WO2017179646A1 - アミロスフェロイド(aspd)様構造体及び医薬組成物 - Google Patents
アミロスフェロイド(aspd)様構造体及び医薬組成物 Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0312—Animal model for Alzheimer's disease
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4709—Amyloid plaque core protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the present disclosure relates to an amylospheroid (ASPD) -like structure, a production method and use thereof, a pharmaceutical composition, a production method and use thereof, and a screening method.
- ABD amylospheroid
- Amylospheroids (a mylo sp heroi d s ( ASPD)) is amyloid ⁇ protein (A [beta]) is an A [beta] aggregates of spherical about 30 gathered diameter of about 10 nm, important in the irreversible stage of Alzheimer's disease will develop It is a structure that is thought to play an important role.
- ASPD was isolated as an A ⁇ aggregate (ie, synthetic ASPD) synthesized in vitro showing strong neurotoxicity (Non-patent Document 1).
- An antibody specific to this synthetic ASPD was produced (Patent Documents 1 and 2), and using this antibody, an ASPD (ie, native ASPD) actually formed in vivo from the brain of a human patient with Alzheimer's disease was produced. It was isolated (Non-patent Document 1).
- NAKarufa3 a subunit of K + -ATPase pump
- ⁇ ⁇ Nerve cell loss is most correlated with clinical symptoms in Alzheimer's disease.
- the amount of native ASPD in the cerebral cortex of Alzheimer's disease patients with neuronal loss is correlated with the severity of Alzheimer's disease, and the amount of native ASPD in the cerebellum of Alzheimer's disease patients with little neuronal loss is It became clear that only a trace amount exists (nonpatent literature 3). Thus, amylospheroids are thought to play an important role in the irreversible stage of Alzheimer's disease.
- native ASPD has also been detected in the brains of patients with dementia with Lewy bodies (Non-patent Document 3), and it is considered that ASPD plays an important role in the onset of dementia with Lewy bodies.
- Synthetic ASPD considered to be equivalent to native ASPD can be produced by slowly stirring a liquid containing A ⁇ (Non-patent Document 1, Patent Document 4).
- amylospheroid plays an important role in Alzheimer's disease and dementia with Lewy bodies.
- it is very difficult to purify native ASPD from the patient's brain and use it in the development of therapeutic drugs and the like. Therefore, if it becomes easy to produce an ASPD-like structure equivalent to or partially equivalent to native ASPD present in the patient's brain, research on Alzheimer's disease and dementia with Lewy bodies, and prevention methods and preventive drugs for these diseases And, it is thought to contribute greatly to the development of therapeutic methods and preventive / therapeutic drugs.
- Synthetic ASPD almost equivalent to native ASPD can be produced in vitro by slowly stirring a liquid containing amyloid ⁇ protein (A ⁇ ) (Non-patent Document 1 and Patent Document 4).
- a ⁇ amyloid ⁇ protein
- Synthetic ASPD was found to be able to induce immunity in rabbits without an adjuvant, and to have a certain vaccine effect and safe in old monkeys.
- synthetic ASPD has also found problems such as the amount of ASPD rising and falling to some extent for each production lot, difficulty in long-term storage of the manufactured ASPD, and difficulty in scaling up the production amount.
- the present disclosure provides an ASPD-like structure and a method for manufacturing the same.
- the present disclosure provides a method for producing a cell-secreted ASPD-like structure, in which cells expressing a part thereof containing an APP or A ⁇ sequence are cultured in a culture solution, and cell secretion is performed in the culture solution.
- the present invention relates to a manufacturing method including a step of obtaining a type ASPD-like structure.
- the present disclosure provides a structure obtained in a culture solution obtained by culturing cells into which an expression system that expresses a part of an APP or A ⁇ sequence is introduced, the antibody being specific for ASPD.
- the present invention relates to a cell-secreted ASPD-like structure that exhibits antigenicity.
- the present disclosure provides a pharmaceutical composition comprising a cell secreted ASPD-like structure as an active ingredient.
- the present disclosure relates to a vaccine comprising a cell secreted ASPD-like structure.
- the present disclosure relates to the use of cell secreted ASPD-like structures as active vaccines.
- the present disclosure in another aspect, relates to the use of cell secreted ASPD-like structures in vaccine manufacture.
- the present disclosure relates to the use of a cell secreted ASPD-like structure as a standard substance in ASPD measurement.
- the present disclosure provides a method for the prevention, amelioration, and / or treatment of a disease caused by ASPD, which is directed to a cell-secreted ASPD-like structure, the pharmaceutical composition, or the vaccine.
- a method comprising administering to a method comprising administering.
- the present disclosure provides a method for causing a subject to acquire immunity against ASPD, a disease caused by ASPD, or Alzheimer's disease and / or dementia with Lewy bodies, the cell-secreted ASPD-like structure, It relates to a pharmaceutical composition, or a method comprising administering said vaccine to a subject.
- the present disclosure relates to a method for producing a pharmaceutical composition or vaccine comprising combining a cell secreted ASPD-like structure and a pharmaceutically acceptable excipient.
- the present disclosure relates to a kit comprising a cell secreted ASPD-like structure and an anti-ASPD antibody.
- the present disclosure relates to a method for producing a kit, comprising combining a cell secreted ASPD-like structure with an anti-ASPD antibody.
- the present disclosure relates to a non-human animal having cells into which an expression system that expresses a portion thereof including an APP or A ⁇ sequence has been introduced.
- the present disclosure provides antigenicity to an antibody specific for ASPD formed in a culture medium in which cells into which an expression system expressing a part thereof including APP or A ⁇ sequence has been introduced are cultured.
- the present invention relates to a method for screening a substance that affects the formation of ASPD, which comprises using as an index the formation efficiency of a structure showing The present disclosure is selected from the group consisting of A ⁇ 40, A ⁇ 41, A ⁇ 42, A ⁇ 43 and combinations thereof secreted into a culture medium in which cells into which an expression system expressing an APP or a part thereof including an A ⁇ sequence has been introduced are cultured.
- the present invention relates to a method for screening a substance that affects C-terminal cleavage of A ⁇ , using at least one of them as an index.
- FIG. 1 is a schematic diagram illustrating the variants of APP expressed and the mutation sites of those mutants.
- FIG. 2 is a graph showing the measurement results of ASPD concentrations in the culture medium of cells overexpressed in the APP expression system of Test Examples 1 to 14.
- FIG. 3 is a graph showing the measurement results of A ⁇ 1-40 concentration in the culture medium of cells overexpressed in the APP expression system of Test Examples 1 to 14.
- FIG. 4 is a graph showing the measurement results of A ⁇ 1-42 concentration in the culture medium of cells overexpressed in the APP expression system of Test Examples 1 to 14.
- FIG. 5 is a graph showing the measurement results of ASPD concentration in the culture medium of cells overexpressing hAPP695sw-G33X.
- FIG. 6 is a graph showing the measurement results of A ⁇ 1-40 concentration in the culture medium of cells overexpressing hAPP695sw-G33X.
- FIG. 7 is a graph showing the measurement results of A ⁇ 1-42 concentration in the culture medium of cells overexpressing hAPP695sw-G33X.
- FIG. 8 is a graph showing an example of the results of evaluating the immunogenicity of synthetic ASPD.
- FIG. 9 is a graph showing an example of the results of confirming the neurotoxicity of synthetic ASPD (wild type) and synthetic ASPD (G33L type).
- FIG. 10 is a graph showing the measurement results of ASPD concentration in the culture medium of cells overexpressed in the APP expression system of Test Examples 15 to 36.
- FIG. 10 is a graph showing the measurement results of ASPD concentration in the culture medium of cells overexpressed in the APP expression system of Test Examples 15 to 36.
- FIG. 11 is a graph showing the measurement results of A ⁇ 1-40 concentration in the culture medium of cells overexpressed in the APP expression system of Test Examples 15 to 36.
- FIG. 12 is a graph showing the measurement results of A ⁇ 1-42 concentration in the culture medium of cells overexpressed in the APP expression system of Test Examples 15 to 36.
- the present disclosure is based on confirmation of the presence of an ASPD-like structure in a culture medium in which cells expressing amyloid precursor protein (APP) are cultured.
- the present disclosure relates to providing a cell-secreted ASPD-like structure that can be easily and scaled up.
- the cell-secreted ASPD-like structure in the present disclosure is excellent in storage stability in one or a plurality of embodiments.
