contain a surfactant or other agent to the extent that it does not interfere with the analysis.

[0176] In one or more embodiments, the ASPD analysis method of the present disclosure may further include the following step (3).

[0177] (3) A step of reacting the anti-mouse antibody linked to the reporter gene with the second antibody bound to ASPD in step (2), or a step of reacting the labeled binding partner linked to the reporter gene with the label of the second antibody bound to ASPD in step (2).

[0178] In one or more embodiments, examples of reporter genes include substances utilizing chemiluminescence or bioluminescence, fluorescent proteins, and the like. From the viewpoint of improving detection sensitivity, substances utilizing chemiluminescence or bioluminescence are preferred.

[0179] In one or more embodiments, examples of substances utilizing chemiluminescence or bioluminescence include enzymes such as bioluminescent enzymes or mutants thereof.

[0180] In one or more embodiments, examples of the enzyme include HRP (horseradish peroxidase) and alkaline phosphatase.

[0181] In one or more embodiments, when the label is biotin, examples of the binding partner include streptavidin and the like.

[0182] In one or more embodiments, luminescence measurement can be performed using a luminometer or a multifunctional microplate reader.

[0183] In one or more embodiments of the ASPD analysis method disclosed herein, the Aβ cross-linked product disclosed herein can be used as a standard substance.

[0184] Therefore, the present disclosure may relate to the following embodiments;

[0185] [E1] An analysis method for ASPD, comprising the following steps (1) and (2).

[0186] Step (1): A step of contacting a sample derived from a living organism that may contain ASPD with an antibody (primary antibody) that is bound to an insoluble carrier and capable of binding to ASPD, and allowing ASPD to bind to the primary antibody if ASPD is contained.

[0187] Step (2): a step of reacting an anti-ASPD monoclonal antibody (secondary antibody) with ASPD bound to the primary antibody in step (1).

[0188] Here, the first antibody is the humanized anti-ASPD monoclonal antibody huASD2 or the monoclonal antibody BAN50a.

[0189] The second antibody is the mouse monoclonal anti-ASPD antibody mASD3 or its labeled antibody.

[0190] [E2] The analysis method according to [E1], which utilizes the principle of immunoassay.

[0191] [E3] The analysis method according to [E2], wherein the method utilizing the principle of immunoassay is ELISA or CLEIA.

[0192] [E4] The analysis method according to any one of [E1] to [E3], wherein the sample derived from a living organism is blood, a plasma component, cerebrospinal fluid, or a dilution thereof.

[0193] [E5] The analysis method according to any one of [E1] to [E4], comprising the step (0) of preparing a sample derived from a living organism before the step (1).

[0194] [E6] The analysis method according to [E5], wherein the sample preparation step (0) includes a step of diluting a biological sample selected from blood, plasma, and cerebrospinal fluid in a solvent or a buffer.

[0195] [E7] The analysis method according to any one of [E1] to [E6], further comprising the following step (3).

[0196] Step (3): a step of reacting the anti-mouse antibody linked to the reporter gene with the second antibody bound to ASPD in step (2), or a step of reacting the labeled binding partner linked to the reporter gene with the label of the second antibody bound to ASPD in step (2).

[0197] [E8] The analysis method according to any one of [E1] to [E7], wherein the substance described in [1] below is used as the ASPD standard substance.

[0198] [E9] The analysis method according to any one of [E1] to [E7], comprising the step of using the cross-linked Aβ according to any one of [A1] to [A10] as a standard substance.

[0199] [E10] The analysis method according to any one of [E1] to [E9], which is used for diagnosis of a disease accompanied by accumulation of ASPD.

[0200] [E11] The analysis method according to [E10], wherein the disease accompanied by accumulation of ASPD is Alzheimer's disease and / or dementia with Lewy bodies.

[0201] [E12] The analysis method according to any one of [E1] to [E11], which is used for companion diagnosis of a therapeutic drug targeting ASPD.

[0202] [E13] A method for diagnosing a disease accompanied by accumulation of ASPD, comprising the step of performing the analysis method described in any one of [E1] to [E12].

[0203] [E14] The diagnostic method according to [E13], wherein the disease accompanied by accumulation of ASPD is Alzheimer's disease and / or dementia with Lewy bodies.

[0204] [E15] A companion diagnostic method for a therapeutic drug targeting ASPD, comprising the step of performing the analytical method according to any one of [E1] to [E12].

[0205] [E16] The diagnostic method according to [E15], wherein the therapeutic drug targeting ASPD is a pharmaceutical composition for preventing, ameliorating and / or treating a disease accompanied by accumulation of ASPD.

