WO2023107830A1 - Amyloid oligomer compositions - Google Patents

Amyloid oligomer compositions Download PDF

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WO2023107830A1
WO2023107830A1 PCT/US2022/080564 US2022080564W WO2023107830A1 WO 2023107830 A1 WO2023107830 A1 WO 2023107830A1 US 2022080564 W US2022080564 W US 2022080564W WO 2023107830 A1 WO2023107830 A1 WO 2023107830A1
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amyloid
polypeptide
composition according
peptide
cells
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PCT/US2022/080564
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French (fr)
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Carla J. SHATZ
Kenan Christopher GARCIA
Taeho Kim
Michael Edward Birnbaum
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The Board Of Trustees Of The Leland Stanford Junior University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • G01N33/5005—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
    • G01N33/502—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 for testing non-proliferative effects
    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • 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
    • 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
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • 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
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/38—Pediatrics
    • G01N2800/385—Congenital anomalies
    • G01N2800/387—Down syndrome; Trisomy 18; Trisomy 13

Definitions

  • This invention pertains to amyloid polypeptide compositions.
  • Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Prion disease, Huntington's disease, and amyotrophic lateral sclerosis, and neurodevelopmental disorders such as Down’s Syndrome are increasingly being realized to have common cellular and molecular mechanisms including protein aggregation and inclusion body formation in selected brain regions. It is believed that small intermediates termed as ‘soluble oligomers’ or ‘amyloid oligomers’ in the aggregation process might influence cellular activity and induce cellular dysfunction (Gadad BS, et al. (2011 ) Targeting oligomers in neurodegenerative disorders: lessons from a-synuclein, tau, and amyloid-p peptide. J Alzheimers Dis;24 Suppl 2:223-32). See also, Kim et al. (2013) Science 341 , 1399-1404.
  • Amyloid oligomer compositions and amyloid monomer compositions used to make amyloid oligomer compositions, are provided. Also provided are reagents and kits thereof that find use in preparing these compositions. These amyloid oligomer compositions have a high affinity and specificity for amyloid oligomer binding partners on cells. As such, the subject amyloid oligomer compositions find many uses in research and drug development.
  • FIG. 1 A
  • 340 peptides (1 :4 ratio) were analyzed by Western blotting in parallel with oligomeric A
  • 340 predominantly form tetrameric complexes with SAv (A
  • 342- SAvT selectively binds to PirB comparably to A
  • HEK293 cells were transfected with PirB and treated with monomeric A
  • 342 (Scr-SAvT; 25 nM) or SAv alone (SAvT; 25 nM) were used as negative controls for A
  • Scale bar 10 pm. See also Fig.
  • 342-SAvT (25 nM) does not bind HEK293 cells expressing PirA1 , PirA4 or control vector, (e) Quantification of A
  • 342-SAvT binding shown in (c) and (d) (Mean ⁇ SEM; n 3).
  • FIG. 1 A
  • 342- SAvT binding to PirB. In control treatments, the same amount of control IgG was applied. Mean ⁇ SEM, n 4, ***P ⁇ 0.001 (Student t-test).
  • FIG. 3 A
  • 342-SAvT but not Scr-SAvT (25 nM each; Red) enriched in punctate patterns along the dendrites of EGFP (Green) -express! ng cortical neurons (cultured 12 days in vitro; DIV12); representative images are shown. Scale bar 5 pM.
  • 342-SAvT (Alexa 647). Green: immunostaining for the dendrite-specific marker MAP2.
  • Anti-Tuj1 (pill-tubulin) antibodies detect neuron-specific tubulin
  • FIG. 4 A
  • 342 were conjugated with streptavidin and analyzed by Coomassie gel staining. Both A
  • FIG. 5 A
  • HEK293 cells transfected with LilrB2 (a), or its homolog LilrBI or LilrB3 (b) were treated with A
  • Scale bars 10 pm
  • 342-SAvT binding shown in (a) and (b). Mean ⁇ SEM, n 3.
  • Anti-PirB (6C1 ) or anti-LilrB2 (AF2078) antibodies effectively block oligo- A
  • These antibodies exhibit the strongest inhibition for oligo-A
  • Mean ⁇ SEM, n 6, **P ⁇ 0.01 (t-test).
  • FIG. 7 A
  • Anti-Ap (4G8) antibody but not control IgG 50 nM each
  • 342- SAvT (10 nM) binding to LilrB2 is markedly diminished by anti-LilrB2 (AF2078) antibody (50 nM).
  • Mean ⁇ SEM, n 5, ***P ⁇ 0.001 , t-test.
  • FIG. 8 Graphic representation of the Ap tetramer approaches that recapitulate Ap oligomer binding and signaling.
  • A Ap oligomer binding to PirB/LilrB2 elicits hyperactivation of cofilin and synapse loss (i.e. decrease of PSD-95), leading to synaptic dysfunction in adult hippocampus as well as in developing visual cortex of Alzheimer’s mice.
  • B Ap-streptavidin tetramer (Ap-SAvT) binds to PirB/LilrB2 with enhanced affinity and activates downstream signaling, similar to Ap42 oligomer.
  • Ap-SAvT can be used to drug screening that antagonizes Ap oligomer effects, to identify novel receptors/binding partners for Ap oligomer, to study molecular mechanisms of Ap oligomer binding and signaling.
  • Amyloid oligomer compositions and amyloid monomer compositions used to make amyloid oligomer compositions are provided. Also provided are reagents and kits thereof that find use in preparing these compositions. These amyloid oligomer compositions have a high affinity and specificity for amyloid oligomer binding partners on cells. As such, the subject amyloid oligomer compositions find many uses in research and drug development.
  • amino acids described herein are preferred to be in the "L" isomeric form.
  • the amino acid sequences are given in one-letter code (A: alanine; C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine; X: any residue).
  • NH2 refers to the free amino group present at the amino terminus (the N terminus) of a polypeptide
  • COOH refers to the free carboxy group present at the carboxy terminus (the C terminus) of a polypeptide.
  • amyloid oligomer compositions are provided.
  • an “amyloid oligomer composition” it is meant a composition of amyloid oligomers, each oligomer comprising two or more amyloid monomer subunits, each amyloid monomer subunit comprising an amyloid peptide/polypeptide and an oligomerization domain.
  • an “amyloid peptide/polypeptide” it is meant a peptide or polypeptide that folds inappropriately in nature and, as a misfolded structure, interact with other amyloid peptide/polypeptides having the same sequence or other cell components to form insoluble fibrous protein aggregates, or “amyloids”.
  • an “oligomerization domain” it is meant a domain that promotes the oligomerization of the amyloid peptide/polypeptide.
  • amyloid monomer compositions By an amyloid monomer composition it is meant the amyloid monomer subunit that oligomerizes to form the amyloid oligomer compositions of the present disclosure.
  • an “amyloid peptide/polypeptide” it is meant a peptide or polypeptide that folds inappropriately in nature and, as a misfolded structure, interacts with other like peptides/polypeptides or other cell components to form insoluble fibrous protein aggregates, or “amyloids”.
  • a number of peptide and polypeptides are known in the art that fold inappropriately in nature and, as misfolded structures, interact with one another or other cell components to form insoluble fibrous protein aggregates, any of which may be used in the amyloid subunits that form the subject amyloid oligomer compositions.
  • Non-limiting examples include beta amyloid (“A
  • Bovine spongiform encephalopathy Huntingtin, implicated in Huntington's Disease; Calcitonin (“ACal”), implicated in Medullary carcinoma of the thyroid; Atrial natriuretic factor (“AANF”), implicated in cardiac arrhythmias and isolated atrial amyloidosis; Apolipoprotein Al (“AApoAl ”), implicated in Atherosclerosis; Serum amyloid A (“AA”), implicated in Rheumatoid arthritis; Medin (“AMed”), implicated in aortic medial amyloid; Prolactin (“APro”), implicated in prolactinomas; Transthyretin (“ATTR”), implicated in Familial amyloid polyneuropathy; Lysozyme (“ALys”), implicated in Hereditary non-neuropathic systemic amyloidosis; Beta 2 microglobulin (Ap2M), implicated in Dialysis related amyloidosis; Gelsolin (“AGel”),
  • amyloid peptide/polypeptide is Ap.
  • Ap “Abeta”, “betaamyloid”, “amyloid beta, or amyloid p, it is meant a peptide of about 28-55 amino acids that is derived from the carboxy terminus of amyloid precursor protein.
  • Amyloid precursor protein, or “APP” GenBank Accession Nos.
  • NM_000484.3 isoform a
  • NM_201413.2 isoform b
  • NM_201414.2 isoform c
  • NM_001136016.3 isoform d
  • NM_001136129.2 isoform e
  • NM_001136130.2 isoform f
  • NM_001136131 .2 isoform g
  • NM_001136131 .2 isoform h
  • NM_001204302.1 isoform i
  • NM_001204303.1 isoform j)
  • Ap is the main component of deposits, called amyloid plaques, found in the brains and vascular walls of patients with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) and associated with retinal ganglion cells in patients with glaucoma.
  • AD Alzheimer's disease
  • CAA cerebral amyloid angiopathy
  • tetramers of Ap peptide bind with specificity to the PirB/LilrB2 receptor on neurons and induced changes in cellular activity.
  • Api -40 (“Ap40”) (SEQ ID NO:1 ) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) and Api -42 (“Ap42”) (SEQ ID NO:2) (DAEFRHDSGYEVHHQKLVFAAEDVGSNKGAIIGLMVGGVVIA), are produced by alternative carboxy-terminal processing of APP (Selkoe et al. (1988) Proc. Natl. Acad. Sci. USA 85:7341 -7345; Selkoe, (1993) Trends Neurosci 16:403-409).
  • Api -42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA.
  • Other naturally occurring variants include, e.g., Api -28 (SEQ ID NO:3) (DAEFRHDSGYEVHHQAAVFAAEDVGSNK), AP12-28 (SEQ ID NO:4) (VHHQKLVFFAEDVGSNKC), Api -37 (SEQ ID NO:5) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG), Api -38 (SEQ ID NO:6) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG), A 1-39 (SEQ ID NO:7) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV), A 1 -43 (SEQ ID N0:8) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT), A 1 -44 (SEQ
  • the Ap peptide comprises native APP sequence.
  • the Ap of the subject compositions comprises a sequence that is found in wild type APP as it exists in nature, i.e. it is “native Ap”.
  • native APP sequences upon which native Ap peptides of the subject compositions may be based include native human APP polypeptide sequences, e.g. as may be found at GenBank Accession Nos.
  • NM_000484 isoform a
  • NM_201413 isoform b
  • NM_201414 isoform c
  • NM_001136016 isoform d
  • NM_001136129 isoform e
  • NM_001136130 isoform f
  • NM_001136131 isoform g
  • NM_001204301 isoform h
  • NM_001204302 isoform i
  • NM_001204303 isoform j
  • native mouse APP polypeptide sequences e.g. as may be found at GenBank Accession Nos.
  • NM_001198823.1 (isoform 1 ); NM_007471.3 (isoform 2); NM_001198824.1 (isoform 3); NM_001198825.1 (isoform 5); and NM_001198826.1 (isoform 6); and native rat APP polypeptide sequences, e.g. as may be found at GenBank Accession No. NM_019288.2.
  • Other native APP sequences may be readily determined by methods known in the art, e.g. in silica by employing publicly available databases, or empirically by isolating and sequencing the native APP of interest.
  • a native Ap can have the amino acid sequence comprised by, e.g. naturally occurring human APP polypeptide, murine APP polypeptide, or polypeptide from any other mammalian species, or from non-mammalian species, e.g. Drosophila, C. elegans, and the like.
  • the Ap peptide comprises variant APP sequence.
  • the Ap of the subject compositions comprises a sequence having less than 100% sequence identity with a native wild type APP sequence over the length of the fragment, i.e. it is “variant Ap”.
  • Variant Ap peptides include peptides wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the native sequence. In such instances, from about one to 20 amino acid residues may be deleted, and optionally substituted by one or more amino acid residues.
  • 3 peptides are derivatives of the above native polypeptides, wherein an amino acid residue has been substituted or covalently modified so that the resulting product has a non-naturally occurring amino acid.
