EP0493470A1 - Techniques d'analyse et reactifs servant a detecter les depots d'amyloide - Google Patents

Techniques d'analyse et reactifs servant a detecter les depots d'amyloide

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Publication number
EP0493470A1
EP0493470A1 EP90914284A EP90914284A EP0493470A1 EP 0493470 A1 EP0493470 A1 EP 0493470A1 EP 90914284 A EP90914284 A EP 90914284A EP 90914284 A EP90914284 A EP 90914284A EP 0493470 A1 EP0493470 A1 EP 0493470A1
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EP
European Patent Office
Prior art keywords
amyloid
preamyloid
val
protein
beta
Prior art date
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Ceased
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EP90914284A
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German (de)
English (en)
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EP0493470A4 (en
Inventor
Barbara Cordell
David Wolf
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Scios LLC
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Scios Nova Inc
California Biotechnology Inc
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Publication of EP0493470A1 publication Critical patent/EP0493470A1/fr
Publication of EP0493470A4 publication Critical patent/EP0493470A4/en
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to assays and re ⁇ agents useful for the chemical intervention of amyloidosis in Alzheimer's disease.
  • AD Alzheimer's disease
  • NFT neurofibrillary tangles
  • the major protein component of amyloid is an ⁇ 4 kilodalton (kd) protein, designated the beta-protein or A4 protein due to a partial beta pleated structure or its molecular weight, respectively.
  • the A4 protein accumulates extracellularly, both in brain parenchyma and in the walls of blood vessels, generally as amyloid plaques which form aggregate fibril structures and are insoluble on SDS-polyacrylamide gels.
  • the fibrils are generally identified as amyloid based on their green birefringence after staining with Congo red and their 40- to 90-A diameter.
  • the second protein mentioned previously, ac ⁇ cumulates intracellularly in neurons of Alzheimer's brains (Castano and Frangione, (1988) Lab Invest 58:122-132) and forms tangles composed of structures resembling paired helical filaments (PHFs).
  • PHFs paired helical filaments
  • the primary structure and number of proteins comprising PHFs are unknown.
  • PHF-containing neurites are found in the periphery of the plaque, whereas deposits of beta-amyloid protein form the central core of mature plaques, surrounded by degenerated neurites and glial cells.
  • the present invention provides a method for determining the ability of a potential therapeutic agent to intervene in the amyloid deposition process associated with Alzheimer's disease in a cellular environment, which method utilizes a recombinantly produced amyloid substrate in a screening assay.
  • the present invention also allows for the development and use of immunological reagents to detect the formation of preamyloid protein aggregation in the cell lines provided by the invention.
  • a method of screening agents capable of intervention in Alzheimer's disease amyloidosis comprises: a) culturing a cell line capable of expressing a gene encoding beta-amyloid protein under conditions suit- able to produce the beta-amyloid protein as an insoluble, preamyloid aggregate; b) combining a known quantity of the agent to be tested to the cell culture; and c) monitoring the combination to determine whether preamyloid aggregate formation is reduced.
  • preamyloid formation can be induced through infection of a cell line with a recombinant virus capable of expressing the beta-amyloid protein as an insoluble preamyloid aggregate.
  • recombinant viruses carry expression vectors comprising DNA encoding the beta-amyloid protein.
  • Immunoassay kits employing the reagents useful to screen potential amyloid intervening agents are also provided by the present invention.
  • FIG. 1 is a schematic illustration of two amyloid expression constructs employing the vaccinia pUVl insertion vector.
  • FIG 2. illustrates the results of immunoprecipitation of 35S-methionine labeled W:A99 infected CV-1 cell lysates using APCP antibodies.
  • FIG 3. are fluorescent photomicrographs of infected CV-1 cells stained with APCP antibodies.
  • FIG. 3A is a Mock control;
  • FIG. 3B is a W:CONT control;
  • FIG. 3C is the W:99 construct; and
  • FIG. 3D is the W:42 construct.
  • the magnification is 200x with a 0.4 second exposure time for each photo.