[0206] [E17] The diagnostic method according to [E16], wherein the disease accompanied by accumulation of ASPD is Alzheimer's disease and / or dementia with Lewy bodies.

[0207] [F1] An ASPD analysis kit comprising humanized anti-ASPD monoclonal antibody huASD2 or monoclonal antibody BAN50a (first antibody), and mouse monoclonal anti-ASPD antibody mASD3 or a labeled antibody thereof (secondary antibody).

[0208] [F2] The ASPD analysis kit according to [F1], further comprising the cross-linked Aβ according to any one of [A1] to [A10].

[0209] [F3] The ASPD analysis kit according to [F1] or [F2], which is used for the analysis method according to any one of [E1] to [E12] or the diagnosis method according to any one of [E13] to [E17].

[0210] The present disclosure may relate to the following embodiments;

[0211] [1] A substance that competes with ASPD-specific antibodies and ASPD, wherein Aβ is cross-linked by a cross-linking agent, wherein the cross-linking agent has a spacer arm length of Above and The following crosslinking agents, or a crosslinking agent having 1 or more and 13 or less EO groups and / or PO groups as a spacer arm.

[0212] [2] A cross-linked Aβ, wherein Aβ is cross-linked with a spacer arm having a length of Above and The following crosslinking agent or a crosslinking agent having 1 or more and 13 or less EO groups and / or PO groups as a spacer arm is crosslinked.

[0213] [3] A kit for analyzing ASPD, comprising the substance described in [1] or the cross-linked product described in [2], and an anti-ASPD antibody.

[0214] [4] Use of the substance described in [1] or the cross-linked product described in [2] as a standard substance in ASPD analysis.

[0215] [5] Use of the substance described in [1] or the cross-linked product described in [2] as a standard substance in the diagnosis of diseases accompanied by accumulation of ASPD.

[0216] [6] The use according to [5], wherein the diagnosis is a companion diagnosis for a therapeutic drug targeting ASPD.

[0217] [7] The use according to [5] or [6], wherein the disease accompanied by accumulation of ASPD is Alzheimer's disease and / or Lewy body dementia.

[0218] [8] An ASPD analysis method comprising the following steps:

[0219] (1) a step of contacting a sample derived from an organism that may contain ASPD with an antibody (primary antibody) that is bound to an insoluble carrier and capable of binding to ASPD, and allowing ASPD to bind to the primary antibody if ASPD is contained, and

[0220] (2) a step of reacting an anti-ASPD monoclonal antibody (second antibody) with ASPD bound to the first antibody in step (1),

[0221] Among them, the first antibody is the humanized anti-ASPD monoclonal antibody huASD2 or the monoclonal antibody BAN50a,

[0222] The second antibody is the mouse monoclonal anti-ASPD antibody mASD3 or its labeled antibody.

[0223] [9] The analysis method according to [8] further comprising the following steps:

[0224] (3) A step of reacting the anti-mouse antibody linked to the reporter gene with the second antibody bound to ASPD in step (2), or a step of reacting the labeled binding partner linked to the reporter gene with the label of the second antibody bound to ASPD in step (2).

[0225]

[10] The analysis method according to [8] or [9], wherein the substance according to [1] or the cross-linked product according to [2] is used as an ASPD standard substance.

[0226] Hereinafter, one or more embodiments of the present invention will be further described using examples.

[0227] Example

[0228] In the following examples, unless otherwise specified, the concentrations of Aβ cross-linked products and ASPD are based on Aβ monomers.

[0229] 1. Preparation of Aβ cross-linked

[0230] The following Aβ cross-linked products 1-3 were prepared.

[0231] Aβ crosslinker 1: Aβ42, crosslinker is BS (PEG) 5

[0232] Aβ crosslinker 2: Aβ42, crosslinker is BS(PEG)9

[0233] Aβ crosslinker 3: Aβ42, crosslinked by DFDNB

[0234] The cross-linking agents used were as follows.