- the cell-secreted ASPD-like structure in the present disclosure can be produced with a very low content of A ⁇ monomer. Therefore, removal of A ⁇ monomer by ultrafiltration is simplified, and vaccines and the like can be obtained. Manufacturing efficiency can be improved.
- the cell-secreted ASPD-like structure in the present disclosure can reduce the content of A ⁇ monomer in one or a plurality of embodiments, the safety when administered to a living body can be improved from, for example, native ASPD or synthetic ASPD. Can also improve.
- an ASPD-like structure refers to a structure in which an antibody specific to ASPD reacts with an antigen antibody, in other words, a structure that exhibits antigenicity to an antibody specific to ASPD.
- the term amylospheroid (ASPD) may include native ASPD and synthetic ASPD.
- Antibodies specific for ASPD include, in one or more embodiments, polyclonal anti-ASPD antibodies and monoclonal anti-ASPD antibodies disclosed in WO2006 / 016644 and WO2009 / 057664, and in 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-ASD An antibody (huASD2) etc. are mentioned.
- a cell-secreted ASPD-like structure refers to an ASPD-like structure produced by the production method according to the present disclosure. In one or more embodiments, it refers to an ASPD-like structure found in the culture medium of cells into which part of the expression system comprising APP or A ⁇ sequences has been introduced.
- the cell secreted ASPD-like structure according to the present disclosure can be said to be a cell secreted type because it is obtained in a culture medium of cells expressing a part thereof including the APP or A ⁇ sequence.
- the present disclosure is, in one aspect, a structure obtained in a culture medium obtained by culturing cells into which an expression system that expresses a part of an APP or A ⁇ sequence is introduced, the antibody being specific for ASPD.
- the present invention relates to a cell-secreted ASPD-like structure that exhibits antigenicity.
- the cell-secreted ASPD-like structure according to the present disclosure may be equivalent to ASPD in one or a plurality of embodiments, and partially equivalent to ASPD in another or a plurality of embodiments. It may be.
- Partially equivalent means at least a structure in which an antibody specific for ASPD reacts with an antigen antibody, in other words, a structure showing antigenicity to an antibody specific for ASPD.
- the cell-secreted ASPD-like structure according to the present disclosure is an A ⁇ aggregate in one or a plurality of embodiments.
- the term A ⁇ may refer to A ⁇ 40 (A ⁇ 1-40), A ⁇ 41 (A ⁇ 1-41), A ⁇ 42 (A ⁇ 1-42), A ⁇ 43 (A ⁇ 1-43), or all or a part thereof.
- the cell secreted ASPD-like structure according to the present disclosure may have the same molecular weight as ASPD in one or more embodiments, and in one or more embodiments, 100 kDa or more, or 100 to 700 kDa. It is.
- the cell secreted ASPD-like structure according to the present disclosure is such that, in one or more embodiments, the ASPD-like structure induces cytotoxicity of ASPD, that is, cell death selectively in functionally mature neurons.
- Properties may be equivalent to ASPD, may be less toxic than ASPD, may not be cytotoxic, or may be more toxic than ASPD .
- the cell-secreted ASPD-like structure according to the present disclosure may have a shape similar to or different from ASPD in one or a plurality of embodiments.
- the cell-secreted ASPD-like structure according to the present disclosure may have a spherical shape having a diameter of 10 to 15 nm in electron microscope observation, or may have a shape different from the shape.
- the cell-secreted ASPD-like structure includes a cell expressing a part thereof containing an amyloid precursor protein (APP) or amyloid ⁇ protein (A ⁇ ) sequence in a culture solution. It can be obtained in the culture by culturing. Accordingly, the present disclosure, in one aspect, includes the steps of culturing cells expressing a portion thereof comprising an APP or A ⁇ sequence in a culture solution to obtain a cell-secreted ASPD-like structure in the culture solution.
- the present invention relates to a method for manufacturing a type ASPD-like structure. Hereinafter, the manufacturing method is also simply referred to as “manufacturing method according to the present disclosure”.
- the APP expressed in the cultured cell may be a human APP or an APP of a non-human animal in one or a plurality of embodiments.
- Human APP may be any of splicing variants of hAPP770, hAPP751, and hAPP695.
- the sequences of APP and A ⁇ can be obtained from known databases.
- the NCBI accession number of hAPP770 is NP_000475 (VERSION NP_000475.1 GI: 4502167).
- UniProtKB-P05067 (Modified: November (1, 1991 -v3) can be referred to for the splicing variant sequence.
- the APP expressed in the cells to be cultured improves the amount of cell-secreted ASPD-like structures formed. It is preferable to express from the above expression system and / or overexpression in the cells. In one or a plurality of embodiments of the production method according to the present disclosure, the APP expressed in the cultured cell preferably has an APP signal sequence from the viewpoint of improving the amount of cell-secreted ASPD-like structures formed. .
- the APP to be expressed in the cultured cell may be a wild type or a mutant APP.
- Mutant APP includes mutations linked to familial Alzheimer's disease, such as Swedish mutation, Italian mutation, Leuven mutation, Icelandic mutation, London mutation, Egyptian mutation, Australian mutation, German mutation, French mutation, Florida mutation, Examples include Iberian mutation, Australian mutation, Belgian mutation, Flemish mutation, Icelandic mutation, British mutation, Tottori mutation, Italian mutation, Arctic mutation, Osaka mutation, Iowa mutation, and Dutch mutation.
- Swedish mutation is preferably included from the viewpoint of improving the amount of cell-secreted ASPD-like structure formed.
- APP expressed in cells to be cultured improves the formation efficiency of a cell-secreted ASPD-like structure. It is preferable to have one or more substitution mutations in the glycine of the GXXXG motif in the amino acid sequence. That is, it preferably has one or more glycine substitution mutations selected from the group consisting of glycine corresponding to the 25th, 29th, 33rd, and 37th positions of A ⁇ , and has at least the 33rd glycine substitution mutation. It is preferable to have.
- substitution mutations from the point of improving the formation efficiency of the cell secreted ASPD-like structure and reducing the amount of A ⁇ in the culture solution, leucine, isoleucine, phenylalanine, valine, methionine, tyrosine, or cysteine Substitution mutations are preferred.
- one or more is 1, 2, 3, or 4, 1, 2, or 3, or 1 or 2.
- APP expressed in cultured cells has substitution mutations at the 33rd and 37th glycines of A ⁇ from the viewpoint of improving the formation efficiency of cell secreted ASPD-like structures. It is preferable.
- substitution mutations include leucine, isoleucine, phenylalanine, valine, methionine, tyrosine from the viewpoint of improving the formation efficiency of cell-secreted ASPD-like structures and reducing the amount of A ⁇ in the culture medium.
- a substitution mutation to cysteine is preferred, and at least one of the 33rd and 37th positions is more preferably a substitution mutation to isoleucine.
- APP expressed in cells to be cultured improves the formation efficiency of a cell-secreted ASPD-like structure.
- examples thereof include one having a substitution mutation of one or more glycines selected from the group consisting of glycine corresponding to the 29th, 33rd, and 37th positions.
- the APP in the present disclosure is selected from the group consisting of a Swedish mutation in hAPP770 or hAPP695 and a glycine corresponding to the 25th, 29th, 33rd, and 37th A ⁇ . Those having a plurality of glycine substitution mutations.
- APP in the present disclosure includes those having a Swedish mutation in hAPP770 or hAPP695 and a substitution mutation of glycine corresponding to the 33rd position of A ⁇ .
- the APP in the present disclosure includes a Swedish mutation in hAPP770 or hAPP695, and a substitution mutation of glycine corresponding to the 33rd and 37th positions of A ⁇ (preferably at least one is a substitution mutation to isoleucine). The thing which has is mentioned.
- the APP expressed in the cells to be cultured may have other mutations as long as the formation of ASPD-like structures is not significantly prevented.
- modified amino acids may include mutations to modified amino acids, unnatural amino acids, D-amino acids and the like.
- modified amino acids include amino group modification, carboxyl group modification, thiol group modification, hydroxyl group modification, saccharification modification, and PEGylation modification in one or more embodiments.
- the APP to be expressed in cells to be cultured may be a partial peptide that is a part of APP and includes an amyloid ⁇ protein (A ⁇ ) sequence (
- a ⁇ amyloid ⁇ protein
- the partial peptide is hereinafter also simply referred to as “part of APP”). This is because ASPD is a form of an aggregate of A ⁇ cut out from APP.