  • FIG. 4 is a illustration of the modified beta- actin expression selection vector, pAX-neo, that was employed to express the beta-amyloid core constructs in mammalian cells. Detailed Description of the Preferred Embodiments
  • the invention involves a method of screening agents capable of intervention in Alzheimer's disease amyloidosis.
  • beta-amyloid core protein or "A4 protein” refers to an approximately 4 kd protein first identified by Glenner and Wong, (1984) Biochem Biophvs Res Comm 120:885, which is defined at the amino terminus by sequence analysis as a mixture of four peptides with slightly different amino termini, the amino termini of the three smaller peptides being completely encoded by that of the largest.
  • beta-amyloid precursor protein refers to either the amyloid precursor protein of 695 amino acids (Kang et al., (1987) supra) or the 751 amino acid protein (Ponte et al., (1988) supra) containing within their sequence, the beta-amyloid core protein sequence defined above.
  • the A4 core protein begins at amino acid 597 of the 695 amino acid protein and at amino acid 653 of the 751 amino acid sequence.
  • preamyloid aggregation refers to a morphological description — first discovered by Tagliavini et al., (1988) Neurosci Lett 93:191-196 — of spherical, granular deposits which are considerably smaller than pre-plaques and plaques found at a high frequency in the brains of Alzheimer's victims. These deposits can be occasionally detected with silver stain but not with Congo red, a stain to which amyloid proteins demonstrate high binding affinity.
  • insertion vector includes plasmids, cosmids or phages capable of mediating homologous recombination into a viral genome such that the DNA encoding the beta-amyloid protein is stably carried by the resulting recombinant virus.
  • plasmids constructed from vaccinia virus DNA are employed.
  • expression vector includes plasmids, cosmids or phages capable of synthesizing a protein encoded by the respective recombinant gene carried by said vector. Such vectors are independently replicated in or capable of integration into the chromosome of an appropri ⁇ ate host cell for expression of the amyloid protein.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • the cell has been stably transformed when the cell is able to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a cell that is capable of stable growth in vitro for many generations.
  • Beta-Amyloid Coding Sequences The beta-amyloid genes may be synthetic or natural, or combinations thereof.
  • the gene encoding the natural 751 amino acid precursor protein is described in PCT WO88/03951, published 2 June 1988 and assigned to the same assignee of the present application, and the expres- sion of the protein in mammalian cells is provided in
  • Example 4 therein. The relevant portions of this publica ⁇ tion are specifically incorporated herein by reference.
  • the genes encode the A42 core protein or an amyloid protein, A99, which comprises the A42 core protein and the cytoplasmic domain.
  • A99 which comprises the A42 core protein and the cytoplasmic domain.
  • This latter protein consists of the 42 residue core protein and 57 amino acids of the cytoplasmic domain of the 751 precursor protein.
  • the sequence of A99 is as follows:
  • genes are provided for expression of the desired protein using recombinant DNA expression vectors.
  • these genes may be natural, synthetic or combinations thereof.
  • it may be desirable to modify the natural amyloid nucleic acid sequence. For example, it will often be preferred to use codons which are preferentially recognized by the desired host.
  • it may be desirable to further alter the nucleotide sequence, either synthetic or natural, to create or remove restriction sites to, for example, enhance insertion of the gene sequence into convenient expression vectors or to substitute one or more amino acids in the resulting polypeptide to increase stability.
  • a general method for site-specific mutagenesis is described in Noren et al., (1989) Science 244:182-188.
  • Peptides of this precursor protein are also provided herein for the generation of specific immunological re- agents and may also be synthetic or natural.
  • Synthetic oligonucleotides are prepared by either the phosphotriester method as described by Edge et al., (1981) Nature 292:756 and Duckworth et al., (1981) Nuc Acids Res ⁇ :1691 or the phosphoramidite method as described by Beaucage and Caruthers, (1981) Tet Lett 22:1859 and Matteucci and Caruthers, (1981) J Am Chem Soc 103:3185, and can be prepared using commercially available automated oligonucleotide synthesizers.