[0235] BS(PEG)5: Bis(succinimidyl)penta(ethylene glycol)

[0236] (Spacer arm length: )

[0237] BS(PEG)9: Bis(succinimidyl)nona(ethylene glycol)

[0238] (Spacer arm length: )

[0239] DFDNB: 1,5-difluoro-2,4-dinitrobenzene

[0240] (Spacer arm length: )

[0241] [Chemical Formula 1]

[0242]

[0243] Aβ cross-linking protocol

[0244] Dissolve 0.56 mg of Aβ42 in 120 μL of DMSO (1 mM)

[0245] ↓

[0246] 10 μL was added to 90 μL PBS(-) and allowed to stand at 25°C for 15 minutes (100 μM Aβ, equivalent to 0.046667 mg)

[0247] ↓

[0248] After adding 50 times the molar amount of cross-linking reagent BS(PEG)5, BS(PEG)9, or DFDNB, the mixture was allowed to stand at 25°C for 30 minutes.

[0249] ↓

[0250] Add Tris-HCl pH 8.0 (final concentration 50 mM) and let stand at 25°C for 30 minutes

[0251] ↓

[0252] The solution was filtered through an ultrafiltration filter with MWCO 50K to obtain a high molecular weight fraction.

[0253] PBS(-)300μL wash × 3

[0254] Recover membrane residues with 50 μL of PBS (-)

[0255] ↓

[0256] Concentration determination by BCA method

[0257] 2. SDS-PAGE of Aβ Cross-linked

[0258] The obtained Aβ cross-linked products 1 to 3 were subjected to SDSPAGE (10-20% gel), and cross-linking was confirmed. An example of the results is shown in Figure 1 .

[0259] like Figure 1 As shown, it can be seen that Aβ cross-linked bodies 1 to 3 are each highly molecularized, with a molecular weight of 100 kDa or more.

[0260] 3. ASPD CLEIA Reactivity of Aβ Cross-linked Forms

[0261] The obtained Aβ cross-linked products 1 to 3 were confirmed to have ASPD CLEIA reactivity.

[0262] The ASPD CLEIA used is Figure 2 The system shown uses an anti-ASPD polyclonal antibody (primary antibody) immobilized on a solid phase, a mouse anti-ASPD monoclonal antibody mASD3 (secondary antibody) on the detection side, and anti-mouse IgG-HRP. mASD3 is a monoclonal antibody described in WO2006 / 016644 and WO2009 / 057664, and the hybridoma has been deposited with the National Center for Biotechnology Information (NDC) under the deposit number FERM BP-10394. HRP stands for horseradish peroxidase.

[0263] An example of the results of comparing the reactivity of ASPD CLEIA [anti-ASPD polyclonal antibody / mASD3] using Aβ cross-linked forms 1 to 3 as test objects with that of ASPD as the test object is shown in FIG. Figure 3 .

[0264] like Figure 3 As shown, Aβ cross-linked products 1 and 2 have reactivity with ASPD closer to that of Aβ cross-linked product 3. Among them, Aβ cross-linked product 1 exhibits reactivity equivalent to that of ASPD.

[0265] Comparison of the reactivity of Aβ cross-linkers (continued)

[0266] Next, the reactivity of the Aβ cross-linked bodies was compared using a commercially available Aβ oligomer assay kit using the monoclonal antibody 82E1 that specifically recognizes the N-terminus of Aβ.

[0267] As test substances, Aβ cross-linked substance 1, ASPD, and Aβ1-16 dimer were used.

[0268] Furthermore, the reactivity of Aβ cross-linked body 1, ASPD, and Aβ1-16 dimer in ASPD CLEIA [anti-ASPD polyclonal antibody / mASD3] was compared.

[0269] An example of the results is shown in Figure 4 .

[0270] like Figure 4 As shown, similarly to ASPD CLEIA, the Aβ cross-linked body 1 also exhibited reactivity comparable to that of ASPD in the commercially available Aβ oligomer measurement kit.

[0271] When the commercially available Aβ oligomer assay kit 82E1 was used, the reactivity of Aβ cross-linked body 1 and ASPD was significantly weaker than that of Aβ1-16 dimer.

[0272] In contrast, in ASPD CLEIA, the reactivity of Aβ1-16 dimers was significantly weaker than that of Aβ cross-linked form 1 and ASPD.

[0273] Furthermore, it was found that ASPD CLEIA has a characteristically high detection sensitivity for Aβ cross-linked form 1 and ASPD.

[0274] 4. Immunogenicity of Aβ cross-links

[0275] Rabbits (New Zealand white) were immunized six times with Aβ cross-linked product 1 (100 μg / time) in combination with Freund's adjuvant (FCA, primary) and incomplete Freund's adjuvant (FIA, 5 times). After that, all blood was collected and the reactivity of the antibodies in the serum with Aβ cross-linked product 1 and synthetic ASPD was evaluated. An example of the results is shown in Figure 5 .