  • the substitution is naturally occurring, e.g.
  • biologically active variants will have an amino acid sequence having 75% or more sequence identity, 80% or more sequence identity, 85% or more amino acid sequence identity, 90% or more amino acid sequence identity with a native sequence polypeptide, preferably 95% or more, in some instances at least 99% sequence identity.
  • Various methods known in the art may be utilized in developing such variant A
  • the amyloid peptide/polypeptide is a-synuclein.
  • a- synuclein alpha-synuclein
  • non A4 component of amyloid precursor it is meant the polypeptide that is encoded in humans by the SNCA or “aSyn” gene, or an amyloid-forming peptide or variant thereof. Sequence for a-synuclein polypeptide may be found at GenBank Accession Nos. NM_000345.3 (variant 1 ), NM_001146054.1 (variant 2), NM_001146055.1 (variant 3), and NM_007308.2 (variant 4, the “NACP112” variant).
  • Point mutations in A30P, A53T, or E46K, and gene duplication or triplication of the aSyn locus have been identified as causal factors of early onset familial PD; E46K has also been associated with Lewy body dementia.
  • Wang et al. (“A soluble a-synuclein construct forms a dynamic tetramer”, Proc Natl Acad Sci U S A. 2011 , 108(43) :17797-802) and Kalia et al. (“a-Synuclein oligomers and clinical implications for Parkinson disease”, Ann Neurol. 2012 Aug 28), alpha- synuclein forms tetramers in solution. The relevance of these tetramers to disease is unknown.
  • the a-synuclein polypeptide comprises native a-synuclein sequence.
  • the a-synuclein of the subject compositions comprises a sequence that is found in wild type a-synuclein, i.e. it is “native a-synuclein”.
  • Non-limiting examples of native a-synuclein sequences upon which native a-synuclein polypeptides of the subject compositions may be based include native human a-synuclein polypeptide sequences, e.g., NM_000345.3 (variant 1), NM_001146054.1 (variant 2), NM_001146055.1 (variant 3), and NM_007308.2 (variant 4); native mouse a-synuclein sequences, e.g., as may be found at GenBank Accession Nos.
  • NM_001042451 .1 and NM_009221.2 ; and native rat a-synuclein sequences, e.g., as may be found at GenBank Accession No. NM_019169.2.
  • Other native a- synuclein sequences may be readily determined by methods known in the art, e.g. in silico by employing publicly available databases, or empirically by isolating and sequencing the native a-synuclein of interest.
  • the a-synuclein polypeptide comprises variant a-synuclein sequence.
  • the a-synuclein of the subject compositions comprises a sequence having less than 100% sequence identity with a native wild type a-synuclein sequence over the length of the fragment, i.e. it is “variant a-synuclein”.
  • Variant a-synuclein polypeptides include polypeptides wherein one or more amino acid residues are added at the N- or C- terminus of, or within, the native sequence. In such instances, from about one to 20 amino acid residues may be deleted, and optionally substituted by one or more amino acid residues.
  • variant a-synuclein polypeptides are derivatives of the above native polypeptides, wherein an amino acid residue has been substituted or covalently modified so that the resulting product has a non-naturally occurring amino acid.
  • the substitution is naturally occurring, e.g. A30P, A53T, or E46K.
  • biologically active variants will have an amino acid sequence having 75% or more sequence identity, 80% or more sequence identity, 85% or more amino acid sequence identity, 90% or more amino acid sequence identity with a native sequence polypeptide, preferably 95% or more, in some instances at least 99% sequence identity.
  • Various methods known in the art may be utilized in developing such variant a-synuclein.
  • the amyloid peptide/polypeptide is tau.
  • tau also known as “MATP”, or “microtubule-associated protein tau”, it is meant the polypeptide that is encoded in humans by the MAPT gene or an amyloid-forming peptide thereof.
  • the tau polypeptide sequence may be found at GenBank Accession Nos.
  • NM_016835.4 isoform 1
  • NM_005910.5 isoform 2
  • NM_016834.4 isoform 3
  • NM_016841.4 isoform 4
  • NM_001123067.3 isoform 5
  • NM_001123066.3 isoform 6
  • NM_001203251 .1 isoform 7
  • NM_001203252.1 isoform 8
  • Mutations in tau have been associated with several neurodegenerative disorders such as Alzheimer's disease, Pick's disease, frontotemporal dementia, cortico-basal degeneration and progressive supranuclear palsy. As discussed in Lasagna-Reeves et al.
  • tau oligomers as potential targets for immunotherapy for Alzheimer's disease and tauopathies. Curr Alzheimer Res. 2011 , 8(6):659-65), tau and peptides thereof, e.g. peptides of domain R2 (residues V275-V300 of the longest human isoform, htau40), of domain R3 (residues V306-K331 ), and of R4 (V337-H362 ), form tetramers and octamers in solution. The relevance of these tetramers and octamers to disease is unknown.
  • the tau polypeptide comprises native tau sequence.
  • the tau of the subject compositions comprises a sequence that is found in wild type tau, i.e. it is “native tau”.
  • native tau sequences upon which native tau polypeptides of the subject compositions may be based include native human tau polypeptide sequences, e.g., as may be found at GenBank Accession Nos.
  • NM_016835.4 isoform 1
  • NM_005910.5 isoform 2
  • NM_016834.4 isoform 3
  • NM_016841.4 isoform 4
  • NM_001123067.3 isoform 5
  • NM_001123066.3 isoform 6
  • NM_001203251 .1 isoform 7
  • NM_001203252.1 isoform 8
  • native mouse tau sequences e.g., as may be found at GenBank Accession Nos. NM_001038609.1 and NM_010838.3
  • native rat tau sequences e.g., as may be found at GenBank Accession No. 1 ,NM_017212.2.
  • Other native tau sequences may be readily determined by methods known in the art, e.g. in silico by employing publicly available databases, or empirically by isolating and sequencing the native tau of interest.
  • the tau polypeptide comprises variant tau sequence.
  • the tau of the subject compositions comprises a sequence having less than 100% sequence identity with a native wild type tau sequence over the length of the fragment, i.e. it is “variant tau”.
  • Variant tau polypeptides include polypeptides wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the native sequence. In such instances, from about one to 20 amino acid residues may be deleted, and optionally substituted by one or more amino acid residues.
  • variant tau polypeptides are derivatives of the above native polypeptides, wherein an amino acid residue has been substituted or covalently modified so that the resulting product has a non-naturally occurring amino acid.
  • biologically active variants will have an amino acid sequence having 75% or more sequence identity, 80% or more sequence identity, 85% or more amino acid sequence identity, 90% or more amino acid sequence identity with a native sequence polypeptide, preferably 95% or more, in some instances at least 99% sequence identity.
  • Various methods known in the art may be utilized in developing such variant tau.
  • the amyloid monomer subunits of the amyloid oligomers comprise an oligomerization domain.
  • the oligomerization domain comprises a ligand that complexes with a ligand binding partner, which, in turn, promotes the directed oligomerization of the amyloid peptide/polypeptide.
  • a “ligand” and a “ligand binding partner” it is meant a complementary set of molecules that demonstrate specific binding, generally of relatively high affinity (an affinity constant, K a , of about 10 6 M -1 ), e.g., a biotin ligand and its binding partner streptavidin or avidin, a hapten and antibody, an enzyme and inhibitor, a lectin and carbohydrate, a ligand and its receptor.
  • the amyloid peptide/polypeptide is typically covalent bound to the ligand.
  • the oligomerization domain comprises an oligomerization moiety, i.e.
  • the amyloid monomer subunits comprise an amyloid peptide/polypeptide covalently bound to a ligand that, in turn, complexes with a ligand binding partner.
  • the phrase "ligand-ligand binding partner pair" refers to any ligand and its ligand binding partner that are capable of recognizing and binding to each other.
  • the ligand(s) and binding partner can be any moieties that are capable of recognizing and binding to each other to form a complex.
  • the ligand and binding partner are non-covalently bound.
  • the ligand and binding partner form a complex via the binding of a third intermediary substance.
  • the ligand and ligand binding partner can be naturally occurring or artificially produced, and optionally can be aggregated with other species of molecules.
  • the ligand binding partner is an oligomerizing ligand binding partner.
  • the ligand binding partner oligomerizes in solution, thereby promoting the directed oligomerization of the ligand (and hence amyloid peptide/polypeptide) to which it is complexed, e.g. streptavidin.
  • the ligand binding partner is polyvalent.
  • the ligand binding partner that has two or more, e.g. three or four, ligand binding sites, e.g. the F(ab’) 2 of an antibody, thereby promoting the directed oligomerization of the ligand (and hence amyloid peptide/polypeptide) to which it is complexed.
  • the ligand binding partner dimerizes in solution, or is divalent, e.g., an antibody.
  • ligand binding partner promotes the dimerization of the amyloid peptide/polypeptide into homo- or hetero-dimers.
  • a “dimer” it is meant that the oligomer comprises two polypeptides, each comprising an amyloid peptide/polypeptide.
  • the ligand binding partner trimerizes in solution, or is trivalent. In other words, it promotes the trimerization of the amyloid peptide/polypeptide into homo- or heterotrimers.
  • the oligomer comprises three polypeptides, each comprising an amyloid peptide/polypeptide.
  • the ligand binding partner tetramerizes in solution, e.g., streptavidin, or is tetravalent, e.g. a streptavidin complex. In other words, it promotes the tetramerization of the amyloid peptide/polypeptide into homo- or hetero-tetramers.
  • a “tetramer” it is meant that the oligomer comprises four polypeptides, each comprising an amyloid peptide/polypeptide.
  • a /lomo-dimer, /lomo-trimer, homotetramer, and the like it is meant that all polypeptides of the oligomer composition comprise the same amyloid peptide/polypeptide. In other words, the oligomer composition is homogeneous for the type of amyloid peptide/polypeptide comprised therein.
  • a heterodimer, hetero-trimer, hetero-tetramer, and the like it is meant that the polypeptides of the oligomer composition comprise different amyloid peptide/polypeptide. In other words, the oligomer composition is heterogeneous for the type of amyloid peptide/polypeptide comprised therein.
  • ligands and ligand binding partners include, but are not limited to, biotin (i.e. biotin or biotin derivatives, e.g. Biocytin, NHS-Biotin, Sulfo-NHS-Biotin, TFP-PEG3-Biotin, BMCC-Biotin, lodoacetyl-LC-Biotin, HPDP-Biotin, Amine-PEG2-Biotin, Hydrazide-Biotin, Alkoxyamine-PEG4-Biotin, the photoactivatable derivate TFPA-PEG3-Biotin ), avidin, streptavidin, agonists and antagonists for cell membrane receptors, receptors, toxins and venoms, viral epitopes, hormones such as steroids, hormone receptors, peptides, enzymes and other catalytic polypeptides, enzyme substrates, cofactors, drugs including small organic molecules, opiates, opiate receptors, lectin
  • biotin
  • the ligand may be a B cell epitope (e.g., antibody), either naturally-occurring or synthetic, such as for example, a polyoma epitope, a FLAG epitope, a hemagglutinin epitope for the 12CA5 monoclonal antibody, a polyhistidine tract, and the like.
  • the ligand binding partner can be an antibody, such as, for example, an IgG, IgM, and the like, or an antigen binding fragment thereof.
  • ligand-ligand binding partner complexes include the following: biotinstreptavidin; antibody-antigen; lectin-carbohydrate; peptide-cell membrane receptor; protein A-antibody; hapten-anti-hapten; digoxigenin-anti-digoxigenin; enzyme-cofactor; and enzymesubstrate.
  • ligand binding partners can be formed into polyvalent ligand binding partners according to methods known in the art.
  • the amyloid peptide/polypeptide may be covalently bound to the ligand by any convenient method.
  • the amyloid peptide/polypeptide and ligand may be encoded by the same nucleic acid, such that upon translation, the polypeptide that is produced is a fusion polypeptide comprising amyloid peptide/polypeptide covalently bound to the ligand.
  • the ligand may become covalently bound to the amyloid peptide/polypeptide by modification of an amyloid peptide/polypeptide, e.g by use of a modifying enzyme.