  • the coding sequences for the amyloid proteins can be inserted into vaccinia virus plasmid insertion vec ⁇ tors for the purpose of generating recombinant vaccinia viruses using the methods described in Moss et al., (1983) Methods in Gene Amplification, Vol. 3, Elsevier-North Hol ⁇ land, p. 202-213; and in Moss et al., (1984) J Virol 4jJ:857-864.
  • the amyloid-vaccinia recombinants can then be used for (1) expression of the respective amyloid protein and analysis of preamyloid formation, and (2) production of amyloid antibodies.
  • the two vaccinia virus insertion vectors pSCll (Chakrabarti et al., (1985) Mol Cell Biol 5:3403-3409 and pUVl (Falkner et al., (1987) Nuc Acids Res 15:7192) were used for the expression of the amyloid proteins and generation of amyloid-vaccinia recombinants. Both vectors are of the co-insertion variety and each contains two vaccinia virus promoters. One promoter (PI) is used to drive the expression of a selectable marker gene (in this case, beta-galactosidase) while the other promoter (P2) is used to drive expression of the heterologous amyloid DNA insert.
  • PI promoter
  • P2 is used to drive expression of the heterologous amyloid DNA insert.
  • the pSCll vector utilizes a vaccinia early-late promoter (P7.5) to drive heterologous gene expression and has a single S al cloning site.
  • the pUVl vector utilizes a vaccinia late promoter (Pll) to drive heterologous gene expression and is designed for the expression of fusion proteins behind the ATG of the Pll late gene. In all cases, amyloid-pUVl constructs were made using the most 5' (after the ATG) cloning site (EcoRI) in order to avoid introduction of additional amino terminal amino acids into the native amyloid protein sequence.
  • procaryotic and eucaryotic systems may be used to express the amyloid genes described herein.
  • Procaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used.
  • Plasmid vectors which contain replication sites, selectable markers and control sequences derived from a species compatible with the host are used; for example, E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species by Bolivar et al. , (1977) Gene 2:95.
  • pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides multiple selectable markers which can be either retained or destroyed in constructing the desired vector.
  • Commonly used procaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., (1977) Nature 198:1056), the tryptophan (trp) promoter system (Goeddel et al. , (1980) Nucleic Acids Res 8:4057), the lambda-derived P-. promoter (Shimatake et al. , (1981)
  • eucaryotic microbes such as yeast
  • yeast may also be used as hosts.
  • Laboratory strains of Saccharomyces cerevisiae, Baker's yeast are most used although a number of other strains or species are commonly available.
  • Vectors employing, for example, the 2 micron origin of replication of Broach, (1983) Meth Enz 101:307, or other yeast compatible origins of replica- tion (see, for example, Stinchcomb et al., (1979) Nature 282:39; Tschumper et al., (1980) Gene 10:157 and Clarke et al., (1983) Meth Enz 101:300) may be used.
  • Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess et al. , (1968) J Adv Enzyme Reg 7:149; Holland et al. , (1978) Biochemistry
  • promoters known in the art include the promoter for 3-phosphoglycerate kinase (Hitzeman et al., (1980) J Biol Chem 255:2073).
  • Other promoters which have the additional advantage of transcription controlled by growth conditions and/or genetic background are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, the alpha factor system and enzymes responsible for maltose and galactose utilization. It is also believed terminator sequences are desirable at the 3' end of the coding sequences. Such terminators are found in the 3' untranslated region fol ⁇ lowing the coding sequences in yeast-derived genes.
  • eucaryotic host cell cultures derived from multicellular organisms. See, for example, Axel et al. , U.S. Patent No. 4,399,216. These systems have the additional advantage of the ability to splice out introns and thus can be used directly to express genomic ragments.
  • Useful host cell lines include VERO, HeLa, baby hamster kidney (BHK), CV-1, COS, MDCK, NIH 3T3, L, and Chinese hamster ovary (CHO) cells. Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and late promoters from Simian Virus 40 (SV40) (Fiers et al., (1978) Nature
  • viral promoters such as those derived from polyoma, herpes virus, Adenovirus 2, bovine papilloma virus, or avian sarcoma viruses.