- a part of APP to be expressed in cells to be cultured may be in a form in which the N-terminus is cleaved, and in one or more further embodiments, The N-terminus may be cleaved at the cleavage site of ⁇ -secretase.
- the part of APP expressed in the cells to be cultured is a part including A ⁇ 1st to 43rd, 1st to 42nd, or 1st to 40th. In one or more further embodiments, it has a portion comprising A ⁇ 1-43, 1-42 or 1-40 and an APP signal sequence.
- Expression of APP or a part thereof in cells to be cultured can be performed in a cell into which an expression system capable of expressing APP or a part thereof is introduced.
- the expression of APP or a part thereof may be by a transient expression system or by a stable expression cell line.
- the expression of APP or a part thereof is preferably high expression or overexpression as long as the formation of the ASPD-like structure is not hindered from the viewpoint of improving the formation amount of the ASPD-like structure.
- APP or a part of its expression system may be controllable.
- an expression system capable of expressing APP or a part thereof is operably linked to a sequence encoding APP or a part thereof, depending on the host cell to be introduced.
- An expression cassette having a nucleic acid can be mentioned. Examples of expression regulatory sequences include promoters, enhancers, transcription terminators, and the like, and other examples include start codons, intron splicing signals, and stop codons.
- APP or a part of its expression system can be introduced by appropriately selecting an expression vector according to a cell (host) to be expressed.
- the expression vector includes a vector having the above-described expression cassette.
- Expression vectors include, in one or more embodiments, plasmids, cosmids, YACS, viral (eg, adenovirus, retrovirus, episomal EBV, etc.) vectors and phage vectors.
- the cell cultured in the production method according to the present disclosure is a cell that expresses APP or a part thereof.
- the cell cultured in the production method according to the present disclosure is preferably a cell into which an expression system capable of expressing APP or a part thereof is introduced from the viewpoint of improving the amount of cell-secreted ASPD-like structure formed. Or it is more preferable that it is a cell which can highly express or overexpress a part thereof.
- the cell expressing APP or a part thereof may be a cell in which the genomic APP gene is overexpressed by gene transfer or genome editing.
- the cell cultured in the production method according to the present disclosure is preferably a cell capable of producing A ⁇ , and more preferably a cell expressing both ⁇ -secretase and ⁇ -secretase. preferable.
- host cells into which APP or a partial expression system thereof is introduced include animal cells, plant cells, insect cells, microorganisms, and the like, and cell lines thereof.
- Animal cells include mammalian cells, human cells, and non-human mammalian cells.
- the host cell into which APP or a partial expression system thereof is introduced is preferably a cell line from the viewpoint of handling.
- Non-limiting specific cell lines include CHO cells, HEK293 cells, Neuro2a cells and the like.
- Culture conditions for cells expressing APP or a part thereof in the production method according to the present disclosure are the type of the cell, the type of expression system introduced into the cell, and the form of introduction of the expression system (transient Depending on the introduction or stable introduction), a person skilled in the art appropriately adjusts the medium, temperature, CO 2 concentration, etc. so that APP or a part thereof incorporated in the expression system is expressed (preferably overexpressed). Can be set.
- the culture in the case of using a transient APP expression system includes culturing and transducing in a growth medium, and then changing to a serum-free medium and culturing for 24 to 48 hours. Can be mentioned. Then, a cell-secreted ASPD-like structure is obtained in the medium after the culture.
- the cells are animal cells, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, Ham'sF12 medium Examples include a medium, Fischer's medium, and a mixed medium thereof. These media may contain serum or serum substitutes, or may be serum-free. Optionally, the medium also contains one or more substances such as lipids, amino acids, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like. obtain.
- EMEM Eagle's Minimum Essential Medium
- DMEM Dulbecco's modified Eagle's Medium
- Ham's F12 medium Ham'sF12 medium
- the medium also contains one or more substances such as lipids, amino acids, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers
- the production method according to the present disclosure further includes recovering a cell-secreted ASPD-like structure from the culture solution.
- the cell-secreted ASPD-like structure in the culture medium is collected by a step of collecting a retentate obtained by ultrafiltration of 50 kDa or 100 kDa with a 0.22 ⁇ m pore size filtrate. Can do.
- the recovery method of ASPD may not be limited to this method.
- the production method according to the present disclosure facilitates the production of a cell-secreted ASPD-like structure
- development of an active vaccine therapy using the cell-secreted ASPD-like structure itself as an antigen in one or more embodiments, development of a pharmaceutical composition that functions as an active vaccine and development of a neuronal cell death inhibitor are facilitated.
- a pharmaceutical composition such as a vaccine using a cell-secreted ASPD-like structure can solve one or a part or more of these problems in one or a plurality of embodiments.
- the present disclosure relates to a pharmaceutical composition comprising a cell secreted ASPD-like structure as an active ingredient.
- a pharmaceutical composition according to the present disclosure include a vaccine comprising a cell secreted ASPD-like structure.
- Pharmaceutical compositions and vaccines according to the present disclosure may include pharmaceutically acceptable excipients and / or adjuvants.
- the pharmaceutical composition and vaccine according to the present disclosure may be used for prevention, amelioration, and / or treatment of a disease caused by ASPD in one or more embodiments.
- the pharmaceutical composition and vaccine according to the present disclosure may be used for prevention, amelioration, and / or treatment of Alzheimer's disease and / or dementia with Lewy bodies in one or more embodiments.
- the pharmaceutical composition and vaccine according to the present disclosure may be administered to a subject who is likely to suffer from a disease caused by ASPD, a subject who may be affected, or a subject who is affected.
- the pharmaceutical composition according to the present disclosure is administered to a subject who is likely to suffer from Alzheimer's disease and / or Lewy body dementia, a subject who may be affected, or a subject who is affected. sell.
- the subject include mammals, humans, and non-human mammals.
- compositions and vaccine according to the present disclosure are acquired for immunity against ASPD, a disease caused by ASPD, or Alzheimer's disease and / or dementia with Lewy bodies.
- the pharmaceutical composition and vaccine administration method according to the present disclosure may be injected intramuscularly, intraperitoneally, intradermally, or subcutaneously, or into the oral tract, digestive tract, airway, urogenital tract. Transmucosal administration may be included.
- Examples of the dose of the cell secreted ASPD-like structure in the pharmaceutical composition and vaccine according to the present disclosure include an amount that induces an immune defense response without causing significant side effects on the administration subject.
- the dosage of the pharmaceutical composition and vaccine according to the present disclosure is 0.01 ⁇ g to 10 mg, 0.1 to 1000 ⁇ g, 1 to 100 ⁇ g, 5 to 50 ⁇ g of the cell secreted ASPD-like structure, Alternatively, the amount is expected to be included in the range of 5 to 25 ⁇ g. Following the initial administration, one or several booster administrations may be given with a sufficient interval.
- the present disclosure is a method for the prevention, amelioration, and / or treatment of a disease caused by ASPD (eg, Alzheimer's disease and / or Lewy body dementia), comprising a cell secreted ASPD-like structure.
- ASPD eg, Alzheimer's disease and / or Lewy body dementia
- the present disclosure provides a method for causing a subject to acquire immunity against ASPD, a disease caused by ASPD, or Alzheimer's disease and / or dementia with Lewy bodies, the cell-secreted ASPD-like structure or the present invention. It relates to a method comprising administering to a subject a pharmaceutical composition or vaccine according to the disclosure.
- this indication is related with the manufacturing method of a pharmaceutical composition or a vaccine including combining a cell secretion type ASPD-like structure, a vaginal shape agent, and / or an adjuvant in one aspect
- the present disclosure is a method for producing a pharmaceutical composition comprising a cell secreted ASPD-like structure as an active ingredient, or a vaccine having a cell secreted ASPD-like structure, the production method according to the present disclosure Relates to a production method comprising a step of producing a cell secreted ASPD-like structure.
- the present disclosure relates to a kit for use in the production method according to the present disclosure, including a cell line into which APP or a partial expression system thereof has been introduced.
- a kit for use in the production method according to the present disclosure including a cell line into which APP or a partial expression system thereof has been introduced.
- the kit according to the present disclosure it is easy to produce a cell-secreted ASPD-like structure.
- the kit according to the present disclosure may further include at least one of a culture container, a medium, and instructions.