  • Suitable modifying enzymes include, for example, BirA, various glycosylases, famesyl transferase protein, and the like.
  • Nonlimiting examples of ligand that may become covalently bound to an amyloid peptide/polypeptide or subunit thereof by a modifying enzyme include biotin, a sugar, a famesyl group, and the like.
  • an amyloid peptide/polypeptide e.g an Ap peptide, includes a modification site (e.g., the BirA recognition sequence GGGLNDIFEAQKIEWH (Castro, C. et al.
  • biotinylated amyloid peptide/polypeptide e.g. Ap peptide
  • streptavidin or avidin which binds biotin with extremely high affinity.
  • the amyloid multimers e.g. “Ap-streptavidin tetramers”, or “Ap-SavT”, can then be stored until needed.
  • the ligand binding partner is free in solution.
  • the ligand binding partner is attached to a solid support.
  • suitable solid supports include beads (e.g., magnetic beads), membranes, microtiter plates, and the like.
  • the support can be glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, PVDF, and the like.
  • the use of a binding partner linked to a solid support can be useful for immobilization and/or isolation of cells that recognize the multimeric amyloid peptide/polypeptide, or the purification of receptor(s) that are bound by the multimeric amyloid peptide/polypeptide.
  • the amyloid monomer subunits that form the subject amyloid oligomer compositions comprise an amyloid peptide/polypeptide covalently bound to an oligomerization moiety.
  • an oligomerization moiety it is meant a functional group - e.g., peptide, polypeptide, small molecule or nucleic acid composition -- that oligomerizes in solution, thereby promoting the directed oligomerization of the amyloid peptide/polypeptide to which it is fused.
  • oligomerizing in solution it is meant that 50% or more of the moiety complexes with itself in solution, e.g. 60% or more, 65% or more, 70% or more, or 75% or more of the moiety binds with itself, in some instances, 80% or more, 85% or more, or 90% or more of the moiety binds with itself, in some cases, 95% or more, 97% or more, or virtually all of the moiety binds with itself.
  • the oligomerization moiety is a dimerization moiety. In other words, it promotes the dimerization of the amyloid peptide/polypeptide into homo- or heterodimers.
  • Non-limiting examples of polypeptides that dimerize in solution include Verde Fluorescent Protein (VFP) and VFP mutant dVFP (Hagan, RP et al. (2010) FEBS J. 277(8):1967-19780, Midoriishi Cyan (MBL International), Turbo Yellow Fluorescent Protein (TurboYFP; Evrogen), dimerizationdependent red fluorescent protein (ddRFP; Alford S.C. et al.
  • the oligomerization moiety is a trimerization moiety. In other words, it promotes the trimerization of the amyloid peptide/polypeptide into homo- or hetero-trimers.
  • polypeptides that form trimers in solution that may be used as oligomerization moieties in the subject compositions include the C-terminal domain of T4 fibritin (foldon) (Guthe, S., et al.
  • the oligomerization moiety is a tetramerization moiety. In other words, it promotes the tetramerization of the amyloid peptide/polypeptide into homo- or heterotetramers.
  • Non-limiting examples of polypeptides that form tetramers in solution that may be used as oligomerization moieties in the subject compositions include DsRed (US Application No. US2004/0137642), HcRed, ZsGreen, (Clontech), cOFP (Stratagene), cFP484, and streptavidin.
  • the ligand or oligomerization moiety is covalently bound directly to the amyloid peptide/polypeptide.
  • the ligand or oligomerization moiety is covalently bound to the amyloid peptide/polypeptide indirectly, i.e. via a linker region.
  • the ligand or oligomerization moiety is separated from the amyloid peptide/polypeptide by a linker region.
  • linker region it is meant a sequence of amino acids, e.g. polar or amphipathic amino acids, that links the amyloid peptide/polypeptide to the ligand or oligomerization moiety.
  • Linker regions are typically about 3 to about 25 amino acids in length, e.g. 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids or more in length, although longer linker regions may also be employed in the subject composition.
  • the linker region allows a flexible, unconstrained solution conformation, in which the geometry of the amyloid oligomer, or its subunits, is unrestricted relative to other domains. In other words, it is a “conformationally flexible linker region”.
  • a modification site such as a BirA modification site, may be included in a linker region, e.g. for the conjugation of ligand to the linker region.
  • the ligand or oligomerization moiety is covalently bound to the N- terminus of the amyloid peptide/polypeptide. In other words, it is covalently bound, either directly or indirectly, to the N-terminal amino acid of the peptide. In other instances, the ligand or oligomerization moiety is covalently bound to the C-terminus of the amyloid peptide/polypeptide. In other words, it is covalently bound, either directly or indirectly, to the C-terminal amino acid of the peptide. In other instances, the ligand is covalently bound to an amino acid of the amyloid peptide/polypeptide other than the N-terminal or C-terminal amino acid. In other words, it is covalently bound, either directly or indirectly, to an internal amino acid of the peptide.
  • the ligand binding domain or oligomerization moiety is labeled with a detectable label.
  • label or “labeled” refer to a molecule or groups of molecules which can provide a detectable signal when the label is incorporated into, or attached to, a polypeptide such as an oligomerization moiety or a ligand binding partner.
  • an oligomerization moiety or a ligand binding partner can be labeled with a radioactive molecule, a luminescent molecule, a fluorescent molecule, a chemi-luminescent molecule, an enzyme, biotinyl moieties, and the like.
  • detectable labels include, but are not limited to, radioisotopes (e.g., 3 H, 14 C, 32 P, 3 5 S, 125 l, 131 l, and the like); fluorescent molecules (e.g., Alexa 488, Alexa 598, Alexa 647, fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin (PE), phycocyanin, allophycocyanin, ortho-phthaldehyde, fluorescamine, peridinin-chlorophyll a (PerCP), Cy3 (indocarbocyanine), Cy5 (indodicarbocyanine), lanthanide phosphors, and the like); enzymes (e.g., horseradish peroxidase, p-galactosi
  • detectable labels are attached by spacer arms or linkers of various lengths to reduce potential steric hindrance.
  • amyloid oligomer compositions may be prepared following any convenient method for the preparation of polypeptides and polypeptide complexes.
  • amyloid peptide/polypeptide is synthesized in vitro or in vivo.
  • the amyloid peptide/polypeptide is subsequently covalently bound to ligand, e.g. biotin, by chemical reaction to produce an amyloid peptide/polypeptide-ligand composition, e.g. biotinylated amyloid peptide/polypeptide.
  • the amyloid peptide/polypeptide-ligand composition is incubated with binding partner, e.g.
  • amyloid peptide/polypeptide is synthesized in vitro or in vivo. The amyloid peptide/polypeptide is subsequently covalently bound to an oligomerization domain by chemical reaction to produce an amyloid monomer composition, which is allowed to assemble into oligomers.
  • amyloid peptide/polypeptide is synthesized in vitro or in vivo already covalently bound to a ligand, i.e., no modification of the amyloid peptide/polypeptide is required to produce the amyloid-ligand fusion polypeptide.
  • the amyloid-ligand fusion polypeptide is incubated with binding partner and allowed to assemble into oligomers.
  • amyloid peptide/polypeptides are synthesized in vitro or in vivo covalently fused to an oligomerization moiety, i.e., no modification of the amyloid peptide/polypeptide is required to produce the amyloid-oligomerization moiety fusion polypeptide.
  • the amyloid-oligomerization moiety fusion polypeptide are then allowed to assemble into oligomers at physiologic pH.
  • Amyloid peptide/polypeptides and amyloid fusion polypeptides may be produced by any convenient method.
  • amyloid peptide/polypeptides and amyloid fusion polypeptides may be chemically synthesized, using conventional methods as known in the art.
  • Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied BioSystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
  • various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules, e.g.
  • amyloid peptide/polypeptides and amyloid fusion polypeptides may be synthesized in a cell, for example by natural or recombinant synthesis of the peptide or fusion polypeptide, and isolated and purified in accordance with conventional methods of recombinant synthesis.
  • amyloid peptide/polypeptide may be synthesized by synthesizing the precursor protein, e.g. APP, in a cell, and subsequently processing the precursor protein in vitro to produce the amyloid peptide/polypeptide.
  • a lysate may be prepared of an expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
  • the expression host i.e. the cell, will endogenously express the amyloid peptide/polypeptide or precursor thereof or amyloid fusion polypeptide.
  • the expression host will ectopically express the amyloid peptide/polypeptide or amyloid fusion polypeptide, e.g., the host will be transformed with a nucleic acid vector that comprises nucleic acid sequence that encodes for the amyloid peptide/polypeptide, precursor protein of the amyloid peptide/polypeptide, or amyloid fusion protein, and the host will ectopically express the amyloid peptide/polypeptide, amyloid precursor protein, or amyloid fusion protein from the vector.
  • compositions which are used will comprise at least 50% by weight of the desired product, more usually at least about 75%, 80%, 85%, or 90% by weight, preferably at least about 95% by weight, and in some instances 97%, 98%, 99%, or 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification.
  • percentages will be based upon total protein.
  • the subject amyloid oligomer compositions of the present disclosure may be readily recognized by virtue of both their structure and function.
  • the subject compositions comprise a controlled amount of amyloid peptide/polypeptide per oligomer.
  • the amyloid oligomer compositions of the subject disclosure are typically a substantially homogeneous composition of oligomers comprising the same number of amyloid peptide/polypeptide per oligomer.
  • a “substantially homogeneous” composition of oligomers it is meant that about 60% or more of the oligomers in the composition comprise the same number of amyloid monomer subunits, e.g. 65% or more, 70% or more, 75% or more, 80% or more, for example, 85% or more, 90% or more, 95% or more, in some instances 97% or more, comprise the same number of amyloid monomer subunits.
  • an amyloid tetramer composition created by the oligomerization of a tetramerization domain would be substantially homogenous for amyloid tetramers, i.e.
  • amyloid dimer composition created by the oligomerization of a dimerization domain would be substantially homogenous for amyloid dimers, i.e. oligomers comprising 2 amyloid monomer subunits. This is in contrast to current methods in the art, which rely upon a random, low-efficiency chemical process that creates a range of oligomerization states simultaneously, with no method to control the number of amyloid peptides per oligomer.
  • the number of subunits in the oligomers of the subject oligomer compositions and the extent of homogeneity of the subject amyloid oligomer compositions may be detected using any convenient method for visualizing amyloid oligomer content in the sample.
  • oligomeric forms may be detected by gel shift and Coomassie gel staining or Western blotting with an antibody specific for the amyloid peptide or oligomerization domain, where oligomeric forms comprising the same number of amyloid subunits will appear as discrete bands, in contrast to the heterogenous smear patterns of bands seen in Western blots for amyloid oligomers generated using current methods in the art.
  • the subject amyloid oligomer compositions of the present disclosure may also be recognized by their high affinity and specificity for their cognate binding partners, which can be directly attributed to the substantial homogeneity of the subject amyloid oligomer composition as well as the methods by which the subject amyloid compositions are made.
  • affinity as used herein, it is meant the binding strength between an amyloid oligomer and its binding partner, e.g. receptor.
  • receptor specificity By “receptor specificity”, “specific binding,” “specifically bind,” and the like as used herein, it is meant the ability of the subject amyloid oligomer composition to preferentially bind directly to one protein relative to other molecules or moieties on the cell.
  • the affinity between an amyloid oligomer and the protein to which it specifically binds when they are specifically bound to each other in a binding complex is characterized by a KD (dissociation constant) of less than 10' 6 M, less than 10 7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10 -12 M, less than 10 -13 M, less than 10 -14 M, or less than 10 -15 M.
  • this high affinity and specificity may be observed as a significantly larger amount of receptor binding and downstream signaling per molar concentration of composition than an oligomer composition prepared using current methods in the art, i.e. without the guidance of an oligomerization domain.
  • amyloid oligomer compositions have a physiological effect on cells that better recapitulates the physiological effect of A
  • amyloid oligomers that are formed by directed oligomerization more accurately model amyloid oligomers that form naturally in vivo than amyloid oligomers that are allowed to freely aggregate.