  • the controllable promoter, hMTII Kerin et al., (1987) Nature 299:797-802
  • hMTII Kerin et al., (1987) Nature 299:797-802
  • Insect expression systems may also be employed to express the amyloid genes.
  • the baculovirus polyhedrin gene has been employed for high- level expression of heterologous proteins (Smith et al., (1983) Mol Cell Biol 3(12) :2156-2165; Summers et al., "Genetic Engineering of the Genome of the Autographa Californica Nuclear Polyhedrosis Virus", Banbury Report: Genetically Altered Viruses in the Environment, 22:319- 339, Cold Spring Harbor Laboratory, 1985).
  • the amyloid DNA clones expressed in vaccinia can also be used to generate stably transfected cell lines expressing the amyloid proteins.
  • these cell lines are generated by first constructing one of two expression plasmids.
  • the selectable marker is provided by a G418 neomycin expres ⁇ sion cassette (neo) consisting of the SV40 early promoter, the bacterial kanamycin-resistance gene also containing its own promoter, the SV40 intervening sequence, and the SV40 polyadenylation site from the early region.
  • the amyloid DNA cloning site is flanked at the 5' end by the human metallothionein gene promoter, pMtlla, modified with an SV40 enhancer, and at the 3' end by the SV40 polyadenylation site from the early region.
  • the amyloid DNA cloning site is flanked at the 5' end by a beta-actin promoter, and at the 3' end by a sequence encoding a use ⁇ ful polyadenylation site, such as that of the SV40 early region or the beta-actin gene.
  • Each of the vectors described above can be transformed into a mammalian cell line such as, but not limited to, those described in the following examples by either calcium phosphate-DNA coprecipitation or electro- poration.
  • the cells are subjected to 1 mg/ml G418 to provide pools of G418-resistant colonies.
  • Suc ⁇ cessful transformants also having a stable inheritance of the DNA contained in the expression construct, are then plated at low density for purification of clonal isolates. Clonal isolates are then analyzed for maximum production of the amyloid protein of interest and high-producing clones are expanded to serve as stock.
  • the amyloid protein may need to be conjugated to a carrier protein such as bovine serum albumin or keyhole limpet hemacyanin using a chemical process which employs carbodiimide, glutaraldehyde, or other cross-linking agents.
  • a carrier protein such as bovine serum albumin or keyhole limpet hemacyanin
  • the protein may be administered without being conjugated to a carrier protein.
  • Vaccinia virus re ⁇ combinants which are expressing amyloid proteins may also be used to prepare antibodies. The vaccinia virus re ⁇ combinants are injected into an animal and then the animal is boosted several weeks after the initial immunization. Ten days to two weeks later the animals are bled and antiserum is collected and analyzed for titer.
  • Monoclonal antibodies are commonly prepared by fusing, under appropriate conditions, B-lymphocytes of an animal which is making polyclonal antibodies with an im ⁇ mortalizing myeloma cell line.
  • the B-lymphocytes can be spleen cells or peripheral blood lymphocytes.
  • Techniques for fusion are also well known in the art, and in general, involve mixing the cells with a fusing agent such as poly ⁇ ethylene glycol.
  • Successful hybrid ⁇ ma formation is as ⁇ sessed and selected by standard procedures such as, for example, HAT medium. From among successful hybridomas, those secreting the desired antibody are screened by as ⁇ saying the culture medium for their presence.
  • Standard immunological techniques such as ELISA (enzyme-linked immun ⁇ assay) , RIA (radioimmunoassay) , IFA (immunofluorescence assay) and Western blot analysis, which are well known in the art, can be employed for diagnostic screening for amyloid expression.
  • ELISA enzyme-linked immun ⁇ assay
  • RIA radioimmunoassay
  • IFA immunofluorescence assay
  • Western blot analysis which are well known in the art, can be employed for diagnostic screening for amyloid expression.
  • One typical arrangement utilizes competition, between labeled antigen (e.g. amyloid protein) and the analyte, for the antibody, followed by physical separation of bound and unbound fractions.