- the kit used for the production method according to the present disclosure is a vector having APP or a partial expression system thereof instead of the cell line into which APP or a partial expression system thereof is introduced. May be.
- the present disclosure relates to a non-human animal having cells into which an expression system that expresses a portion thereof including an APP or A ⁇ sequence is introduced.
- the non-human animal can be a model animal for diseases caused by ASPD, such as Alzheimer's disease and / or Lewy body dementia.
- the cell is preferably a brain cell or a nerve cell from the viewpoint of the model animal.
- the method for introducing the expression system is not particularly limited, and examples thereof include a method using gene introduction technology and a method using genome editing technology.
- Non-human animals include non-human mammals and non-human primates.
- One or more embodiments of this aspect are genetically modified animals having cells that have been genetically modified to express a portion thereof comprising APP or A ⁇ sequences.
- the APP expressed in the genetically modified cell may be, for example, a mutant of APP or a mutant of A ⁇ as disclosed in the present disclosure. That is, the APP expressed in the cells has a substitution mutation of one or more glycines selected from the group consisting of glycine corresponding to the 25th, 29th, 33rd, and 37th of A ⁇ , as described above. It preferably has at least the 33rd glycine substitution mutation.
- substitution mutation substitution mutation to leucine, isoleucine, phenylalanine, valine, methionine, tyrosine, or cysteine is preferable.
- the APP expressed in the cell preferably has a substitution mutation of the 33rd and 37th glycine of A ⁇ .
- the substitution mutation is preferably a substitution mutation to leucine, isoleucine, phenylalanine, valine, methionine, tyrosine, or cysteine, and at least one of the 33rd and 37th positions is a substitution mutation to isoleucine. More preferred.
- the cell secreted ASPD-like structure can be used as a standard substance of ASPD in ASPD measurement.
- the cell-secreted ASPD-like structure can be used in a kit for measuring ASPD. That is, in one aspect, the present disclosure relates to a kit comprising a cell secreted ASPD-like structure and an anti-ASPD antibody. Examples of anti-ASPD antibodies include antibodies specific for the above-mentioned ASPD. Moreover, this indication is related with the manufacturing method of a kit including combining a cell secretion type ASPD-like structure and an anti- ASPD antibody in one aspect
- the present disclosure is, in one aspect, a method for screening a substance that affects the formation of ASPD, which is formed in a culture medium in which cells into which an expression system that expresses a part thereof including an APP or A ⁇ sequence is introduced are cultured.
- the present invention relates to a screening method comprising, as an index, the formation efficiency of a structure showing antigenicity to an antibody specific for ASPD (that is, a cell-secreted ASPD-like structure).
- the index may be the amount of cell secreted ASPD-like structures formed, or may be A ⁇ 40, A ⁇ 41, A ⁇ 42, A ⁇ 43, or a relative amount with respect to the total amount thereof.
- This form of screening method is a substance that affects the formation of ASPD by comparing the formation efficiency of cell-secreted ASPD-like structures with and without the addition of a test substance to the culture medium. Judging may be included. For example, if the formation efficiency decreases when a test substance is added to the culture solution, it can be determined that the substance has an effect of inhibiting the formation of ASPD. In addition, for example, if the formation efficiency is enhanced when a test substance is added to the culture solution, it can be determined that the substance has an effect of promoting the formation of ASPD.
- APP expressed in cells to be cultured may be, for example, a variant of APP or a variant of A ⁇ as disclosed in the present disclosure.
- the present disclosure provides a method for screening a substance that affects C-terminal cleavage of A ⁇ , in a culture solution in which cells into which an expression system that expresses APP or a part thereof including an A ⁇ sequence is introduced are cultured.
- the present invention relates to a screening method including using as an index at least one A ⁇ selected from the group consisting of A ⁇ 40, A ⁇ 41, A ⁇ 42, A ⁇ 43, and combinations thereof formed in the above.
- the indicator may be a secretion amount or a concentration in a culture solution of A ⁇ 40, A ⁇ 41, A ⁇ 42, A ⁇ 43, and a combination thereof, or, for example, a ratio of A ⁇ 42 to A ⁇ 40 (A ⁇ 42 / A relative amount such as A ⁇ 40) may be used.
- the index is compared to determine whether the substance affects the C-terminal cleavage of A ⁇ , or to ⁇ -secretase. It may include determining whether or not the substance has an influence.
- APP expressed in cells to be cultured may be, for example, a variant of APP or a variant of A ⁇ as disclosed in the present disclosure.
- the present disclosure relates to a mutant synthetic ASPD.
- the mutant synthetic ASPD refers to a synthetic ASPD-like structure synthesized using a mutant A ⁇ by the same production method as the synthetic ASPD. That is, in the present disclosure, the mutant synthetic ASPD is a synthesis formed by stirring a liquid containing a mutant A ⁇ having one or more substitution mutations in glycine of the GXXXG motif in the amino acid sequence of the 25th to 37th A ⁇ .
- An ASPD-like structure may contain, for example, a plasticizer such as a phthalate ester.
- the mutant synthetic ASPD comprises dissolving the mutant A ⁇ in an organic solvent containing the plasticizer, diluting the solution of the mutant A ⁇ with an aqueous solution, and the dilution. It can synthesize
- the mutant A ⁇ preferably has one or a plurality of glycine substitution mutations selected from the group consisting of glycine corresponding to the 25th, 29th, 33rd, and 37th positions of A ⁇ . It preferably has a glycine substitution mutation.
- the mutant A ⁇ preferably has a substitution mutation of the 33rd and 37th glycine of A ⁇ .
- the substitution mutation is preferably a substitution mutation to leucine, isoleucine, phenylalanine, valine, methionine, tyrosine, or cysteine from the viewpoint of improving the formation efficiency of synthetic ASPD, and at least one of the 33rd and 37th positions is More preferably, it is a substitution mutation to isoleucine.
- the mutant ASPD according to the present disclosure exhibits antigenicity against an ASPD-specific antibody. In addition, in one or a plurality of other embodiments, the mutant ASPD according to the present disclosure exhibits cytotoxicity against mature neurons.
- the present disclosure may relate to the following: [1] A method for producing a cell-secreted amylospheroid (ASPD) -like structure, wherein cells expressing a part thereof containing an amyloid precursor protein (APP) or amyloid ⁇ protein (A ⁇ ) sequence are cultured in a culture solution A production method comprising a step of culturing and obtaining a cell-secreted ASPD-like structure in the culture solution. [2] The production method according to [1], further comprising recovering a cell secreted ASPD-like structure from the culture solution. [3] The production method according to [1] or [2], wherein the cell is a cell into which part of an expression system containing an APP or A ⁇ sequence has been introduced.
- a method for producing a cell-secreted amylospheroid (ASPD) -like structure wherein cells expressing a part thereof containing an amyloid precursor protein (APP) or amyloid ⁇ protein (A ⁇ ) sequence are cultured in a culture solution
- a pharmaceutical composition comprising the cell secreted ASPD-like structure according to [6] as an active ingredient.
- a vaccine comprising the cell secreted ASPD-like structure according to [6].
- a kit comprising the cell secreted ASPD-like structure according to [6] and an anti-ASPD antibody.
- [13] A method for preventing, ameliorating, and / or treating a disease caused by ASPD, the cell-secreted ASPD-like structure according to [6], the pharmaceutical composition according to [7], or [8] A method comprising administering to the subject the vaccine according to [8].
- the method according to [13], wherein the disease caused by ASPD is Alzheimer's disease and / or dementia with Lewy bodies.
- a method for causing a subject to acquire immunity against ASPD, a disease caused by ASPD, or Alzheimer's disease and / or dementia with Lewy bodies, the cell-secreted ASPD-like structure according to [6], [ [7] A method comprising administering to a subject the pharmaceutical composition according to [7] or the vaccine according to [8].
- a method for producing a pharmaceutical composition or vaccine comprising combining the cell-secreted ASPD-like structure according to [6] and a pharmaceutically acceptable excipient.
- a method for producing a kit comprising combining the cell secreted ASPD-like structure according to [6] and an anti-ASPD antibody.
- a non-human animal having cells into which an expression system that expresses a part of an APP or A ⁇ sequence is introduced.