  • nucleic acids that encode for amyloid monomer compositions of the present disclosure e.g. nucleic acids that encode for amyloid peptide/polypeptide fused to a ligand, nucleic acids that encode for amyloid peptide/polypeptide fused to an oligomerization moiety; and nucleic acids that encode for peptides/polypeptides that may be used to generate the amyloid monomer compositions of the present disclosure, e.g. nucleic acids that encode for amyloid peptide/polypeptides, nucleic acids that encodes for precursor proteins of the amyloid peptide/polypeptide, etc. These nucleic acid compositions are described in greater detail below.
  • nucleic acids that encode for amyloid monomer compositions of the present disclosure or peptides/polypeptides that may be used to generate the amyloid monomer compositions of the present disclosure are provided.
  • a nucleic acid composition is meant a composition comprising a sequence of DNA, i.e. a DNA molecule, having an open reading frame, or coding sequence, that encodes for an amyloid monomer composition of the present disclosure or a peptide/polypeptide that may be used to generate the amyloid monomer compositions of the present disclosure.
  • DNA molecule By a “DNA molecule” it is meant the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form or a double-stranded helix.
  • This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. Also encompassed in this term are DNA molecules that are homologous, substantially similar or identical to the nucleic acids of the present invention.
  • coding sequence it is meant a DNA sequence which is transcribed and translated into a peptide or polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences, e.g. non-naturally occurring fusion polypeptides, e.g.
  • amyloid peptide/polypeptide-ligand fusion polypeptide or amyloid peptide/polypeptide- oligomerization moiety fusion polypeptide By “isolated” is meant to describe a polynucleotide, a polypeptide, an antibody, or a host cell that is in an environment different from that in which the polynucleotide, the polypeptide, the antibody, or the host cell naturally occurs.
  • the subject nucleic acids are present in other than their natural environment, e.g., they are isolated, present in enriched amounts, etc., from their naturally occurring environment, e.g., the organism from which they are obtained.
  • the subject invention provides coding sequences encoding the amyloid monomer compositions of the present disclosure or peptides/polypeptides that may be used to generate the amyloid monomer compositions of the present disclosure, as well as homologs thereof.
  • the subject nucleic acid compositions may comprise one or more DNA regulatory sequences.
  • DNA regulatory sequences are transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for and/or regulate expression of a coding sequence in a host cell.
  • the coding sequence may be associated with a polyadenylation signal and transcription termination sequence 3' to the coding sequence.
  • the coding sequence may be operably linked to a promoter sequence 5’ to the coding sequence.
  • promoter sequence a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Various promoters may be used to drive the various vectors of the present invention.
  • the promoter may be a constitutively active promoter, i.e. a promoter that is active in the absence externally applied agents, e.g. the CMV IE1 promoter, the SV40 promoter, GAPDH promoter, Actin promoter.
  • the promoter may be an inducible promoter, i.e. a promoter whose activity is regulated upon the application of an agent to the cell, e.g. doxycycline, the tet-on or tet-off promoter, the estrogen receptor promoter, etc.
  • the promoter may be a tissue-specific promoter, i.e. a promoter that is active on certain types of cells.
  • the coding sequence is inserted into a vector.
  • a vector it is meant a nucleic acid that is a replicon, such as plasmid, minicircle, phage, cosmid, etc, to which another DNA segment, i.e. an “insert”, e.g. a coding sequence, an expression cassette, etc., may be attached so as to bring about the replication of the attached segment.
  • an “insert” e.g. a coding sequence, an expression cassette, etc.
  • the subject invention also provides constructs comprising the subject nucleic acids inserted into a vector, where such constructs may be used for a number of different applications, including propagation, protein production, etc. Viral and non-viral vectors may be prepared and used, including plasmids.
  • vector will depend on the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal or person. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially.
  • the partial or full-length polynucleotide is inserted into a vector typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector.
  • the desired nucleotide sequence can be inserted by homologous recombination in vivo.
  • Regions of homology are added by ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence, for example.
  • expression cassettes or systems that find use in, among other applications, the synthesis of the subject proteins.
  • an “expression cassette” it is meant a DNA coding sequence operably linked to a promoter and/or other DNA regulatory sequences.
  • the peptide or polypeptide encoded by a polynucleotide of the invention is expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. Suitable vectors and host cells are described in U.S. Patent No. 5,654,173.
  • a subject polynucleotide for example, a polynucleotide encoding an amyloid monomer composition, e.g.
  • an amyloid peptide/polypeptide fused to a ligand, an amyloid peptide/polypeptide fused to an oligomerization moiety, or a polynucleotide encoding a peptide/polypeptide that may be used to generate an amyloid monomer composition is linked to a regulatory sequence as appropriate to obtain the desired expression properties.
  • regulatory sequences can include promoters (attached either at the 5' end of the sense strand or at the 3' end of the antisense strand), enhancers, terminators, operators, repressors, and inducers.
  • the promoters can be regulated or constitutive.
  • conditionally active promoters such as tissue-specific or developmental stage-specific promoters.
  • tissue-specific or developmental stage-specific promoters are linked to the desired nucleotide sequence using the techniques described above for linkage to vectors. Any techniques known in the art can be used.
  • the expression vector will provide a transcriptional and translational initiation region, which may be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region.
  • These control regions may be native to the subject species from which the subject nucleic acid is obtained, or may be derived from exogenous sources.
  • Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins.
  • a selectable marker operative in the expression host may be present.
  • Expression vectors may be used for, among other things, the production of fusion proteins, as described above.
  • Expression cassettes may be prepared comprising a transcription initiation region, the gene or fragment thereof, and a transcriptional termination region.
  • sequences that allow for the expression of functional epitopes or domains usually at least about 8 amino acids in length, more usually at least about 15 amino acids in length, to about 25 amino acids, and up to the complete open reading frame of the gene.
  • the cells containing the construct may be selected by means of a selectable marker, the cells expanded and then used for expression.
  • the above described expression systems may be employed with prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression.
  • a unicellular organism such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, e.g. COS 7 cells, HEK 293, CHO, Xenopus Oocytes, etc.
  • vertebrates e.g. COS 7 cells, HEK 293, CHO, Xenopus Oocytes, etc.
  • Small peptides can also be synthesized in the laboratory. Polypeptides that are subsets of the complete protein sequence may be used to identify and investigate parts of the protein important for function.
  • Specific expression systems of interest include bacterial, yeast, insect cell and mammalian cell derived expression systems. Representative systems from each of these categories is are provided below:
  • yeast Expression systems in yeast include those described in Hinnen et al., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J. Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142; Kunze et al., J. Basic Microbiol. (1985) 25:141 ; Gleeson et al., J. Gen. Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986) 202:302; Das et al., J. Bacteriol.
  • Insect Cells Expression of heterologous polypeptides in insects is accomplished as described in U.S. Patent No. 4,745,051 ; Friesen et al., “The Regulation of Baculovirus Gene Expression”, in: The Molecular Biology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0 155,476; and Vlak et al., J. Gen. Virol. (1988) 69:765 776; Miller et al., Ann. Rev. Microbiol.
  • Mammalian Cells Mammalian expression is accomplished as described in Dijkema et al., EMBO J. (1985) 4:761 , Gorman et al., Proc. Natl. Acad. Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41 :521 and U.S. Patent No. 4,399,216. Other features of mammalian expression are facilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44, Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.
  • the resulting replicated nucleic acid, RNA, expressed protein or polypeptide is within the scope of the invention as a product of the host cell or organism.
  • the product is recovered by any appropriate means known in the art.
  • nucleic acids that hybridize to the above described nucleic acid under stringent conditions.
  • stringent hybridization conditions is hybridization at 50°C or higher and O.IxSSC (15 mM sodium chloride/1 .5 mM sodium citrate).
  • Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least about 90% as stringent as the above specific stringent conditions.
  • nucleic acids may be introduced into cells by any convenient method.
  • nucleic acids may be introduced via electroporation, calcium chloride transfection, or lipofection.
  • nucleic acids may be provided to cells via a virus.
  • the cells are contacted with viral particles comprising the nucleic acid of interest.
  • Retroviruses for example, lentiviruses, and adenoviruses are particularly suitable to the method of the invention. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection.
  • the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line.
  • Different packaging cell lines provide a different envelope protein to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells. Such methods are well known in the art.
  • the subject nucleic acid may be maintained episomally, e.g. as plasmids, minicircle DNAs, virus-derived vectors such cytomegalovirus, adenovirus, etc..
  • the subject nucleic acids may be integrated (covalently linked) into the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell 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 primary cell that is capable of stable growth in vitro for many generations.
  • Transgenic cells of the subject invention include one or more amyloid oligomer nucleic acids according to the subject invention present as a transgene, where included within this definition are the parent cells transformed to include the transgene and the progeny thereof.
  • Transgenic organisms of the subject invention include cells and multicellular organisms, e.g. animals, in which the subject composition is expressed in cells or tissues where amyloid peptide/polypeptide, e.g. A
  • Transgenic organisms of the subject invention also include cells and multicellular organisms, e.g. animals, in which the subject composition is expressed in cells or tissues where amyloid peptide/polypeptide, e.g. A
  • Transgenic organisms of the subject invention may be made through any convenient method.
  • transgenic organisms may be made by homologous recombination, in which a particular locus, e.g. the locus from which the precursor protein of the amyloid peptide/polypeptide is produced (for example, for the A
  • the subject nucleic acid may replace the targeted gene, e.g. the APP gene, or a coding region thereof.
  • the subject nucleic acid may be inserted into the targeted gene locus, e.g.
  • a subject nucleic acid composition may be randomly integrated into the genome, e.g. as a nucleic acid construct comprising the subject nucleic acid in operable linkage with a promoter, or as a nucleic acid composition not operably linked to a promoter .
  • Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like.
  • DNA constructs for homologous recombination will comprise at least a portion of the APP-oligomer nucleic acid of the subject invention, wherein the DNA construct includes regions of homology to the target locus.
  • DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection may be included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990), Meth. Enzymol. 185:527-537.
  • an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF).
  • LIF leukemia inhibiting factor
  • ES or embryonic cells may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct.
  • Blastocysts are obtained from 4 to 6 week old superovulated females.
  • the ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting offspring screened for the construct.
  • chimeric progeny can be readily detected.
  • the chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vitro culture.
  • the transgenic animals may be any non-human mammal, such as laboratory animals, domestic animals, etc.
  • the transgenic animals may be used in functional studies, drug screening, etc. Representative examples of the use of transgenic animals include those described infra.
  • the subject amyloid oligomer compositions find many uses in research and drug discovery.
  • amyloid oligomer compositions can be used to model amyloid binding to a cell or amyloid signaling in a cell that expresses an amyloid receptor, e.g. in a cell that expresses PirB/LilrB2, e.g. to study the effects of amyloid oligomer binding on intracellular signaling pathways, to screen for agents that inhibit amyloid oligomer signaling, etc.
  • the subject amyloid oligomer compositions can also be used to identify novel amyloid oligomer binding partners, e.g. new receptors for amyloid oligomers.
  • the subject amyloid oligomer compositions can be used to generate animal models of diseases and disorders associated with amyloid formation, e.g.
  • Alzheimer’s disease cerebral amyloid angiopathy, Down’s Syndrome, Diabetes mellitus type 2, Parkinson's disease, tauopathies, spongiform encephalopathies, Huntington's Disease, Medullary carcinoma of the thyroid, cardiac arrhythmias and isolated atrial amyloidosis, Atherosclerosis, Rheumatoid arthritis, Aortic medial amyloid, prolactinomas, Familial amyloid polyneuropathy, Hereditary non-neuropathic systemic amyloidosis, Dialysis related amyloidosis, Finnish amyloidosis, Lattice corneal dystrophy, systemic AL amyloidosis, and Sporadic Inclusion Body Myositis.
  • the subject amyloid oligomer compositions are used to promote amyloid peptide/polypeptide signaling in a cell.
  • a cell expressing an amyloid receptor is contacted with amyloid oligomer in a concentration, or amount, that is effective to promote amyloid oligomer receptor signaling, e.g. to increase signaling by 25%, 50%, 75%, 90%, 95%, or more, relative to the signaling in the absence of the amyloid oligomer.