  • labeled antigen e.g. amyloid protein
  • Analyte competes for the binding of the labeled antigen; hence more label will remain in the unbound fraction when larger amounts of analyte are present.
  • this competitive-binding type assay the sample is incubated with a known titer of labeled amyloid protein and amyloid protein antibody.
  • Antibody-protein complex is then separated from uncomplexed reagents using known techniques and the amount of label in the co plexed material is measured, e.g.
  • the amount of amyloid protein in the sample is determined by comparing the measured amount of label with a standard curve.
  • the present assay provides one of the first steps in addressing the question whether preamyloid corti- cal deposits herald organic dementia.
  • Down's syndrome is the one known disease closely related to the proposed etiology of AD.
  • NFTs congophilic angiopathy
  • senile plaques As reported in Giacione et al.
  • the expression of the amyloid protein is initiated by culturing the transformed cell line under conditions which are suitable for cell growth and expression of the amyloid protein.
  • high level expression of the protein is preferred.
  • a CHO cell line transformed with a beta-actin vector comprising the DNA encoding the A42 or A99 amyloid protein is grown in a mammalian culture medium such as, for example, a 1:1 mixture of F12 medium and DME medium with 10% fetal calf serum for 5-72 hr at 37°C.
  • Transfected viral monolayers are selected and plaque purified, and stocks of amyloid-vaccinia recombinant viruses are prepared.
  • preamyloid aggregates can be monitored by standard immunocytochemical methods using, for example, beta-amyloid primary antibodies which are detected using a secondary, labeled anti-antibody. If one is interested in testing whether the compound of interest can inhibit preamyloid formation, the compound is introduced to the culture medium before monitoring for preamyloid aggregation. Alternatively, the compound is introduced to the culture medium after preamyloid forma ⁇ tion has been established and this reaction mixture is monitored to see whether the compound induces amyloid resorption.
  • Potential therapeutic compounds for use in the present invention include, for example, amyloid-fibril denaturing agents such as dimethyl sulfoxide, and cytotoxic agents such as colchicine and chlorambucil.
  • amyloid-fibril denaturing agents such as dimethyl sulfoxide
  • cytotoxic agents such as colchicine and chlorambucil.
  • the efficacy of these agents may be monitored through observa ⁇ tion of reduced antibody binding to the amyloid deposit. Reduction in such binding is indicative of reduced preamyloid deposition.
  • preamyloid formation in the host cell may trigger other cellular events which could be employed as markers unrelated to the etiology of Alzheimer's disease, but correlative with the presence of preamyloid deposits.
  • an increase in the level of certain enzymes, specifically proteases may be measured in lieu of the preamyloid deposition.
  • an increase in the concentration levels of these enzymes is observed when cultured cells are subjected to stress.
  • kits suitable for the above diagnostic or screening methods contain the appropriate reagents and are constructed by packaging the appropriate materials, including the preamyloid protein aggregates immobilized on a solid support with labeled antibodies in suitable containers, along with any other reagents (e.g., wash solutions, enzyme substrate, anti-amyloid antibodies) or other materials required for the conduct of the assay.
  • the reagents are usually premeasured for ease of use.
  • An optional component of the kit is a set of instructions describing any of the available immunoassay methods.
  • a kit for a direct assay can comprise preamyloid proteins aggregates immobilized on a solid immunoassay support and a container comprising labeled antibody to the amyloid protein, as well as the other reagents mentioned above.
  • the following examples describe the expression vectors containing the 42 amino acid plaque core region (A42), and the 42 amino acid plaque core region including the 57 amino acid adjacent carboxy-terminal region of the beta-amyloid precursor protein (A99).
  • Alternative constructs for the A42 and A99 constructs were prepared which included a 17 amino acid amyloid signal sequence. As these constructs did not express the amyloid protein well, further experimentation with these vectors was not performed.