[0276] exist Figure 5 In the table, “pre-serum” indicates the reactivity of serum before immunization with the Aβ cross-linker 1 immobilized plate, “immobilized Aβ cross-linker” indicates the reactivity of the obtained serum with the Aβ cross-linker 1 immobilized plate, “immobilized synthetic ASPD” indicates the reactivity of the obtained serum with the synthetic ASPD immobilized plate, “control” indicates the reactivity of the obtained serum with the F12 protein immobilized plate, and “blank” indicates the reactivity of the obtained serum with a blank plate.

[0277] Depend on Figure 5 The results showed that Aβ cross-linked body 1 is immunogenic and can induce antibodies that react with ASPD.

[0278] 5. Determination of ASPD in Cerebrospinal Fluid (CSF)

[0279] ASPD in human CSF samples was measured using the ASPD CLEIA system [anti-ASPD polyclonal antibody / mASD3]. Specifically, the following procedure was used.

[0280] (1) Add a known concentration of ASPD to CSF ​​and dilute with sample diluent (1 to 16 times).

[0281] (2) Primary reaction: 100 μL / well was added to a multi-well plate immobilized with rabbit anti-ASPD polyclonal antibody and allowed to stand at 25°C for 60 minutes.

[0282] Cleaning × 3

[0283] (3) Secondary reaction: mASD3 (1 μg / ml) was added at 100 μL / well and incubated at 25°C for 60 minutes.

[0284] Cleaning × 3

[0285] (4) Tertiary reaction: Anti-mouse IgG-HRP (1 / 20000) was added at 100 μL / well and allowed to stand at 25°C for 60 minutes.

[0286] Cleaning × 3

[0287] (5) Measurement: 100 μL / well of HRP chemiluminescent substrate (trade name: SuperSignal pico, manufactured by ThermoFisher) was added, and luminescence was measured using a microplate reader (trade name: infinite M200pro, manufactured by TECAN).

[0288] Aβ aggregate 1 or cell-secreted ASPD-like structure (WO2017 / 179646) was used as a standard for the measurement.

[0289] As the sample diluent, 3% BSA in PBS(-) with 0.05% preservative (trade name: ProClin, manufactured by Sigma-Aldrich) was used.

[0290] PBS(-)0.05% Tween20 was used as the washing solution.

[0291] Table 1 below shows the results of adding known concentrations of ASPD (high concentration H: 108 pM, medium concentration M: 54 pM, low concentration L: 27.5 pM, no N: 0 pM) to human CSF (age 84 / Caucasian / M) and quantifying the concentrations by 4-fold dilution.

[0292] [Table 1]

[0293] Table 1: Recovery rate of ASPD added to human CSF (dilution ratio × 4)

[0294]

[0295] The recovery rate was approximately 100% as shown in Table 1. Similar results were obtained when human CSF (age 66 / Caucasian / M) and human CSF (age 69 / Caucasian / M) were used.

[0296] Next, the sample at which the ASPD peaked in human CSF was diluted 2-fold, 4-fold, and 8-fold with a sample diluent, and the samples at dilution ratios of 1-fold, 1 / 2, 1 / 4, and 1 / 8 were measured. The results are shown in FIG. Figure 6 .

[0297] like Figure 6 As shown, the results of the diluted samples were plotted, and the dilution linearity was obtained.

[0298] 6. Determination of ASPD in Human Plasma

[0299] ASPD in human plasma samples was measured using ASPD CLEIA [anti-ASPD polyclonal antibody / mASD3]. Specifically, the assay was performed according to the following procedure.

[0300] (1) Add known concentrations of ASPD to human plasma and dilute with sample diluent (1 to 16 times).

[0301] (2) Primary reaction: 100 μL / well was added to a multi-well plate immobilized with rabbit anti-ASPD polyclonal antibody and shaken at 25°C for 60 minutes.

[0302] Cleaning × 3

[0303] (3) Secondary reaction: mASD3 (1 μg / ml) was added at 100 μL / well and shaken at 25°C for 60 minutes.

[0304] Cleaning × 3

[0305] (4) Tertiary reaction: Anti-mouse IgG-HRP (1 / 20000) was added at 100 μL / well and shaken at 25°C for 60 minutes.

[0306] Cleaning × 3

[0307] (5) Measurement: 100 μL / well of HRP chemiluminescent substrate (trade name: SuperSignal pico, manufactured by ThermoFisher) was added, and luminescence was measured using a microplate reader (trade name: infinite M200pro, manufactured by TECAN).