  • the subject amyloid oligomer compositions find use in screens to identify agents that modulate the effects of amyloid peptide/polypeptides on a cell.
  • an agent that modulates the binding of Ap to Ap receptor may be desirable to identify an agent that modulates the binding of Ap to Ap receptor, and/or that modulates intracellular signaling events promoted by the binding of Ap to Ap receptor.
  • a candidate agent that is identified as having activity in disrupting Ap binding to its receptor or disrupting Ap-mediated intracellular signaling will be an agent that finds use in the treatment of an Ap-associated disease or disorder, e.g., Alzheimer’s disease, Down’s Syndrome, etc.
  • the ability of the candidate agent to modulate e.g.
  • binding of Ap to Ap receptor and/or Ap-mediated intracellular signaling may be accurately determined by assessing the ability of the subject composition to bind receptor and/or promote intracellular signaling in the presence of candidate agent and comparing that ability to its ability in the absence of candidate agent.
  • the ability of an agent to inhibit amyloid receptor binding may be assessed by employing the subject amyloid oligomer composition in a cell-free assay, e.g. using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • an Ap receptor or domain thereof e.g. the extracellular domain of PirB/LILRB2, the receptor amy3, etc.
  • an Ap receptor or domain thereof e.g. the extracellular domain of PirB/LILRB2, the receptor amy3, etc.
  • the receptor is contacted with the Ap tetramer and the candidate agent
  • the affinity of the Ap tetramer for the immobilized receptor is compared to the affinity of the Ap tetramer for the immobilized receptor in the absence of candidate agent.
  • a reduced affinity of Ap tetramer for the receptor in the presence of candidate agent indicates that the candidate agent will disrupt, i.e. inhibit, reduce, or otherwise suppress, Ap binding to a cell expressing that receptor, e.g. PirB/LILRB2.
  • the ability of an agent to inhibit amyloid receptor binding can be assessed by employing the subject amyloid oligomer composition in a cell-based assay.
  • a cell expressing an Ap receptor or domain thereof, e.g. PirB/LILRB2 may be contacted with Ap tetramer and candidate agent, and the effect of the agent on the ability of the Ap tetramer to bind the cell and/or promoter intracellular signaling is assessed by assessing one or more output parameters.
  • Parameters are quantifiable components of cells, particularly components that can be accurately measured, desirably in a high throughput system.
  • a parameter can be any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc.
  • one such method may comprise contacting a cell that expresses an A
  • An output parameter that may be quantified when screening for agents that modulate the effects of A
  • 3 tetramer to bind to a cell can be readily visualized by, for example, flow cytometry, immunohistochemistry, fluorescence resonance energy transfer (FRET), and the like.
  • Another example of an output parameter is an increase in cytosolic cAMP and Ca(2+), or the activation of one or more of the signal transduction mediators protein kinase A, MAPK, Akt, and cFos.
  • Other output parameters could include those that are reflective of the function of the cells in the culture.
  • neurons contacted with candidate agent may be assessed for the number of spines, the number of synapses shared with neighboring neurons, the plasticity of the neurons, and the like, where an increase in the number of spines, number of synapses, and plasticity of the neuron in the presence of candidate agent indicates that the agent disrupts the effect of A
  • 3 composition e.g. a 24-48h exposure at low micromolar concentrations, may be used as an output parameter, where a reduction in cell death in the presence of candidate agent indicates that the agent disrupts the effect of A
  • one parameter is measured. In some instances, multiple parameters are measured.
  • Cells useful for screening include any cell that endogenously or ectopically expresses a receptor for the subject amyloid oligomer composition, e.g., for A
  • Cells may be from any mammalian species, e.g. murine, rodent, canine, feline, equine, bovine, ovine, primate, human, etc.
  • Cells may be from established cell lines, e.g.
  • HEK293 cells, 293T cells, NG108 cells, PC-12 cells, etc. may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture.
  • primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage.
  • the primary cell lines of the present invention are maintained for fewer than 10 passages in vitro.
  • the cells may be harvested from an individual by any convenient method.
  • cells may be harvested by biopsy, or by harvesting a tissue in its entirety.
  • An appropriate solution may be used for dispersion or suspension of the harvested cells.
  • Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, DMEM-F12, etc., conveniently supplemented with a synthetic supplement, e.g. B27, NGS, N2, etc., or with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM.
  • Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.
  • the cells may be used immediately, or they may be stored, frozen, for long periods of time, being thawed and capable of being reused.
  • the cells will usually be frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells.
  • Candidate agents of interest for screening include known and unknown compounds that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc.
  • An important aspect of the invention is to evaluate candidate drugs, including toxicity testing; and the like.
  • Candidate agents include organic molecules comprising functional groups necessary for structural interactions, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Included are pharmacologically active drugs, genetically active molecules, etc.
  • Compounds of interest include chemotherapeutic agents, hormones or hormone antagonists, etc.
  • Exemplary of pharmaceutical agents suitable for this invention are those described in, "The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition. Also included are toxins, and biological and chemical warfare agents, for example see Somani, S. M. (Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992).
  • Candidate agents of interest for screening also include nucleic acids, for example, nucleic acids that encode siRNA, shRNA, antisense molecules, or miRNA, or nucleic acids that encode polypeptides.
  • Many vectors useful for transferring nucleic acids into target cells are available.
  • the vectors may be maintained episomally, e.g. as plasmids, minicircle DNAs, virus-derived vectors such cytomegalovirus, adenovirus, etc., or they may be integrated into the target cell genome, through homologous recombination or random integration, e.g. retrovirus derived vectors such as MMLV, HIV-1 , ALV, etc.
  • Vectors may be provided directly to the subject cells. In other words, the pluripotent cells are contacted with vectors comprising the nucleic acid of interest such that the vectors are taken up by the cells.
  • nucleic acid vectors such as electroporation, calcium chloride transfection, and lipofection
  • the nucleic acid of interest may be provided to the subject cells via a virus.
  • the pluripotent cells are contacted with viral particles comprising the nucleic acid of interest.
  • Retroviruses for example, lentiviruses, are particularly suitable to the method of the invention. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line.
  • the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line.
  • Different packaging cell lines provide a different envelope protein to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells.
  • Envelope proteins are of at least three types, ecotropic, amphotropic and xenotropic.
  • Retroviruses packaged with ecotropic envelope protein, e.g. MMLV are capable of infecting most murine and rat cell types, and are generated by using ecotropic packaging cell lines such as BOSC23 (Pear et al. (1993) P.N.A.S. 90:8392-8396).
  • Retroviruses bearing amphotropic envelope protein are capable of infecting most mammalian cell types, including human, dog and mouse, and are generated by using amphotropic packaging cell lines such as PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431 -437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with xenotropic envelope protein, e.g. AKR env, are capable of infecting most mammalian cell types, except murine cells.
  • Amphotropic packaging cell lines such as PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431 -437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988) PNAS 85
  • the appropriate packaging cell line may be used to ensure that the subject CD33+ differentiated somatic cells are targeted by the packaged viral particles.
  • Methods of introducing the retroviral vectors comprising the nucleic acid encoding the reprogramming factors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art.
  • Vectors used for providing nucleic acid of interest to the subject cells will typically comprise suitable promoters for driving the expression, that is, transcriptional activation, of the nucleic acid of interest.
  • suitable promoters for driving the expression that is, transcriptional activation, of the nucleic acid of interest.
  • This may include ubiquitously acting promoters, for example, the CMV- b-actin promoter, or inducible promoters, such as promoters that are active in particular cell populations or that respond to the presence of drugs such as tetracycline.
  • transcriptional activation it is intended that transcription will be increased above basal levels in the target cell by at least about 10 fold, by at least about 100 fold, more usually by at least about 1000 fold.
  • vectors used for providing reprogramming factors to the subject cells may include genes that must later be removed, e.g. using a recombinase system such as Cre/Lox, or the cells that express them destroyed, e.g. by including genes that allow selective toxicity such as herpe
  • Candidate agents of interest for screening also include polypeptides. Such polypeptides may optionally be fused to a polypeptide domain that increases solubility of the product.
  • the domain may be linked to the polypeptide through a defined protease cleavage site, e.g. a TEV sequence, which is cleaved by TEV protease.
  • the linker may also include one or more flexible sequences, e.g. from 1 to 10 glycine residues.
  • the cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, e.g.
  • Domains of interest include endosomolytic domains, e.g. influenza HA domain; and other polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE domain, and the like.
  • the polypeptide may comprise the polypeptide sequences of interest fused to a polypeptide permeant domain.
  • permeant domains are known in the art and may be used in the non-integrating polypeptides of the present invention, including peptides, peptidomimetics, and non-peptide carriers.
  • a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK.
  • the permeant peptide comprises the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein.
  • Other permeant domains include poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine, octa-arginine, and the like.
  • Patent applications 20030220334; 20030083256; 20030032593; and 20030022831 herein specifically incorporated by reference for the teachings of translocation peptides and peptoids).
  • the nona-arginine (R9) sequence is one of the more efficient PTDs that have been characterized (Wender et al. 2000; Uemura et al. 2002).
  • the polypeptide may be formulated for improved stability.
  • the peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream.
  • the polypeptide may be fused to another polypeptide to provide for added functionality, e.g. to increase the in vivo stability.
  • fusion partners are a stable plasma protein, which may, for example, extend the in vivo plasma half-life of the polypeptide when present as a fusion, in particular wherein such a stable plasma protein is an immunoglobulin constant domain.
  • the stable plasma protein is normally found in a multimeric form, e.g., immunoglobulins or lipoproteins, in which the same or different polypeptide chains are normally disulfide and/or noncovalently bound to form an assembled multichain polypeptide
  • the fusions herein containing the polypeptide also will be produced and employed as a multimer having substantially the same structure as the stable plasma protein precursor.
  • These multimers will be homogeneous with respect to the polypeptide agent they comprise, or they may contain more than one polypeptide agent.
  • the candidate polypeptide agent may be produced by eukaryotic cells or by prokaryotic cells, it may be further processed by unfolding, e.g. heat denaturation, DTT reduction, etc. and may be further refolded, using methods known in the art.
  • Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc.
  • modifications of glycosylation e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g.
  • polypeptides by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes.
  • enzymes which affect glycosylation such as mammalian glycosylating or deglycosylating enzymes.
  • sequences that have phosphorylated amino acid residues e.g. phosphotyrosine, phosphoserine, or phosphothreonine.
  • the polypeptides may have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
  • Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non- naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.
  • the candidate polypeptide agent may be prepared by in vitro synthesis, using conventional methods as known in the art.
  • Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
  • the candidate polypeptide agent may be isolated and purified in accordance with conventional methods of recombinant synthesis.
  • a lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
  • compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification.
  • percentages will be based upon total protein.
  • the candidate polypeptide agents to be screened are antibodies.
  • antibody or “antibody moiety” is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.
  • the specific or selective fit of a given structure and its specific epitope is sometimes referred to as a “lock and key” fit.
  • the archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g.
  • Antibodies utilized in the present invention may be either polyclonal antibodies or monoclonal antibodies. Antibodies are typically provided in the media in which the cells are cultured.
  • Candidate agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • Candidate agents are screened for biological activity by adding the agent to at least one and usually a plurality of cell samples, usually in conjunction with cells not contacted with the agent.
  • the change in parameters in response to the agent is measured, and the result evaluated by comparison to reference cultures, e.g. in the presence and absence of the agent, obtained with other agents, etc.
  • the agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture.
  • the agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution.
  • a flow-through system two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second.
  • a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.
  • a plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
  • determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions.
  • the concentrations may be further refined with a second series of dilutions, if necessary.
  • one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.
  • Various methods can be utilized for quantifying the selected parameters. For example, flow cytometry, immunohistochemistry, or FRET may be employed to measure A
  • Western blotting and kinase assays may be used to measure phosphorylation of PKA, MAPK, Akt, and cFos.
  • the fluorescent indicators Fura-2 and BCECF may be employed to measure changes in cytosolic Ca(+2)
  • Radioimmunoassays or FRET may be employed to measure changes in cytosolic cAMP. Such methods would be well known to one of ordinary skill in the art.