- a structure formed in a culture solution obtained by culturing cells into which an expression system that expresses a part of an APP or A ⁇ sequence is introduced, and has an antigenicity against an antibody specific for ASPD A method for screening a substance that affects the formation of ASPD, comprising using as an index the formation efficiency of a structure to be shown. [20] selected from the group consisting of A ⁇ 40, A ⁇ 41, A ⁇ 42, A ⁇ 43, and combinations thereof secreted into a culture medium in which cells into which an expression system that expresses an APP or A ⁇ sequence is introduced are cultured. A method for screening a substance that affects the C-terminal cleavage of A ⁇ , using at least one of these as an index. [21] A synthetic ASPD-like structure formed by stirring a liquid containing mutant A ⁇ having one or a plurality of substitution mutations in glycine of the GXXXG motif in the amino acid sequence of the 25th to 37th amino acids of A ⁇ .
- FIG. 1 (SEQ ID NO: 1) is a partial amino acid sequence of hAPP containing human A ⁇ .
- the APPs of Test Examples 1 to 7 are hAPP770 wild type, hAPP770 Swedish variant, and hAPP770 Swedish type in which one or two glycines of the GXXXG motif are mutated.
- the APPs of Test Examples 8 to 14 are hAPP695 wild type, hAPP695 Swedish variant, and hAPP695 Swedish type in which one or two glycines of the GXXXG motif are mutated.
- CHO cells overexpressing hAPP of Test Examples 1 to 14 were prepared as follows. CHO / K1 cells were seeded in a 12-well plate at a density of 2.5 ⁇ 10 4 cells / cm 2 in growth medium and maintained at 37 ° C. and 5% CO 2 . After 24 hours, the expression vector was introduced into CHO / K1 cells using a gene introduction reagent (FuGENE HD). In the introduction method, 25 ⁇ L of Opti-MEM and 0.5 ⁇ g of plasmid DNA were mixed per well, 1.5 ⁇ L of FuGENE HD was further added, and the mixture was reacted at room temperature for 10 minutes.
- FuGENE HD gene introduction reagent
- a ⁇ ELISA About the collect
- the kits used are as follows. The measurement was performed according to the instructions attached to the kit.
- a ⁇ 1-40 human ⁇ amyloid (1-40)
- ELISA kit Wako Waako # 292-62301
- a ⁇ 1-42 human ⁇ -amyloid (1-42)
- ELISA kit Wako high-sensitivity product (Wako # 296-64401)
- glycine at position 629 (position 33 of A ⁇ ) of hAPP695 Swedish mutant is substituted with a predetermined amino acid, for example, leucine, isoleucine, phenylalanine, methionine, valine, tyrosine, cysteine.
- a predetermined amino acid for example, leucine, isoleucine, phenylalanine, methionine, valine, tyrosine, cysteine.
- proline or threonine
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Abstract
Description
本開示は、その他の態様において、細胞分泌型ASPD様構造体を含むワクチンに関する。
本開示は、その他の態様において、細胞分泌型ASPD様構造体の能動ワクチンとしての使用に関する。
本開示は、その他の態様において、ワクチン製造における細胞分泌型ASPD様構造体の使用に関する。
本開示は、その他の態様において、細胞分泌型ASPD様構造体の、ASPD測定における標準物質としての使用に関する。
本開示は、その他の態様において、ASPD、ASPDに起因する疾病、又は、アルツハイマー病及び/又はレビー小体型認知症に対する免疫を対象に獲得させる方法であって、細胞分泌型ASPD様構造体、前記医薬組成物、又は、前記ワクチンを対象に投与することを含む方法に関する。
本開示は、その他の態様において、細胞分泌型ASPD様構造体と、薬学的に許容される腑形剤とを組み合わせることを含む、医薬組成物又はワクチンの製造方法に関する。
本開示は、その他の態様において、細胞分泌型ASPD様構造体と、抗ASPD抗体とを組み合わせることを含む、キットの製造方法に関する。
本開示は、その他の態様において、APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を有する非ヒト動物に関する。
本開示は、APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を培養した培養液中に分泌される、Aβ40、Aβ41、Aβ42、Aβ43及びこれらの組合せからなる群から選択される少なくとも1つを指標とする、AβのC末端切断に影響を与える物質のスクリーニング方法に関する。
本開示は、一又は複数の実施形態において、簡便でスケールアップ可能な、細胞分泌型ASPD様構造体の提供に関する。
本開示における細胞分泌型ASPD様構造体は、一又は複数の実施形態において、保存安定性に優れる。
本開示における細胞分泌型ASPD様構造体は、一又は複数の実施形態において、Aβモノマーの含有率を極めて低く製造することもできるため、限外ろ過によるAβモノマーの除去が簡単となり、ワクチン等の製造効率の向上が可能となる。
また、本開示における細胞分泌型ASPD様構造体は、一又は複数の実施形態において、Aβモノマーの含有率を低減できるから、生体に投与した場合の安全性を、例えば、ネイティブASPDや合成ASPDよりも、向上しうる。
本開示において、ASPD様構造体とは、ASPDに特異的な抗体が抗原抗体反応する構造体、言い換えれば、ASPDに特異的な抗体に対して抗原性を示す構造体をいう。
なお、本開示において、単にアミロスフェロイド(ASPD)という場合には、ネイティブASPD及び合成ASPDを含みうる。
本開示において、細胞分泌型ASPD様構造体は、本開示にかかる製造方法によって製造されるASPD様構造体をいう。一又は複数の実施形態において、APP又はAβ配列を含むその一部の発現系が導入された細胞の培養液中に見出されたASPD様構造体をいう。本開示に係る細胞分泌型ASPD様構造体は、APP又はAβ配列を含むその一部を発現する細胞の培養液中に得られるものであるから、細胞分泌型であるといえる。
本開示に係る細胞分泌型ASPD様構造体は、一又は複数の実施形態において、アミロイド前駆体タンパク質(APP)又はアミロイドβタンパク質(Aβ)配列を含むその一部を発現する細胞を培養液中で培養することで、その培養液中に得ることができる。