  • FIG. 1 il ⁇ lustrates the various amyloid expression constructs, all of which were modified at the 5' end to satisfy the clon ⁇ ing constraints of the vaccinia Pll promoter in the pUVl vector. Specifics for each construct are as follows:
  • the A42-encoding sequence (nucleotides 2080 to 2205, numbered in accordance with the 751 amyloid pre- cursor sequence) was synthesized as a 145 basepair (bp) EcoRI-BamHI oligomer, provided below, containing the ap ⁇ intestinalte TGA stop codon and an amino-terminal Asn-Ser adaptor sequence:
  • the synthetic fragment was ligated to EcoRI- and BamHI-digested pGeml (Promega-Biotec) , deriving pGemA42.
  • the EcoRI-BamHI fragment of pGemA42 was subsequently isolated and ligated into the EcoRI-BamHI site of pUVl deriving pUVl:A42.
  • the 590 bp Ddel-PvuII fragment of plasmid 4T4B was isolated from the carboxy-terminal 1 kilobase (kb) EcoRI fragment of 4T4B and ligated with a 27 bp EcoRI-Ddel adaptor sequence and cloned into the EcoRI- and Smal- digested pUVl, deriving pUVl:A99.
  • the 761 bp Xbal-Sall fragment of pUVl:A99 was further subcloned into the Xbal-Sall vector fragment of mpl8 and pGem2. Sequence data confirmed the predicted sequence.
  • the vaccinia insertion vectors described in Example 1 were used to generate amyloid-vaccinia re- combinant viruses as follows.
  • Confluent monolayers of CV-1 cells in 60 mm dishes were infected with vaccinia virus (Wyeth strain) at a multiplicity of infection (moi) of 0.05 pfu/cell.
  • vaccinia virus Wild-type vaccinia virus DNA
  • m multiplicity of infection
  • the cells were transfected with a calcium phosphate precipitate of 10 ug insertion plasmid DNA and 0.5 ug wild-type vaccinia virus DNA.
  • Cells were fed with complete medium and incubated at 37°C for two days.
  • Monolayers were collected and TK- vaccinia viruses were selected on TK-143 cells in the presence of 5- bromodeoxyuridine (BudR) at 25 ug/ l.
  • BudR 5- bromodeoxyuridine
  • the beta-amyloid antibodies were generated from synthetic peptides.
  • the synthetic peptides were prepared using solid phase synthesis according to standard protocols. Purification of the crude peptides was ac ⁇ complished by desalting with gel filtration followed by ion-exchange chromatography and preparative reverse-phase liquid chromatography. Each peptide was fully character ⁇ ized by amino acid composition and sequence analysis. COOH-CORE corresponds to amino acids 653-
  • C00H-B2 and C00H-C2 correspond to amino acids 736-751(NGYENPTYKFFEQMQN)
  • C00H-B3 and C00H-C3 correspond to amino acids 705-719(KKKQYTSIHHGWEV)
  • C00H-C5 corresponds to amino acids 729- 742(HLSKMQQNGYENPT) .
  • Antibody titers against the appropriate peptide were determined by enzyme-linked immunosorbent assays coupled with horseradish peroxidase and found to be 7.4xl0 4 , 2.7xl0 5 , IxlO 5 , 9.1xl0 6 , 8.2xl0 5 , and 2.5xl0 5 for COOH-CORE, C00H-B2, C00H-C2, C00H-B3, C00H-C3, and C00H- C5, respectively.
  • Antibodies to 9523 correspond to amino acids 673-685(AEDVGSKNGAIIG) and 9524 correspond to amino acids 701-712(LVMLKKKQYTSI) .
  • Antibodies to these two peptides were generated by coinjecting New Zealand white rabbits each with 200 ug methylated bovine serum albumin (PBS) plus 200 ug of the respective synthetic peptide in PBS. Rabbits were boosted one, two and three weeks following primary inoculation with identical amounts of peptide.
  • PBS methylated bovine serum albumin
  • CV-1 cells were infected with W:99 at a multiplicity of infection of one.
  • 35S-methionine 250 uCi/ml was added at 20 hr post infection for 4 hr.
  • the expression product of W:A99 demonstrated high level expression of the 99 amino acid core protein and showed evidence of self-aggregation as well as ag ⁇ gregation with other proteins or self-aggregation combined with proteolysis since multi ers of A99 did not always occur in integers of 11.5-17 kd.