  • the subject amyloid oligomer compositions find use in experiments to identify signaling pathways that are activated by binding of an amyloid oligomer of interest to its receptor.
  • cells expressing an amyloid receptor may be contacted with a subject amyloid oligomer composition, and the gene expression profile of the contacted cells compared to the gene expression profile of cells not contacted with the subject composition, e.g. by isolating RNA or protein from contacted cells and cells not contacted with the subject composition, and analyzing the RNA or protein by, e.g., RNA or protein microarray, qRT-PCR, Northern blotting, Western blotting, and the like.
  • cells expressing an amyloid receptor may be contacted with a subject amyloid oligomer composition, and the activation state (e.g. phosphorylation state, ubiquitination state, acetylation state, proteolytic cleavage state, etc.) of one or more proteins in the contacted cells may be compared to the activation state of one or more proteins in cells not contacted with the subject composition, e.g. by Western blotting or flow cytometry using antibodies that are specific for the activation state of the protein(s) of interest.
  • the activation state e.g. phosphorylation state, ubiquitination state, acetylation state, proteolytic cleavage state, etc.
  • Such experiments may be used to identify changes in gene expression or protein activity in the presence of the amyloid oligomer, which may in turn be used to identify which signaling pathways have become more or less active in the cells as a result of contacting with the amyloid oligomer.
  • the subject amyloid oligomer compositions find use in experiments to identify a novel receptor for an amyloid oligomer of interest.
  • the amyloid oligomer composition may be immobilized on a solid support (e.g. polystyrene bead, microplate, etc), and a cell lysate passed over the immobilized oligomer composition.
  • Bound receptor may be identified by, e.g. 2D-electrophoresis, MALDI-TOF, mass spectrometry, etc.
  • the specificity of the protein that is identified for the amyloid oligomer may be confirmed by, for example, co-immunoprecipitation of labeled amyloid oligomer and receptor, e.g. ectopically expressed by cells, or endogenously expressed in vivo, e.g. using receptor-specific antibodies or antibodies specific for a receptor tag.
  • the subject amyloid oligomer compositions find use in creating an animal model of an amyloid-associated disease.
  • an animal model of an amyloid-associated disease may be created by ectopically expressing a subject amyloid monomer composition comprising a disease-associated amyloid peptide/polypeptide from the genome in the animal, e.g. as a transgene in the animal, e.g. as described above.
  • the animal model may be created by administering to the animal a subject amyloid oligomer composition comprising the disease-associated amyloid peptide/polypeptide in an amount effective to induce the disease.
  • the administration of the subject composition can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration.
  • the subject composition may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.
  • the subject composition may be formulated for immediate activity or it may be formulated for sustained release. For some conditions, particularly central nervous system conditions, it may be necessary to formulate the subject composition to cross the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • BBB blood-brain barrier
  • osmotic means such as mannitol or leukotrienes
  • vasoactive substances such as bradykinin.
  • a BBB disrupting agent can be co-administered with the subject composition when the composition is administered by intravascular injection.
  • a syringe e.g. intravitreally or intracranially
  • continuous infusion e.g. by cannulation, e.g. with convection
  • implanting a device upon which the subject composition has been reversably affixed see e.g. US Application Nos. 20080081064 and 20090196903, incorporated herein by reference).
  • reagents, devices and kits thereof for practicing one or more of the above-described methods.
  • the subject reagents and kits thereof may vary greatly.
  • reagents and kits may include one or more of the following: amyloid peptide/polypeptide, ligand, ligand binding protein, reagents for the conjugation of ligand to amyloid peptide/polypeptide, oligomerization moiety, reagents for the conjugation of oligomerization moiety to amyloid peptide/polypeptide, amyloid monomers, amyloid oligomers, reagents to test the purity of an amyloid monomer or oligomer composition, and the like.
  • the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
  • Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded.
  • Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
  • Soluble beta-amyloid (Ap) oligomers are highly heterogeneous among different preparations, limiting their use in quantitative cell biological and biochemical studies.
  • Ap-streptavidin tetramers Ap-SAvT
  • Ap receptors such as PirB and LilrB2
  • Ap-SAvT is a reproducible Ap mimetic that can act as a potent, biologically active ligand for PirB/LilrB2
  • AD Alzheimer’s disease
  • Ap p-amyloid
  • PirB Paned Immunoglobulin-like Receptor B
  • LilrB2 Leukocyte Immunoblobulin-like Receptor B2
  • Ap oligomer binding to PirB/LilrB2 perturbs downstream signaling cascades implicated in neuronal actin organization (e.g.
  • Ap oligomer preparations consist of heterogeneous mixes of various aggregates and monomers of Ap peptides, it has been difficult to generate reproducible, synaptotoxic species of Ap oligomers, and to study how they interact with receptors and engage downstream pathways.
  • Ap oligomers we created a chemically defined Ap tetramer amenable to quantitative cell biological and biochemical studies by using streptavidin (SAv) that is site-specifically labeled with a fluorophore (i.e. Alexa Fluor 647 or Fluorescein) (Fig. 1a).
  • SAv streptavidin
  • the Ap42-SAv conjugates also contain small amounts of 8-mer and 16-mer forms as detected by Western blot for Ap as discrete bands; this pattern is distinct from the broad, heterogeneous distribution of bands seen in Western blots for A
  • 342-SAvT can recapitulate the behavior of A
  • HEK 293 cells expressing PirB, a high-affinity receptor for A
  • 342- SAvT to PirB expressing HEK293 cells is comparable to that of A
  • 342-SAvT also exhibits prominent binding to LilrB2-expressing HEK293 cells, in comparison to barely detectable binding to LilrBI - or LilrB3-expressing cells (Fig. 5), again consistent with A
  • 340 which is more abundantly produced in Alzheimer's-affected brain tissue, can also bind to PirB when it is tetramerized.
  • 340- SAvT (Fig. 1b), but not monomeric A
  • the observed interactions are saturable, with an apparent dissociation constant ( d) of 33 nM for A
  • 3-SAvT recapitulates the receptor binding characteristics of A
  • 3-SAvT binding to PirB is competitive with that of known blocking reagents or competitors for A
  • 342-SAvT binding is effectively prevented by co-incubation with an anti-Ap antibody, 4G8 (Fig. 2a).
  • 342-SAvT binding is blocked by the anti-PirB antibody (6C1 ) (Fig. 2b). This antibody recognizes the D1 D2 domain of PirB, where A
  • 342- SAvT binding is significantly reduced when cells are co-treated with A
  • 342-SAvT to LilrB2 is inhibited by anti-Ap (4G8) or anti-LilrB2 (AF2078) antibodies (Fig. 6b and 7).
  • 3 oligomers can be pulled down using Fc reagents such as soluble PirB (D1 D6)-Fc and LilrB2 (D1 D4)-Fc, which lack the transmembrane and intracellular domains.
  • Fc reagents such as soluble PirB (D1 D6)-Fc and LilrB2 (D1 D4)-Fc, which lack the transmembrane and intracellular domains.
  • These reagents but not a control A
  • 3-SAvT can pull down other known receptors for A
  • 342 oligomers are known to alter neuronal signaling pathways. To examine if A
  • 342-SAvT can affect neuronal signaling upon binding to PirB, cortical neurons cultured in vitro were studied. Cofilin activation and PSD-95 loss are induced following application of A
  • PirB/_ cortical neurons
  • Fig. 3c, d cortical neurons
  • 3 streptavidin tetramers can thus be used to study signaling pathways associated with Amyloid-
  • Amyloid-p streptavidin tetramers can be used in assays to screen small molecules or to discover drugs that block Ap binding to target receptors/proteins. Since Ap-SAv tetramers bind reproducibly to PirB/LilrB2 and recapitulate Ap oligomer binding (i.e. Ap42-SAvT competes with Ap42 oligomers for PirB binding), this interaction can be used to screen for blocking compounds including those from large compound libraries, antibodies etc.
  • Reagents were obtained from the following sources: antibodies to amyloid- P 17-24 (4G8; #SIG39220) and neuronal class III tubulin (Tuj1 ; #PRB-435P) are from Covance (Emerville, GA); blocking antibody to LilrB2 (#AF2078), as well as LilrB2-Fc (#2078- T4) and EphB2-Fc (#467-B2-200) from R&D Systems (Minneapolis, MN); antibodies to cofilin (#3312, #5175) and pCofilin (#3313) from Cell Signaling Technology (Danvers, MA); blocking antibody to PirB (6C1 ; #550348) from BD Biosciences (San Jose, CA); antibody to MAP2 (#AB15452) from Millipore (Temecula, CA); antibody to PSD-95 (#MA1 -045) and Fluorescein maleimide (Pierce #62245) from Ther
  • Lipofectamine and Alexa Fluor 647 C2-maleimide are from Invitrogen (Grand Island, NY).
  • Protease inhibitor cocktail (#P8340), phosphatase inhibitor cocktails (#P5726, #P0044) and all other chemicals are obtained from Sigma (St. Louis, MO).
  • Plasmids encoding Myc-tagged human LilrBI (accession #: NM_001081637.1 ), LilrB2 (accession #: NM_001080978.1 ) and LilrBS (accession #: NM_006864.2), as well as murine PirA1 (accession #: NM_011087.1 ), PirA4 (accession #: NM_011091.1) are obtained from OriGene Technologies (Rockville, MD); PirB 8 , PirB-Fc and LilrB2-Fc constructs 6 were generated as described previously.
  • Ap-SAvT generation To generate tetrameric Ap streptavidin conjugates (Ap-SAvT), a custom-engineered streptavidin containing a short linker with the C-terminal Cysteine was expressed in E.coli, refolded in the presence of reducing agent as previously described, and site specifically coupled to Alexa Fluor 647 or fluorescein maleimide.
  • the resulting labeled streptavidin was then mixed with monomeric N-terminal biotinylated A
  • HEK 293 cells maintained in DMEM containing 10% fetal bovine serum, supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM glutamine were transiently transfected with expression vectors encoding PirB, PirA1 , PirA4, LilrBI , LilrB2, LilrB3, or control plasmids, and plated onto 8 chamber slides (Thermo Scientific).
  • the bound mono-Ap, oligo- Ap, or Ap-SAvT was visualized with streptavidin-Alexa fluorophore (Alexa 488, 598 or 647; wavelength choice depending on experiments) or SAv-conjugated Alexa 647/FITC; DAPI was used to counterstain cell nuclei. Fluorescent images were captured with fixed illumination and exposure time using a Nikon 20X objective of numerical aperture 0.75, and then fluorescence intensity was quantified using Imaged software (NIH). In each experiment, values from control vector transfected cells were used as background, and subtracted from those of experimental cells.
  • 3-SAvT immunoprecipitation with Fc-fusion proteins To determine the levels of A

Abstract

Amyloid oligomer compositions and amyloid monomer compositions used to make amyloid oligomer compositions are provided. Also provided are reagents and kits thereof that find use in preparing these compositions. These amyloid oligomer compositions have a high affinity and specificity for amyloid oligomer binding partners on cells. As such, the subject amyloid oligomer compositions find many uses in research and drug development.

Description

AMYLOID OLIGOMER COMPOSITIONS
CROSS REFERENCE TO OTHER APPLICATIONS
[0001] This application claims the benefit of U.S. provisional application No. 63/286,451 , filed December 6, 2021 , the contents of which are hereby incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE
[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “S21 -285_STAN- 978WO_Seqlist” created on November 29, 2022, and having a size of 16 KB. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
GOVERNMENT RIGHTS
[0003] This invention was made with government support under contract EY002858 awarded by the National Institutes of Health. The Government has certain rights in the invention.
FIELD OF THE INVENTION
[0004] This invention pertains to amyloid polypeptide compositions.
BACKGROUND OF THE INVENTION
[0005] Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Prion disease, Huntington's disease, and amyotrophic lateral sclerosis, and neurodevelopmental disorders such as Down’s Syndrome are increasingly being realized to have common cellular and molecular mechanisms including protein aggregation and inclusion body formation in selected brain regions. It is believed that small intermediates termed as ‘soluble oligomers’ or ‘amyloid oligomers’ in the aggregation process might influence cellular activity and induce cellular dysfunction (Gadad BS, et al. (2011 ) Targeting oligomers in neurodegenerative disorders: lessons from a-synuclein, tau, and amyloid-p peptide. J Alzheimers Dis;24 Suppl 2:223-32). See also, Kim et al. (2013) Science 341 , 1399-1404.