したがって、本開示は一態様において、APP又はAβ配列を含むその一部を発現する細胞を培養液中で培養し、該培養液中に細胞分泌型ASPD様構造体を得る工程を含む、細胞分泌型ASPD様構造体の製造方法に関する。以下、前記製造方法を単に「本開示に係る製造方法」ともいう。
その他の一又は複数の実施形態において、培養する細胞内で発現させるAPPは、細胞分泌型ASPD様構造体の形成効率を向上させる点から、Aβの33番目及び37番目のグリシンの置換変異を有することが好ましい。本実施形態において、置換変異としては、細胞分泌型ASPD様構造体の形成効率を向上する点、及び、培養液中のAβ量を低減する点から、ロイシン、イソロイシン、フェニルアラニン、バリン、メチオニン、チロシン、又はシステインへの置換変異が好ましく、33番目及び37番目の少なくとも一方が、イソロイシンへの置換変異であることがより好ましい。
本開示に係る製造方法の一又は複数の実施形態において、培養する細胞内で発現させるAPPの一部は、N末端が切断された形態であってもよく、さらなる一又は複数の実施形態において、N末端がβシクレターゼの切断部位で切断された形態であってもよい。
本開示に係る製造方法の一又は複数の実施形態において、培養する細胞内で発現させるAPPの一部は、Aβの1~43番目、1~42番目又は1~40番目を含む部分であることが挙げられ、さらなる一又は複数の実施形態において、Aβの1~43番目、1~42番目又は1~40番目を含む部分、及び、APPのシグナル配列を有することが挙げられる。
APP又はその一部の発現系が導入される宿主細胞は、一又は複数の実施形態において、取扱性の点から、細胞株が好ましい。限定されない具体的な細胞株としては、CHO細胞、HEK293細胞、Neuro2a細胞などが挙げられる。
培養液中の細胞分泌型ASPD様構造体の回収は、一又は複数の実施形態において、孔径0.22μmフィルターのろ液を50kDa又は100kDaの限外ろ過をした保持液を回収する工程により行うことができる。但し、ASPDの回収方法はこの方法に限定されなくてもよい。
ASPDを構成するAβそのものを生体内に導入して免疫を付与する試みの結果、Aβの配列の一部(16~33番目の残基)がT細胞を活性化し炎症性反応を引き起こすことが報告された(Monsonego, A. et al. J. Clin. Invest. 112:415-422 (2003), Monsonego, A. et al. PNAS 103:5048-5053 (2006))。したがって、Aβそのものをワクチンとして用いることは難しい。
一方、ASPDは、T細胞を活性化するAβの配列(16~33番目の残基)が球状構造の内部に折り畳まれてほぼ隠され(Ohnishi et al. PNAS 112:E4465-E4474 (2015))、Aβワクチンで問題となる炎症性T細胞応答を惹起しないというデータが得られている。
そして、合成ASPDについては、アジュバントなしでウサギに免疫がつくこと、その免疫量は通常のペプチド抗原の1/5~1/10と少量で充分であることが見出された。さらに、ASPDを用いて老齢カニクイザルの皮下投与で免疫すると、脳の糖代謝(神経活動)が高まることも見出された。
これらの知見から、ASPDは、安全性が高い能動ワクチンとして使用できるといえる。そして、本開示にかかる細胞分泌型ASPD様構造体も、能動ワクチンとして使用でき、また、ワクチンの製造に使用できる。
したがって、本開示は、一態様において、ASPDを有効成分とする医薬組成物に関し、或いは、ASPDを含むワクチンに関する。
本開示に係る細胞分泌型ASPD様構造体を用いたワクチン等の医薬組成物であれば、一又は複数の実施形態において、これらの問題の1つ若しくは一部又はそれ以上を解消しうる。
本開示に係る医薬組成物及びワクチンは、一又は複数の実施形態において、ASPDに起因する疾病を罹患するおそれのある対象、罹患したおそれのある対象、又は、罹患した対象に投与されうる。本開示に係る医薬組成物は、一又は複数の実施形態において、アルツハイマー病及び/又はレビー小体型認知症を罹患するおそれのある対象、罹患したおそれのある対象、又は、罹患した対象に投与されうる。該対象としては、哺乳類、ヒト、ヒト以外の哺乳類が挙げられる。
本開示は、その他の態様において、ASPD、ASPDに起因する疾病、又は、アルツハイマー病及び/又はレビー小体型認知症に対する免疫を対象に獲得させる方法であって、細胞分泌型ASPD様構造体又は本開示に係る医薬組成物若しくはワクチンを対象に投与することを含む方法に関する。
本開示は、その他の態様において、細胞分泌型ASPD様構造体を有効成分とする医薬組成物、又は、細胞分泌型ASPD様構造体を有するワクチンの製造方法であって、本開示に係る製造方法により細胞分泌型ASPD様構造体を製造する工程を含む製造方法に関する。
本開示は、その他の態様において、APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を有する非ヒト動物に関する。前記細胞は、細胞分泌型ASPD様構造体を分泌するから、前記非ヒト動物は、ASPDに起因する疾病、例えば、アルツハイマー病及び/又はレビー小体型認知症のモデル動物となり得る。前記細胞としては、該モデル動物とする観点から、脳の細胞が好ましく、或いは、神経細胞が好ましい。前記発現系の導入方法は、特に制限されないが、遺伝子導入技術を用いる方法、ゲノム編集技術を用いる方法などが挙げられる。非ヒト動物としては、ヒト以外の哺乳類、ヒト以外の霊長類が挙げられる。本態様の一又は複数の実施形態は、APP又はAβ配列を含むその一部を発現するように遺伝子改変された細胞を有する遺伝子改変動物である。
本態様のモデル動物において、遺伝子改変された細胞内で発現されるAPPは、例えば、本開示で開示されるような、APPの変異体又はAβの変異体であってもよい。すなわち、前記細胞で発現されるAPPは、上述と同様に、Aβの25番目、29番目、33番目、及び37番目に相当するグリシンからなる群から選択される1又は複数のグリシンの置換変異を有することが好ましく、少なくとも33番目のグリシンの置換変異を有することが好ましい。置換変異としては、ロイシン、イソロイシン、フェニルアラニン、バリン、メチオニン、チロシン、又はシステインへの置換変異が好ましい。
その他の一又は複数の実施形態において、前記細胞で発現されるAPPは、Aβの33番目及び37番目のグリシンの置換変異を有することが好ましい。本実施形態において、置換変異としては、ロイシン、イソロイシン、フェニルアラニン、バリン、メチオニン、チロシン、又はシステインへの置換変異が好ましく、33番目及び37番目の少なくとも一方が、イソロイシンへの置換変異であることがより好ましい。
細胞分泌型ASPD様構造体は、一又は複数の実施形態において、ASPD測定におけるASPDの標準物質として使用できる。その他の一又は複数の実施形態において、細胞分泌型ASPD様構造体は、ASPDを測定するキットに使用できる。すなわち、本開示は、一態様において、細胞分泌型ASPD様構造体と抗ASPD抗体を含むキットに関する。抗ASPD抗体としては、上述のASPDに特異的な抗体が挙げられる。また、本開示は、一態様において、細胞分泌型ASPD様構造体と、抗ASPD抗体とを組み合わせることを含む、キットの製造方法に関する。これらのキットは、例えば、ASPDの測定に使用できる。
本開示は、一態様において、ASPDの形成に影響を与える物質のスクリーニング方法であって、APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を培養した培養液中に形成される、ASPDに特異的な抗体に対して抗原性を示す構造体(すなわち、細胞分泌型ASPD様構造体)の形成効率を指標とすることを含むスクリーニング方法に関する。
前記指標は、一又は複数の実施形態において、細胞分泌型ASPD様構造体の形成量であってもよく、Aβ40、Aβ41、Aβ42、Aβ43又はこれらの合計量に対する相対量であってもよい。この形態のスクリーニング方法は、テスト物質を培養液に添加した場合と添加しない場合とにおける細胞分泌型ASPD様構造体の形成効率を比較して、ASPDの形成に影響を与える物質であるか否かを判断すること含んでもよい。例えば、テスト物質を培養液に添加した場合に該形成効率が低下すれば、該物質がASPDの形成を阻害する影響があると判断できる。また、例えば、テスト物質を培養液に添加した場合に該形成効率が亢進すれば、該物質がASPDの形成を促進する影響があると判断できる。
本態様のスクリーニング方法において、培養する細胞内で発現されるAPPは、例えば、本開示で開示されるような、APPの変異体又はAβの変異体であってもよい。
本開示は、一態様において、AβのC末端切断に影響を与える物質のスクリーニング方法であって、APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を培養した培養液中に形成される、Aβ40、Aβ41、Aβ42、Aβ43及びこれらの組合せからなる群から選択される少なくとも1つのAβを指標とすることを含むスクリーニング方法に関する。