[0308] Aβ aggregate 1 or cell-secreted ASPD-like structure (WO2017 / 179646) was used as a standard for the measurement.

[0309] As the sample diluent, 3% BSA in PBS(-) with 0.05% preservative (trade name: ProClin, manufactured by Sigma-Aldrich) was used.

[0310] PBS(-)0.05% Tween20 was used as the washing solution.

[0311] Table 2 below shows an example of the results of adding known concentrations of ASPD to human plasma (age 29 / Caucasian / F), followed by quantification by dilution 4-fold with 3% BSA PBS(-).

[0312] [Table 2]

[0313] Table 2: Recovery of ASPD spiked into human plasma (dilution factor × 4)

[0314]

[0315] As shown in Table 2, ASPD can be measured using human plasma instead of human CSF. However, the recovery rate is approximately half, which is believed to be due to the influence of plasma components.

[0316] Next, the sample with the peak ASPD in human plasma was diluted 2-fold, 4-fold, 8-fold, and 16-fold with the sample diluent, and the samples with dilution ratios of 1-fold, 1 / 2, 1 / 4, 1 / 8, and 1 / 16 were measured. The results are shown in FIG. Figure 7 .

[0317] like Figure 7 As shown, the results for the diluted samples were plotted, and a dilution linearity was obtained. However, the slope was half that of the buffer solution. This is believed to be due to the influence of plasma components.

[0318] It should be noted that the influence on the recovery rate in the human plasma sample can be eliminated by reducing the dilution ratio of the sample to 1 / 16.

[0319] 7.ASPD CLEIA [huASD2 / mASD3]

[0320] As the antibody immobilized on the solid phase, humanized anti-ASPD monoclonal antibody huASD2 was used, and ASPD was measured by ASPD CLEIA using mouse anti-ASPD monoclonal antibody mASD3 and anti-mouse IgG-HRP. Figure 8 ).

[0321] huASD2 is described in WO2009 / 057664.

[0322] An example of the results is shown in Figure 9 .like Figure 9As shown, four independent ASPD measurements were performed, and the results showed substantially the same luminescence values.

[0323] 8.ASPD CLEIA [huASD2 / biotinylated mASD3]

[0324] To improve the detection sensitivity of ASPD, a system using a mASD3 antibody biotinylated with HRP conjugated to streptavidin (SA) was constructed ( Figure 10 ).

[0325] The results of measuring ASPD using the huASD2 / biotinylated mASD3 system showed that the detection sensitivity was improved compared with the huASD2 / mASD3 system ( Figure 11 ).

[0326] In addition, the huASD2 / biotinylated mASD3 system has high specificity for ASPD, and a multi-antigen peptide (MAP16, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) bound to 16 molecules of Aβ1-10 molecules was hardly detected ( Figure 12 On the other hand, the BAN50 / biotinylated BAN50 system (High-molecular-weight amyloid β oligomer ELISA kit, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a control. The results showed that the system detected MAP16 with good sensitivity and almost no ASPD was detected. Figure 12 ).

[0327] 9. Combination of antibodies that can reduce the effects of plasma

[0328] A combination of an immobilized primary antibody and a detection-side secondary antibody was found that could reduce the influence of plasma.

[0329] An ASPD CEILA system was constructed using the following combination of [primary antibody / secondary antibody] to measure ASPD in plasma samples.

[0330] The primary / secondary antibody combinations used were [huASD2 / biotinylated mASD3], [mASD3 / biotinylated mASD3], [82E1 / biotinylated mASD3], [BAN50a / biotinylated mASD3], and [6E10 / biotinylated mASD3]. Plasma samples containing ASPD at a 1 / 4 dilution ratio were prepared as samples for comparison with buffer samples.

[0331] An example of the results is shown in Figure 13 .

[0332] like Figure 13As shown, it was confirmed that the combination of [BAN50a / biotinylated mASD3] can most effectively avoid the influence of plasma components.

[0333] 10. Measurement using blood samples from AD patients

[0334] ASPD CLEIA was performed on 10 plasma samples from patients diagnosed with Alzheimer's disease (AD) (○) and 10 plasma samples from healthy individuals (△) using the combination of [huASD2 / biotinylated mASD3]. ASPD was detected in 4 of the AD patients. The results are shown in Figure 14 .