[0006] Current methods for studying the effect of amyloid oligomers on cells rely on the use of compositions that are created by allowing amyloid peptide/polypeptide to oligomerize randomly in vitro. However, these compositions are highly heterogeneous, making it difficult to the study their interactions with cellular proteins and to establish which form(s) of amyloid are responsible for inducing cellular dysfunction (Benilova, I., et al. (2012). The toxic Abeta oligomer and Alzheimer's disease: an emperor in need of clothes. Nat Neurosci 15, 349-357). To study systematically the interaction between amyloid oligomers and their receptors, a chemically defined version of amyloid oligomers is required. The present invention addresses these issues.
SUMMARY OF THE INVENTION
[0007] Amyloid oligomer compositions, and amyloid monomer compositions used to make amyloid oligomer compositions, are provided. Also provided are reagents and kits thereof that find use in preparing these compositions. These amyloid oligomer compositions have a high affinity and specificity for amyloid oligomer binding partners on cells. As such, the subject amyloid oligomer compositions find many uses in research and drug development.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.
[0009] Figure 1 . A|3-streptavidin conjugation generates reproducible tetrameric A|3 (A|3-SAvT), which selectively binds PirB with nanomolar affinity, (a) Schematic representation of A|3-SAvT fluorescently-tagged with Alexa Fluor 647 or Fluorescein (M.W. = 72kDa). (b) A|3-SAvT generated by conjugating SAv-Alexa 647 to biotinylated monomeric A|342 or A|340 peptides (1 :4 ratio) were analyzed by Western blotting in parallel with oligomeric A|342. A|342 or A|340 predominantly form tetrameric complexes with SAv (A|342- and A|340-SAvT); A|342 also generates small amounts of 8-mer or 16-mer with SAv (arrowheads). In contrast, A|342 oligomers are highly heterogeneous. See also online methods for more details, (c-e) A|342- SAvT selectively binds to PirB comparably to A|342 oligomers, (c) HEK293 cells were transfected with PirB and treated with monomeric A|342 (100 nM), oligomerized A|342 (100 nM), or A|342-SAvT (25 nM); bound A|3 or A|3-SAvT was then visualized. SAv-conjugated to scrambled peptide for A|342 (Scr-SAvT; 25 nM) or SAv alone (SAvT; 25 nM) were used as negative controls for A|342-SAvT. Scale bar = 10 pm. See also Fig. 4a for A|3-SAvT fluorescently-tagged with Fluorescein, (d) Similar to A|342 oligomers, A|342-SAvT (25 nM) does not bind HEK293 cells expressing PirA1 , PirA4 or control vector, (e) Quantification of A|342-SAvT binding shown in (c) and (d) (Mean ± SEM; n = 3). Expression of control (EV), PirA1 , PirA4 or PirB was verified by Western blotting (inset), (f) A|340-SAvT (25 nM) but not monomeric A|340 (100 nM) binds to PirB expressed in HEK293 cells, (g) Dose-dependent binding of A|340- and A|342-SAvT to PirB-expressing HEK293 cells. To measure specific binding, values from corresponding control cells (empty vector transfected) were subtracted and saturation curves were plotted using GraphPad Prism software (Mean ± SEM; n = 8). (h) Scatchard plot analysis of (g). Calculated d for A|342-SAvT = 33 nM and d for A|340-SAvT = 33 nM.
[0010] Figure 2. A|3-SAvT competes with and recapitulates A|3 oligomers for receptor binding, (a) Co-incubation of A|342-SAvT (10 nM) with anti- Ap (4G8) antibodies (50 nM) prevents A|342- SAvT binding to PirB. In control treatments, the same amount of control IgG was applied. Mean ± SEM, n = 4, ***P<0.001 (Student t-test). (b) Co-treatment of anti-PirB (6C1 ) antibodies but not control IgG (50 nM each) to PirB-expressing HEK 293 cells markedly diminishes A|342- SAvT (10 nM) binding (Mean ± SEM, n = 4, ***P<0.001 , t-test). See also Supplementary Fig. 3a. (c) Co-treatment of A|342 oligomers (200 and 500 nM) significantly diminishes A|342-SAvT (10 nM) binding to PirB; as negative controls, vehicle or the same amount of scrambled peptides for A|342 (A|3-Scr) was applied. Mean ± SEM, n = 5, **P<0.01 ; ***P<0.001 (t-test). (d) Soluble PirB-, LilrB2-, and EphB2-ectodomains fused to IgG-Fc, which were previously shown to interact with A|342 oligomers, can immunoprecipitate A|342-SAvT. In contrast, PirB (D5D6)- Fc does not pull down A|342-SAvT. The results were repeated in two independent experiments, (e) Immunoprecipitation of A|340-SAvT with PirB-, LilrB2-, or EphB2-Fc proteins, (f) Quantification of A|340-SAvT binding represented in (e). Average band intensities ± SEM (n = 3), *P<0.05; **P<0.01 , t-test (relative to PirB (D5D6)-Fc).
[0011] Figure 3. A|342-SAvT binds neuronal PirB and activates downstream pathways, (a) A|342-SAvT but not Scr-SAvT (25 nM each; Red) enriched in punctate patterns along the dendrites of EGFP (Green) -express! ng cortical neurons (cultured 12 days in vitro; DIV12); representative images are shown. Scale bar = 5 pM. (b) A|342-SAvT (25 nM) bind in punctate patterns (arrowheads) along the dendrites of WT cortical neurons (DI V21 ) ; binding is markedly diminished in cortical neurons from PirB-/- mice (right). Red: A|342-SAvT (Alexa 647). Green: immunostaining for the dendrite-specific marker MAP2. (c) A|342-SAvT (10 nM) induces a decrease in cofilin phosphorylation (= activation; 1 hr treatment) as well as loss of postsynaptic protein PSD-95 (24hr treatment) in WT cortical neurons but not in PirB-/- neurons (DIV18-22). Anti-Tuj1 (pill-tubulin) antibodies detect neuron-specific tubulin, (d) Quantification of cofilin phosphorylation (pCofilin/Cofilin, n = 7) and PSD-95 levels (n = 4) represented in (c). Mean ± SEM, *P< 0.05, D-test.
[0012] Figure 4. A|3-SAvT mimics A|3-oligomers for PirB binding, (a) A|342- SAvT fluorescently-tagged with fluorescein but not control SAvT (25 nM) binds to PirB expressing HEK293 cells. Scale bar = 10 pm. (b) N-terminally biotinylated A|342 or scrambled peptides for A|342 were conjugated with streptavidin and analyzed by Coomassie gel staining. Both A|342 and scrambled peptides for A|342 form tetramers efficiently, (c) Schematic representation of A|3 or A|3-SAvT binding to PirB. Tetramerization with streptavidin permits not only A|342 but also A|340 to bind PirB with nanomolar affinities, which are significantly higher than that of A|342 oligomers.
[0013] Figure 5. A|342-SAvT selectively binds to LilrB2, a human ortholog of PirB. HEK293 cells transfected with LilrB2 (a), or its homolog LilrBI or LilrB3 (b) were treated with A|342- SAvT (25 nM), or monomeric or oligomeric A|342 (100 nM each), and bound A|342-SAvT or A|342 was visualized. Scale bars = 10 pm (c) Quantification of A|342-SAvT binding shown in (a) and (b). Mean ± SEM, n = 3.
[0014] Figure 6. Anti-PirB (6C1 ) or anti-LilrB2 (AF2078) antibodies effectively block oligo- A|342 binding to PirB or LilrB2. (a) Co-treatment of anti-PirB (6C1 ) antibody prevents oligo- A|342 binding to PirB-expressing HEK293 cells in a dosedependent fashion, (b) Anti-LilrB2 (AF2078) antibodies block oligo-A|342 binding to LilrB2. These antibodies exhibit the strongest inhibition for oligo-A|342 binding to PirB and LilrB2, among currently available antibodies. Mean ± SEM, n = 6, **P<0.01 (t-test).
[0015] Figure 7. A|342-SAvT binding to LilrB2 is blocked by anti- Ap and anti- Lil rB2 antibodies, (a) Anti-Ap (4G8) antibody but not control IgG (50 nM each) prevents A|342-SAvT (10 nM) binding to LilrB2-expressing HEK293 cells. Mean ± SEM, n = 4, ***P<0.001 , t-test. (b) A|342- SAvT (10 nM) binding to LilrB2 is markedly diminished by anti-LilrB2 (AF2078) antibody (50 nM). Mean ± SEM, n = 5, ***P<0.001 , t-test.
[0016] Figure 8. Graphic representation of the Ap tetramer approaches that recapitulate Ap oligomer binding and signaling. (A) Ap oligomer binding to PirB/LilrB2 elicits hyperactivation of cofilin and synapse loss (i.e. decrease of PSD-95), leading to synaptic dysfunction in adult hippocampus as well as in developing visual cortex of Alzheimer’s mice. (B) Ap-streptavidin tetramer (Ap-SAvT) binds to PirB/LilrB2 with enhanced affinity and activates downstream signaling, similar to Ap42 oligomer. Ap-SAvT can be used to drug screening that antagonizes Ap oligomer effects, to identify novel receptors/binding partners for Ap oligomer, to study molecular mechanisms of Ap oligomer binding and signaling.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Amyloid oligomer compositions and amyloid monomer compositions used to make amyloid oligomer compositions are provided. Also provided are reagents and kits thereof that find use in preparing these compositions. These amyloid oligomer compositions have a high affinity and specificity for amyloid oligomer binding partners on cells. As such, the subject amyloid oligomer compositions find many uses in research and drug development. These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the compositions and methods as more fully described below. [0018] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0019] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.
[0021] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
[0022] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.
[0023] The amino acids described herein are preferred to be in the "L" isomeric form. The amino acid sequences are given in one-letter code (A: alanine; C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine; X: any residue). In keeping with standard polypeptide nomenclature, NH2 refers to the free amino group present at the amino terminus (the N terminus) of a polypeptide, while COOH refers to the free carboxy group present at the carboxy terminus (the C terminus) of a polypeptide.
[0024] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
Compositions
Polypeptides
[0025] In some aspects of the invention, amyloid oligomer compositions are provided. By an “amyloid oligomer composition” it is meant a composition of amyloid oligomers, each oligomer comprising two or more amyloid monomer subunits, each amyloid monomer subunit comprising an amyloid peptide/polypeptide and an oligomerization domain. By an “amyloid peptide/polypeptide” it is meant a peptide or polypeptide that folds inappropriately in nature and, as a misfolded structure, interact with other amyloid peptide/polypeptides having the same sequence or other cell components to form insoluble fibrous protein aggregates, or “amyloids”. By an “oligomerization domain”, it is meant a domain that promotes the oligomerization of the amyloid peptide/polypeptide. Also provided in some aspects of the invention are amyloid monomer compositions. By an amyloid monomer composition it is meant the amyloid monomer subunit that oligomerizes to form the amyloid oligomer compositions of the present disclosure.