  • SK-N-MC ATCC # HTB10
  • IMR-32 ATCC # CCL127
  • the culture medium for each host was as follows:
  • CV-1 The medium was Eagle MEM supplemented with 10% FBS, penicillin, streptomycin and L- Gin-
  • SK-N-MC Eagle MEM supplemented with 10% FBS, non- essential amino acids, penicillin, streptomycin and L-Gln.
  • PC-12 DMEM21, 5% DHS, 5% DFBS and L-Gln;
  • IMR-32 Eagle MEM (Hank's BSS) and 10% deltaFBS plus nonessential amino acids, penicillin, streptomycin and L-Gln.
  • Each cell line was grown to confluency on a microscope slide divided into 4 individual chambers (Lab Tech) .
  • One chamber was mock infected, the second infected with a control recombinant virus lacking A4 sequences
  • FIG. 3 shows fluorescent photomicrographs of CV-
  • a number of constructs expressing the beta- amyloid core protein were constructed using a derivative of the beta-actin expression/selection vector designated pHbetaAPr-1-neo.
  • This vector illustrated in FIG. 4, is a combination of the following elements: a) bp 1-4300 is the 4.3 kb EcoRI-AluI fragment from the human beta-actin gene isolate pl4Tbeta-17 (Leavitt et al. , (1984) Mol Cell Biol 4:1961-1969). For sequencing details of the promoter see Ng et al. , (1985) Mol Cell Biol 5:2720-2732. The cap site, 5' untranslated region and IVS 1 positions are indicated in FIG. 4.
  • bp 4300-4320 is in part derived from pSP64 polylinker (Melton et al., (1984) Nuc Acids Res 12:7035- 7056); c) bp 4320-6600 is derived from pcDVl (Okayama & Berg, (1983) Mol Cell Biol 3:280-289); and d) bp 6600-10000 is the PvuII-EcoRI fragment from pSV-neo (Southern & Berg, (1982) J Mol App Genet 1:327-341) containing the bacterial neo ycin gene linked to the SV40 origin plus early promoter.
  • Beta-actin A42 was constructed by excising the EcoRI-BamHI 145 bp fragment from pGEM-A42, adding a Sall- EcoRI adaptor sequence (5'-TCG ACA TGG ATG CAC AAT TA-3') and cloning into the pAX-neo expression vector at the Sall-and BamHI sites.
  • the beta-actin A99 plasmid was constructed by excising the 670 bp EcoRI-Hindlll fragment of pGEM_-A99, adding the above-described Sall-EcoRI adap ⁇ tor sequence and cloning into the pAX-neo vector at the Sail and HindiII sites.
  • Each construct was introduced into CHO cells by the calcium phosphate precipitation method using 7 ug of each DNA per 10 cells, and a resistant population was selected with G418-neomycin. The efficiency of
  • transfection for the A99 or A42 constructs was over 10 for 10 cells and pools of cells transfected with either beta-actin A99 or with beta-actin A42 were selected using G418-neomycin resistance (500 ug/ml).
  • Cells infected with W:99 or W:42 which are capable of forming amyloid deposits are plated in a 96- well microtiter plate.
  • an automated pipetter is used to introduce the drug to be tested to the cells.
  • a range of concentrations of the drug is incubated in a tissue culture incubator (or preincubated) with the cells at 37 C for a predetermined time period, or alternatively, for 3 to 72 hours.
  • the culture media is removed, and the cells are prepared for preamyloid measurement as follows.
  • the cells are fixed for immunocytochemical staining with amyloid antibodies.
  • the primary antibodies are introduced followed by incubation with labeled, secondary anti-antibodies and the level of binding between the primary and secondary antibodies is measured using an ELISA plate reader to record the optical density of the labeled antibody.
  • a smaller optical density reading as compared to a control sample of cells grown in the absence of the test drug is indicative of that drug's ability to inhibit amyloid deposition.
  • This procedure may be modified to permit detection of preamyloid dissolution using a correlative enzyme marker.