前記指標は、一又は複数の実施形態において、Aβ40、Aβ41、Aβ42、Aβ43及びこれらの組合せの分泌量若しくは培養液中の濃度であってもよく、或いは、例えば、Aβ40に対するAβ42の割合(Aβ42/Aβ40)といった相対量であってもよい。この形態のスクリーニング方法は、テスト物質を培養液に添加した場合と添加しない場合とにおける前記指標を比較して、AβのC末端切断に影響を与える物質であるか否か、或いは、γセクレターゼに対する影響を与える物質であるか否かを判断すること含んでもよい。例えば、テスト物質を培養液に添加した場合に「Aβ42/Aβ40」が低下すれば、該物質がAβ42の産生を抑制する影響があると判断できる。
本態様のスクリーニング方法において、培養する細胞内で発現されるAPPは、例えば、本開示で開示されるような、APPの変異体又はAβの変異体であってもよい。
本開示は、さらにその他の態様において、変異型合成ASPDに関する。本開示において、変異型合成ASPDは、変異型のAβを用いて、合成ASPDの製造方法と同様の製法で合成される合成ASPD様構造体をいう。すなわち、本開示において、変異型合成ASPDは、Aβの25~37番目のアミノ酸配列におけるGXXXGモチーフのグリシンに1又は複数の置換変異を有する変異型Aβを含む液体を撹拌することで形成される合成ASPD様構造体をいう。前記液体には、変異型合成ASPDの形成効率向上の観点から、例えば、フタル酸エステルなどの可塑剤が含まれていてもよい。一又は複数の実施形態において、変異型合成ASPDは、前記変異型Aβを、前記可塑剤を含む有機溶媒に溶解すること、前記変異型Aβの溶液を水性溶液で希釈すること、及び、前記希釈後の溶液を撹拌することを含む製造方法で合成できる。
前記変異型Aβとしては、Aβの25番目、29番目、33番目、及び37番目に相当するグリシンからなる群から選択される1又は複数のグリシンの置換変異を有することが好ましく、少なくとも33番目のグリシンの置換変異を有することが好ましい。置換変異としては、合成ASPDの形成効率向上の観点から、ロイシン、イソロイシン、フェニルアラニン、バリン、メチオニン、チロシン、システインへの置換変異が好ましい。
前記変異型Aβとしては、その他の一又は複数の実施形態において、Aβの33番目及び37番目のグリシンの置換変異を有することが好ましい。本実施形態において、置換変異としては、合成ASPDの形成効率向上の観点から、ロイシン、イソロイシン、フェニルアラニン、バリン、メチオニン、チロシン、又はシステインへの置換変異が好ましく、33番目及び37番目の少なくとも一方が、イソロイシンへの置換変異であることがより好ましい。
本開示に係る変異型ASPDは、一又は複数の実施形態において、ASPD特異的抗体に対して抗原性を示す。また、本開示に係る変異型ASPDは、その他の一又は複数の実施形態において、成熟神経細胞に対して細胞毒性を示す。
[1] 細胞分泌型アミロスフェロイド(ASPD)様構造体の製造方法であって、アミロイド前駆体タンパク質(APP)又はアミロイドβタンパク質(Aβ)配列を含むその一部を発現する細胞を培養液中で培養し、該培養液中に細胞分泌型ASPD様構造体を得る工程を含む、製造方法。
[2] さらに、前記培養液中から細胞分泌型ASPD様構造体を回収することを含む、[1]記載の製造方法。
[3] 前記細胞が、APP又はAβ配列を含むその一部の発現系が導入された細胞である、[1]又は[2]に記載の製造方法。
[4] 前記APPは、野生型、又は、1若しくは複数のアミノ酸変異を有する前記野生型の変異体である、[1]から[3]のいずれかに記載の製造方法。
[5] 前記APPは、Aβアミノ酸配列のGXXXGモチーフにおけるグリシンにアミノ酸変異を有する野生型又は変異体のAPPである、[1]から[4]のいずれかに記載の製造方法。
[6] APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を培養した培養液中に得られる構造体であって、ASPDに特異的な抗体に対して抗原性を示す、細胞分泌型ASPD様構造体。
[7] [6]に記載の細胞分泌型ASPD様構造体を有効成分とする医薬組成物。
[8] [6]に記載の細胞分泌型ASPD様構造体を含むワクチン。
[9] [6]に記載の細胞分泌型ASPD様構造体、及び、抗ASPD抗体を含むキット。
[10] [6]に記載の細胞分泌型ASPD様構造体の能動ワクチンとしての使用。
[11] ワクチン製造における[6]に記載の細胞分泌型ASPD様構造体の使用。
[12] [6]に記載の細胞分泌型ASPD様構造体の、ASPD測定における標準物質としての使用。
[13] ASPDが起因となる疾病の予防、改善、及び/又は治療の方法であって、[6]に記載の細胞分泌型ASPD様構造体、[7]に記載の医薬組成物、又は、[8]に記載のワクチンを対象に投与することを含む、方法。
[14] ASPDが起因となる疾病が、アルツハイマー病及び/又はレビー小体型認知症である、[13]に記載の方法。
[15] ASPD、ASPDに起因する疾病、又は、アルツハイマー病及び/又はレビー小体型認知症に対する免疫を対象に獲得させる方法であって、[6]に記載の細胞分泌型ASPD様構造体、[7]に記載の医薬組成物、又は、[8]に記載のワクチンを対象に投与することを含む、方法。
[16] [6]に記載の細胞分泌型ASPD様構造体と、薬学的に許容される腑形剤とを組み合わせることを含む、医薬組成物又はワクチンの製造方法。
[17] [6]に記載の細胞分泌型ASPD様構造体と、抗ASPD抗体とを組み合わせることを含む、キットの製造方法。
[18] APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を有する、非ヒト動物。
[19] APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を培養した培養液中に形成される構造体であって、ASPDに特異的な抗体に対して抗原性を示す構造体の形成効率を指標とすることを含む、ASPDの形成に影響を与える物質のスクリーニング方法。
[20] APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を培養した培養液中に分泌される、Aβ40、Aβ41、Aβ42、Aβ43及びこれらの組合せからなる群から選択される少なくとも1つを指標とする、AβのC末端切断に影響を与える物質のスクリーニング方法。
[21] Aβの25~37番目のアミノ酸配列におけるGXXXGモチーフのグリシンに1又は複数の置換変異を有する変異型Aβを含む液体を撹拌することで形成される合成ASPD様構造体。
(1-1)ヒトアミロイド前駆体タンパク質(hAPP)
発現させるアミロイド前駆体タンパク質(APP)として、下記表1及び図1に示す試験例1~14のAPPを用いた。図1の配列(配列番号1)は、ヒトのAβを含むhAPPの部分アミノ酸配列である。
試験例1~7のAPPは、hAPP770野生型、hAPP770Swedish変異型、及びGXXXGモチーフの1又は2個のグリシンが変異したhAPP770Swedish型である。
試験例8~14のAPPは、hAPP695野生型、hAPP695Swedish変異型、及びGXXXGモチーフの1又は2個のグリシンが変異したhAPP695Swedish型である。
上記試験例1~14のhAPPを過剰発現するCHO細胞を以下のように作製した。
CHO/K1細胞を増殖培地にて2.5x104cells/cm2の密度で12ウェルプレートに播種し、37℃、5%CO2で維持した。24時間後、遺伝子導入試薬(FuGENE HD)を用いて発現ベクターをCHO/K1細胞に導入した。導入方法は、1ウェルあたりOpti-MEM 25μL及びプラスミドDNA 0.5μgを混合し、さらに1.5μLのFuGENE HDを加え、室温で10分間反応させた。その間に0.5mL増殖培地を交換しておき反応終了後、反応液を25μL添加した。24時間後、新しい1mL増殖培地に交換してさらに培養した。
増殖培地:10%FBS含有Ham’s F-12培地に、100ユニット/mL penicillin、及び、100μg/mL streptomycinを加えたもの
(2-1)hAPPを過剰発現する培養細胞の培養と上清の回収
遺伝子導入から48時間後、細胞を1xPBS(-)にて2回洗浄したのち1mL無血清培地に交換した。無血清培地に交換24時間後、0.22μmフィルターでろ過した培養上清を回収し、直ちに液体窒素で凍結させて-80℃にて保存した。
無血清培地:DMEM/F-12に1xITS-Xを加えたもの
APP過剰発現細胞を培養した培養液を回収し、培養液中のASPD,Aβ1-40、及びAβ1-42の含有量を下記条件のELISA法で確認した。その結果を図2~4に示す。
[ASPD ELISA]
回収した培養上清について、抗ASPD抗体2種類(rpASD1、MASD3)を用いたサンドイッチELISAで定量した。
詳細には、測定前日に白色96ウェルプレートに抗ASPDポリクローナル抗体(rpASD1)を500ng/ウェルとなるように分注し、4℃で一晩固相化した。翌日、ウェルをPBS-Tで3回洗浄した後、3%BSAにて30分間ブロッキングを行い、再度3回洗浄した。各ウェルに100μLの標準液またはサンプルを分注し、1時間室温でインキュベーション後、3回洗浄し、100ng/ウェルとなるように抗ASPDモノクローナル抗体(MASD3)を添加し、さらに1時間室温でインキュベーションした。再度3回洗浄を行い、1/10,000希釈した二次抗体(抗マウスIgG-HRP)を100μL/ウェルで分注し、1時間室温でインキュベーションした。3回洗浄した後、100μLの発光基質を添加して1分遮光で反応させ、ルミノメーターで発光を検出した。
[Aβ ELISA]