[0335] These results demonstrated that ASPD in blood can be measured using ASPD CLEIA. Sequence Listing <110> Kobe Medical Industry City Promotion Organization <120> Cross-linked amyloid β (Aβ) as a surrogate for amyloid spheroids (ASPD), and analysis of ASPD <130> H4801 <150> JP2019209136 <151> 2019-11-19 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Gly Ile Glu Ile Lys Ser Tyr Phe Tyr Ala Thr Tyr Tyr Phe Gly 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Asn Arg Glu Val Gly Gly Leu Asp Asn Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Humanized Antibody <400> 2 Gln Ser Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala 1 5 10 15 Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Ser Val Gly Gly 20 25 30 Lys Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr 35 40 45 Leu Leu Lys Tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile 65 70 75 80 Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95 Ser Ile His Glu Ser Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu 100 105 110 Thr Val Leu Gly Arg 115 <210> 3 <211> 120 <212> PRT <213> Artificial sequence <220> <223> recombinant antibodies <220> <221> Xaa <222> (49)..(49) <223> Gly or Ala <220> <221> Xaa <222> (81)..(81) <223> Leu or Val <220> <221> Xaa <222> (100)..(100) <223> Thr or Arg <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Xaa Gly Ile Glu Ile Lys Ser Tyr Phe Tyr Ala Thr Tyr Tyr Tyr Phe Gly 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Xaa Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Xaa Asn Arg Glu Val Gly Gly Leu Asp Asn Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 116 <212> PRT <213> Artificial sequence <220> <223> recombinant antibodies <220> <221> Xaa <222> (2)..(2) <223> Ser or Ala <220> <221> Xaa <222> (8) <223> Ser or Ala <220> <221> Xaa <222> (48)..(48) <223> Tyr or Phe <220> <221> Xaa <222> (49)..(49) <223> Leu or Phe <220> <221> Xaa <222> (51)..(51) <223> Lys, Phe or Arg <220> <221> Xaa <222> (74)..(74) <223> Ala or Thr <220> <221> Xaa <222> (79)..(79) <223> Gly or Ala <400> 4 Gln Xaa Val Leu Thr Gln Pro Xaa Ser Leu Ser Ala Ser Pro Gly Ala 1 5 10 15 Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Ser Val Gly Gly 20 25 30 Lys Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Xaa 35 40 45 Xaa Leu Xaa Tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Lys Asp Xaa Ser Ala Asn Ala Xaa Ile 65 70 75 80 Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95 Ser Ile His Glu Ser Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu 100 105 110 Thr Val Leu Gly 115

Claims

1. An ASPD analysis method comprising the following steps: (1) a step of contacting a sample derived from a living organism that may contain ASPD with an antibody capable of binding to ASPD, i.e., a first antibody, which is bound to an insoluble carrier, and allowing ASPD to bind to the first antibody if ASPD is contained; and (2) a step of reacting an anti-ASPD monoclonal antibody, i.e., a second antibody, with ASPD bound to the first antibody in step (1); in, The first antibody is the humanized anti-ASPD monoclonal antibody huASD2 or the monoclonal antibody BAN50a, The second antibody is the mouse monoclonal anti-ASPD antibody mASD3 or its labeled antibody.

2. The analysis method according to claim 1, further comprising the following steps: (3) A step of reacting the anti-mouse antibody linked to the reporter gene with the second antibody bound to ASPD in step (2), or a step of reacting the labeled binding partner linked to the reporter gene with the label of the second antibody bound to ASPD in step (2).

3. The analysis method according to claim 1 or 2, wherein As a standard substance for ASPD, a substance that competes with ASPD-specific antibodies and amyloid spheres was used. The substance is a substance formed by cross-linking beta-amyloid protein (Aβ) with a cross-linking agent. The cross-linking agent is a spacer arm with a length of Above and The following crosslinking agents, or crosslinking agents having 1 or more and 13 or less oxyethylene groups, i.e., -CH2CH2O- and / or oxypropylene groups, i.e., -CH2CH2CH2O-, as spacer arms.

4. The analysis method according to claim 3, wherein The reactive ends at both ends of the cross-linking agent are NHS esters.

5. A substance for use in the analytical method according to claim 3 or 4, wherein the substance competes with ASPD for an ASPD-specific antibody, the substance being a substance formed by cross-linking β-amyloid protein (Aβ) with a cross-linking agent. The cross-linking agent is a spacer arm with a length of Above and The following crosslinking agents, or crosslinking agents having 1 or more and 13 or less oxyethylene groups, i.e., -CH2CH2O- and / or oxypropylene groups, i.e., -CH2CH2CH2O-, as spacer arms. 6 . A kit for use in the analysis method according to claim 3 or 4 , comprising the substance according to claim 5 , the first antibody, and the second antibody.

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