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Abstract

Technique d'analyse à base de culture tissulaire in vitro servant à détecter les dépôts d'amyloïde spécifiques à la maladie d'Alzheimer, adaptée pour procéder à une analyse de routine de triage de médicaments. Des réactifs de diagnostic immunologique de la maladie d'Alzheimer sont également décrits.
EP19900914284 1989-09-18 1990-09-12 Assays and reagents for amyloid deposition Ceased EP0493470A4 (en)

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WO1993013114A1 (fr) 1991-12-24 1993-07-08 Isis Pharmaceuticals, Inc. COMPOSITIONS ET PROCEDES DE MODULATION DE LA β-AMYLOIDE
US6610493B1 (en) 1993-06-17 2003-08-26 Brigham And Women's Hospital Screening compounds for the ability to alter the production of amyloid-β peptide
US5804560A (en) * 1995-01-06 1998-09-08 Sibia Neurosciences, Inc. Peptide and peptide analog protease inhibitors
US5872101A (en) * 1995-01-06 1999-02-16 Sibia Neurosciences, Inc. Methods of treating neurodegenerative disorders using protease inhibitors
US7799535B1 (en) * 1997-12-09 2010-09-21 Arch Development Corporation Methods for identifying factors that control the folding of amyloid proteins of diverse origin
EP1793001B1 (fr) * 2001-02-15 2011-11-09 The University of Chicago Cribles à base de levure pour agents affectant le repliement de protéines
CA2438661C (fr) 2001-02-15 2011-05-31 University Of Chicago Methodes de criblage d'agents modifiant le repliement des proteines utilisant la levure

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EP0274826A1 (fr) * 1986-11-17 1988-07-20 California Biotechnology, Inc. Protéine recombinante amyloide d'alzheimer
WO1989006242A1 (fr) * 1987-10-08 1989-07-13 The Mclean Hospital Corporation Anticorps agissant sur un peptide de l'amyloide de sequence a4
WO1989006693A1 (fr) * 1988-01-13 1989-07-27 The Mclean Hospital Corporation Sequences genetiques codant pour l'amyloide cerebrale d'alzheimer

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FR2403557A1 (fr) * 1977-09-19 1979-04-13 Beljanski Mirko Procede et reactifs de detection des substances cancerigenes et des substances anticancereuses
US4666829A (en) * 1985-05-15 1987-05-19 University Of California Polypeptide marker for Alzheimer's disease and its use for diagnosis
US4919915A (en) * 1987-03-03 1990-04-24 Paul Averback Method for detecting the ability to prevent red-to-green congophilic birefringence
US4912206A (en) * 1987-02-26 1990-03-27 The United States Of America As Represented By The Department Of Health And Human Services CDNA clone encoding brain amyloid of alzheimer's disease
CA1340802C (fr) * 1987-08-15 1999-10-26 Yasuyuki Takahashi Proteine precurseur de l'amyloide senile et anticorps specifique pour cette proteine
WO1989007657A1 (fr) * 1988-02-10 1989-08-24 The Children's Medical Center Corporation Precurseurs de proteines amyloides, sondes genetiques, anticorps, et modes d'utilisation

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Publication number Priority date Publication date Assignee Title
EP0274826A1 (fr) * 1986-11-17 1988-07-20 California Biotechnology, Inc. Protéine recombinante amyloide d'alzheimer
WO1989006242A1 (fr) * 1987-10-08 1989-07-13 The Mclean Hospital Corporation Anticorps agissant sur un peptide de l'amyloide de sequence a4
WO1989006693A1 (fr) * 1988-01-13 1989-07-27 The Mclean Hospital Corporation Sequences genetiques codant pour l'amyloide cerebrale d'alzheimer

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Title
See also references of WO9104339A1 *

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WO1991004339A1 (fr) 1991-04-04
EP0493470A4 (en) 1992-11-25
AU6431190A (en) 1991-04-18
AU641434B2 (en) 1993-09-23
CA2065404C (fr) 2002-12-24
JPH05502368A (ja) 1993-04-28

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