回収した培養上清について、市販で入手可能なAβ monomer ELISA kitを用いて測定した。使用したKitは、以下の通り。測定は、Kit添付の説明書通り行った。
Aβ1-40:ヒトβアミロイド(1-40)ELISAキットワコー(Wako#292-62301)
Aβ1-42:ヒトβアミロイド(1-42)ELISAキットワコー高感度品(Wako#296-64401)
hAPP770とhAPP695を比較した場合、変異による大きな差は見られなかったが、若干hAPP695の方が、培養上清中のASPD濃度が高い傾向があった。また、変異の場所と数については、2カ所でも1カ所でもG704又はG629のG/L変異がASPD形成には重要であると考えられた。さらに、野生型(wt)に比べ、Swedish(sw)変異ではアミロイドがより多く分泌されるが、さらにGXXXGモチーフに変異を入れることによって、ほとんどのアミロイドがASPDの構成要素として使用されることが示唆された。
hAPP695Swedish変異型の629位(Aβの33位)のグリシンを19種のアミノ酸に変異させたAPPを過剰発現する細胞を上記(1-2)と同様に作製し、上記2.と同様に培養してその培養液中に培養液中のASPD,Aβ1-40、及びAβ1-42の含有量を確認した。その結果を表2及び図5~7に示す。
CHOの他に、HEK293及びNeuro2aの細胞株においても、APP又はAβ配列を含むその一部の発現系が導入された細胞とすることで、細胞内でASPD様構造体が形成されることが認められた。
試験例14のhAPP695-sw-G33Iを発現させて得られた細胞分泌型ASPD構造体を0ヶ月、3ヶ月及び6ヶ月間-80℃保存したサンプルを、2種類のASPD特異的抗体(中和抗体)を用いたサンドイッチELISAで測定して検量線を作成した。
その結果、その検量線の一次回帰直線の傾きは、3つのサンプルでほぼ一定の値を示した。したがって、サンドイッチELISAに使用している中和抗体2種類に対する反応性が安定しており、保存による劣化はないと考えられた。
一方、従来の方法で製造された合成ASPDは、-80℃の保存後では一次回帰直線における傾きのバラツキが大きく、ASPD濃度に対しるシグナルの大きさもバラツキが大きく、保存安定性が低いと考えられる。
ニュージーランドホワイト(各条件3羽)に、フロイントアジュバント、水酸化アルミニウムアジュバント、アジュバントなしで、合成ASPDによる100μg免疫を6回行い、その後、全採血を行い、血清中の合成ASPDに対する反応性をドットブロットで評価を行った。その結果の一例を図8に示す。全てのウサギでアジュバントなしでも充分な反応性が得られることがわかった。
野生型Aβ1-42を用いて製造した合成ASPDの神経細胞毒性と、G33L変異型Aβ1-42を用いて製造した変異型合成ASPD(G33L)の神経細胞毒性とを比較した。
具体的には、一定濃度の合成ASPD(0.58μM)及び変異型合成ASPD(0.53μM)をそれぞれ成熟ラット海馬由来初代培養神経細胞に投与し一晩置いた後、蛍光試薬(Thermo Fisher社製CyQUANT)を添加し、共焦点イメージサイトメーターCQ1を用いて細胞生存率を確認した。その結果を図9の右に示す。同図に示す通り、変異型合成ASPD(G33L型)は、合成ASPD(野生型)と同様に、成熟神経細胞に対して同程度の細胞毒性を示した。
合成ASPDまたは変異型合成ASPD(G33L)をリガンドとしてセンサーチップ上に固相化し、アナライトとして抗ASPDポリクローナル抗体(rpASD1)を流して表面プラズモン共鳴(SPR)により解離定数(KD)を求めた。その結果、抗ASPDポリクローナル抗体(rpASD1)に対する反応性は、合成ASPDと変異型合成ASPD(G33L)とで同等であった。
発現させるアミロイド前駆体タンパク質(APP)として、下記表3及び図1に示す試験例15~36のAPPを用いた。上記1及び2の記載と同様にして、APP過剰発現細胞を培養した培養液を回収し、培養液中のASPD,Aβ1-40、及びAβ1-42の含有量を下記条件のELISA法で確認した。その結果を図10~12に示す。
ロイシンへの1アミノ酸又は2アミノ酸置換変異と、イソロイシンのそれらとで、上清中のASPD,Aβ1-40、及びAβ1-42の含有量に大きな差は見られなかった。しかし、Aβの33番目及び37番目のグリシンが二つともイソロイシンに変異したAPP(試験例30)のASPD形成量は、Aβの33番目及び37番目のグリシンが二つともロイシンに変異したAPP(試験例23)と比べて、著しく増加していた。
また、2か所をロイシンとイソロイシンに変異させたAPP(試験例31-36)では、Aβの33番目及び37番目のグリシンをロイシンとイソロイシンに変異させたAPP(試験例35-36)のASPDの形成量が、Aβの33番目及び37番目のグリシンが二つともイソロイシンに変異したAPP(試験例30)と同程度に増加した。
Claims (21)
- 細胞分泌型アミロスフェロイド(ASPD)様構造体の製造方法であって、
アミロイド前駆体タンパク質(APP)又はアミロイドβタンパク質(Aβ)配列を含むその一部を発現する細胞を培養液中で培養し、該培養液中に細胞分泌型ASPD様構造体を得る工程を含む、製造方法。 - さらに、前記培養液中から細胞分泌型ASPD様構造体を回収することを含む、請求項1記載の製造方法。
- 前記細胞が、APP又はAβ配列を含むその一部の発現系が導入された細胞である、請求項1又は2に記載の製造方法。
- 前記APPは、野生型、又は、1若しくは複数のアミノ酸変異を有する前記野生型の変異体である、請求項1から3のいずれかに記載の製造方法。
- 前記APPは、Aβアミノ酸配列のGXXXGモチーフにおけるグリシンにアミノ酸変異を有する変異体のAPPである、請求項1から4のいずれかに記載の製造方法。
- APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を培養した培養液中に得られる構造体であって、ASPDに特異的な抗体に対して抗原性を示す、細胞分泌型ASPD様構造体。
- 請求項6に記載の細胞分泌型ASPD様構造体を有効成分とする医薬組成物。
- 請求項6に記載の細胞分泌型ASPD様構造体を含むワクチン。
- 請求項6に記載の細胞分泌型ASPD様構造体、及び、抗ASPD抗体を含むキット。
- 請求項6に記載の細胞分泌型ASPD様構造体の能動ワクチンとしての使用。
- ワクチン製造における請求項6に記載の細胞分泌型ASPD様構造体の使用。
- 請求項6に記載の細胞分泌型ASPD様構造体の、ASPD測定における標準物質としての使用。
- ASPDが起因となる疾病の予防、改善、及び/又は治療の方法であって、請求項6に記載の細胞分泌型ASPD様構造体、請求項7に記載の医薬組成物、又は、請求項8に記載のワクチンを対象に投与することを含む、方法。
- ASPDが起因となる疾病が、アルツハイマー病及び/又はレビー小体型認知症である
、請求項13に記載の方法。 - ASPD、ASPDに起因する疾病、又は、アルツハイマー病及び/又はレビー小体型
認知症に対する免疫を対象に獲得させる方法であって、請求項6に記載の細胞分泌型ASPD様構造体、請求項7に記載の医薬組成物、又は、請求項8に記載のワクチンを対象に投与することを含む、方法。 - 請求項6に記載の細胞分泌型ASPD様構造体と、薬学的に許容される腑形剤とを組み合わせることを含む、医薬組成物又はワクチンの製造方法。
- 請求項6に記載の細胞分泌型ASPD様構造体と、抗ASPD抗体とを組み合わせることを含む、キットの製造方法。
- APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を有する、非ヒト動物。
- APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を培養した培養液中に形成される構造体であって、ASPD特異的抗体に対して抗原性を示す構造体の形成効率を指標とすることを含む、ASPDの形成に影響を与える物質のスクリーニング方法。
- APP又はAβ配列を含むその一部を発現する発現系が導入された細胞を培養した培養液中に分泌される、Aβ40、Aβ41、Aβ42、Aβ43及びこれらの組合せからなる群から選択される少なくとも1つを指標とする、AβのC末端切断に影響を与える物質のスクリーニング方法。
- Aβの25~37番目のアミノ酸配列におけるGXXXGモチーフのグリシンに1又は複数の置換変異を有する変異型Aβを含む液体を撹拌することで形成される合成ASPD様構造体。
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CN201780023469.9A CN109072274A (zh) | 2016-04-14 | 2017-04-13 | 淀粉样蛋白球体(aspd)样结构体及医药组合物 |
EP17782459.6A EP3444344A4 (en) | 2016-04-14 | 2017-04-13 | AMYLOSPHEROID (ASPD) -LIKE STRUCTURE AND PHARMACEUTICAL COMPOSITION |
KR1020187032344A KR20180134373A (ko) | 2016-04-14 | 2017-04-13 | 아밀로스페로이드(aspd)형 구조체 및 의약 조성물 |
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