[0026] As discussed above, by an “amyloid peptide/polypeptide” it is meant a peptide or polypeptide that folds inappropriately in nature and, as a misfolded structure, interacts with other like peptides/polypeptides or other cell components to form insoluble fibrous protein aggregates, or “amyloids”. A number of peptide and polypeptides are known in the art that fold inappropriately in nature and, as misfolded structures, interact with one another or other cell components to form insoluble fibrous protein aggregates, any of which may be used in the amyloid subunits that form the subject amyloid oligomer compositions. Non-limiting examples include beta amyloid (“A|3”), implicated in Alzheimer’s disease, cerebral amyloid angiopathy, and Down’s Syndrome; IAPP (amylin) (“AIAPP”), implicated in Diabetes mellitus type 2; Alpha- synuclein (a-synuclein), implicated in synucleinopathies including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy; Tau, implicated in Alzheimer’s disease and tauopathies; Prion Protein, Scrapie form (“PrPSc”, or “APrP”), implicated in spongiform encephalopathies e.g. Bovine spongiform encephalopathy; Huntingtin, implicated in Huntington's Disease; Calcitonin (“ACal”), implicated in Medullary carcinoma of the thyroid; Atrial natriuretic factor (“AANF”), implicated in cardiac arrhythmias and isolated atrial amyloidosis; Apolipoprotein Al (“AApoAl ”), implicated in Atherosclerosis; Serum amyloid A (“AA”), implicated in Rheumatoid arthritis; Medin (“AMed”), implicated in aortic medial amyloid; Prolactin (“APro”), implicated in prolactinomas; Transthyretin (“ATTR”), implicated in Familial amyloid polyneuropathy; Lysozyme (“ALys”), implicated in Hereditary non-neuropathic systemic amyloidosis; Beta 2 microglobulin (Ap2M), implicated in Dialysis related amyloidosis; Gelsolin (“AGel”), implicated in Finnish amyloidosis; Keratoepithelin (“AKer”), implicated in Lattice corneal dystrophy; Cystatin (“ACys”), implicated in Cerebral amyloid angiopathy (Icelandic type); Immunoglobulin light chain (“AL”), implicated in systemic AL amyloidosis, and S-IBM, implicated in Sporadic Inclusion Body Myositis; any of which may be used in the subject amyloid oligomer compositions.
[0027] In certain embodiments, the amyloid peptide/polypeptide is Ap. By Ap, “Abeta”, “betaamyloid”, “amyloid beta, or amyloid p, it is meant a peptide of about 28-55 amino acids that is derived from the carboxy terminus of amyloid precursor protein. Amyloid precursor protein, or “APP” (GenBank Accession Nos. NM_000484.3 (isoform a), NM_201413.2 (isoform b), NM_201414.2 (isoform c), NM_001136016.3 (isoform d), NM_001136129.2 (isoform e), NM_001136130.2 (isoform f), NM_001136131 .2 (isoform g), NM_001136131 .2 (isoform h), NM_001204302.1 (isoform i), NM_001204303.1 (isoform j)), is a cell surface receptor and transmembrane precursor protein that is naturally cleaved by p and y secretases to form a number of peptides, including Ap peptides. Ap is the main component of deposits, called amyloid plaques, found in the brains and vascular walls of patients with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) and associated with retinal ganglion cells in patients with glaucoma. As demonstrated in the working examples below, tetramers of Ap peptide bind with specificity to the PirB/LilrB2 receptor on neurons and induced changes in cellular activity.
[0028] Two major variants, Api -40 (“Ap40”) (SEQ ID NO:1 ) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) and Api -42 (“Ap42”) (SEQ ID NO:2) (DAEFRHDSGYEVHHQKLVFAAEDVGSNKGAIIGLMVGGVVIA), are produced by alternative carboxy-terminal processing of APP (Selkoe et al. (1988) Proc. Natl. Acad. Sci. USA 85:7341 -7345; Selkoe, (1993) Trends Neurosci 16:403-409). Api -42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. Other naturally occurring variants include, e.g., Api -28 (SEQ ID NO:3) (DAEFRHDSGYEVHHQAAVFAAEDVGSNK), AP12-28 (SEQ ID NO:4) (VHHQKLVFFAEDVGSNKC), Api -37 (SEQ ID NO:5) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG), Api -38 (SEQ ID NO:6) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG), A 1-39 (SEQ ID NO:7) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV), A 1 -43 (SEQ ID N0:8) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT), A 1 -44 (SEQ ID N0:9) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATV), A 1-45 (SEQ ID NQ:10) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVI), A 1-46 (SEQ ID N0:11) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIV), A 1 -47 (SEQ ID N0:12) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVI), A 1 -48 (SEQ ID N0:13) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVIT), A 1 -49 (SEQ ID N0:14) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITL), A 1-55 (SEQ ID N0:15) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK), A 2-4O (SEQ ID N0:16) (AEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV), and A 3-4O (SEQ ID N0:17) (EFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV), any of which may be used in the subject amyloid oligomer compositions.
[0029] In some embodiments, the Ap peptide comprises native APP sequence. In other words, the Ap of the subject compositions comprises a sequence that is found in wild type APP as it exists in nature, i.e. it is “native Ap”. Non-limiting examples of native APP sequences upon which native Ap peptides of the subject compositions may be based include native human APP polypeptide sequences, e.g. as may be found at GenBank Accession Nos. NM_000484 (isoform a); NM_201413 (isoform b); NM_201414 (isoform c); NM_001136016 (isoform d); NM_001136129 (isoform e); NM_001136130 (isoform f); NM_001136131 (isoform g); NM_001204301 (isoform h); NM_001204302 (isoform i); and NM_001204303 (isoform j); native mouse APP polypeptide sequences, e.g. as may be found at GenBank Accession Nos. NM_001198823.1 (isoform 1 ); NM_007471.3 (isoform 2); NM_001198824.1 (isoform 3); NM_001198825.1 (isoform 5); and NM_001198826.1 (isoform 6); and native rat APP polypeptide sequences, e.g. as may be found at GenBank Accession No. NM_019288.2. Other native APP sequences may be readily determined by methods known in the art, e.g. in silica by employing publicly available databases, or empirically by isolating and sequencing the native APP of interest. Thus, a native Ap can have the amino acid sequence comprised by, e.g. naturally occurring human APP polypeptide, murine APP polypeptide, or polypeptide from any other mammalian species, or from non-mammalian species, e.g. Drosophila, C. elegans, and the like.
[0030] In other embodiments, the Ap peptide comprises variant APP sequence. In other words, the Ap of the subject compositions comprises a sequence having less than 100% sequence identity with a native wild type APP sequence over the length of the fragment, i.e. it is “variant Ap”. Variant Ap peptides include peptides wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the native sequence. In such instances, from about one to 20 amino acid residues may be deleted, and optionally substituted by one or more amino acid residues. Also encompassed as variant A|3 peptides are derivatives of the above native polypeptides, wherein an amino acid residue has been substituted or covalently modified so that the resulting product has a non-naturally occurring amino acid. In some instances, the substitution is naturally occurring, e.g. A|31 -42 H13R, A|31-42 V18A, A[31 -42 F19P, A|31 -42 E22D, A 1-42 E22V, A|31 -42 E22A, A|31 -42 D23A, A|31-42 G25A, A|31 -42 N27A, A|31 -42 K28A, Api -42 G29A, A[31 -42 131 A, A[31 -42 G37A, the English Mutation, the Iowa Mutation, the Tottori-Japanese Mutation, the Flemish Mutation, the Arctic Mutation, the Italian Mutation, etc. Ordinarily, biologically active variants will have an amino acid sequence having 75% or more sequence identity, 80% or more sequence identity, 85% or more amino acid sequence identity, 90% or more amino acid sequence identity with a native sequence polypeptide, preferably 95% or more, in some instances at least 99% sequence identity. Various methods known in the art may be utilized in developing such variant A|3.
[0031] In certain embodiments, the amyloid peptide/polypeptide is a-synuclein. By “a- synuclein”, “alpha-synuclein”, or “non A4 component of amyloid precursor”, it is meant the polypeptide that is encoded in humans by the SNCA or “aSyn” gene, or an amyloid-forming peptide or variant thereof. Sequence for a-synuclein polypeptide may be found at GenBank Accession Nos. NM_000345.3 (variant 1 ), NM_001146054.1 (variant 2), NM_001146055.1 (variant 3), and NM_007308.2 (variant 4, the “NACP112” variant). Point mutations in A30P, A53T, or E46K, and gene duplication or triplication of the aSyn locus have been identified as causal factors of early onset familial PD; E46K has also been associated with Lewy body dementia. As taught in Wang et al. (“A soluble a-synuclein construct forms a dynamic tetramer”, Proc Natl Acad Sci U S A. 2011 , 108(43) :17797-802) and Kalia et al. (“a-Synuclein oligomers and clinical implications for Parkinson disease”, Ann Neurol. 2012 Aug 28), alpha- synuclein forms tetramers in solution. The relevance of these tetramers to disease is unknown.
[0032] In some embodiments, the a-synuclein polypeptide comprises native a-synuclein sequence. In other words, the a-synuclein of the subject compositions comprises a sequence that is found in wild type a-synuclein, i.e. it is “native a-synuclein”. Non-limiting examples of native a-synuclein sequences upon which native a-synuclein polypeptides of the subject compositions may be based include native human a-synuclein polypeptide sequences, e.g., NM_000345.3 (variant 1), NM_001146054.1 (variant 2), NM_001146055.1 (variant 3), and NM_007308.2 (variant 4); native mouse a-synuclein sequences, e.g., as may be found at GenBank Accession Nos. NM_001042451 .1 and NM_009221.2; and native rat a-synuclein sequences, e.g., as may be found at GenBank Accession No. NM_019169.2. Other native a- synuclein sequences may be readily determined by methods known in the art, e.g. in silico by employing publicly available databases, or empirically by isolating and sequencing the native a-synuclein of interest.
[0033] In other embodiments, the a-synuclein polypeptide comprises variant a-synuclein sequence. In other words, the a-synuclein of the subject compositions comprises a sequence having less than 100% sequence identity with a native wild type a-synuclein sequence over the length of the fragment, i.e. it is “variant a-synuclein”. Variant a-synuclein polypeptides include polypeptides wherein one or more amino acid residues are added at the N- or C- terminus of, or within, the native sequence. In such instances, from about one to 20 amino acid residues may be deleted, and optionally substituted by one or more amino acid residues. Also encompassed as variant a-synuclein polypeptides are derivatives of the above native polypeptides, wherein an amino acid residue has been substituted or covalently modified so that the resulting product has a non-naturally occurring amino acid. In some instances, the substitution is naturally occurring, e.g. A30P, A53T, or E46K. Ordinarily, biologically active variants will have an amino acid sequence having 75% or more sequence identity, 80% or more sequence identity, 85% or more amino acid sequence identity, 90% or more amino acid sequence identity with a native sequence polypeptide, preferably 95% or more, in some instances at least 99% sequence identity. Various methods known in the art may be utilized in developing such variant a-synuclein.
[0034] In certain embodiments, the amyloid peptide/polypeptide is tau. By “tau”, also known as “MATP”, or “microtubule-associated protein tau”, it is meant the polypeptide that is encoded in humans by the MAPT gene or an amyloid-forming peptide thereof. The tau polypeptide sequence may be found at GenBank Accession Nos. NM_016835.4 (isoform 1 ), NM_005910.5 (isoform 2), NM_016834.4 (isoform 3), NM_016841.4 (isoform 4), NM_001123067.3 (isoform 5), NM_001123066.3 (isoform 6), NM_001203251 .1 (isoform 7), NM_001203252.1 (isoform 8). Mutations in tau have been associated with several neurodegenerative disorders such as Alzheimer's disease, Pick's disease, frontotemporal dementia, cortico-basal degeneration and progressive supranuclear palsy. As discussed in Lasagna-Reeves et al. (“Tau oligomers as potential targets for immunotherapy for Alzheimer's disease and tauopathies.” Curr Alzheimer Res. 2011 , 8(6):659-65), tau and peptides thereof, e.g. peptides of domain R2 (residues V275-V300 of the longest human isoform, htau40), of domain R3 (residues V306-K331 ), and of R4 (V337-H362 ), form tetramers and octamers in solution. The relevance of these tetramers and octamers to disease is unknown.
[0035] In some embodiments, the tau polypeptide comprises native tau sequence. In other words, the tau of the subject compositions comprises a sequence that is found in wild type tau, i.e. it is “native tau”. Non-limiting examples of native tau sequences upon which native tau polypeptides of the subject compositions may be based include native human tau polypeptide sequences, e.g., as may be found at GenBank Accession Nos. NM_016835.4 (isoform 1 ), NM_005910.5 (isoform 2), NM_016834.4 (isoform 3), NM_016841.4 (isoform 4), NM_001123067.3 (isoform 5), NM_001123066.3 (isoform 6), NM_001203251 .1 (isoform 7), NM_001203252.1 (isoform 8); native mouse tau sequences, e.g., as may be found at GenBank Accession Nos. NM_001038609.1 and NM_010838.3; and native rat tau sequences, e.g., as may be found at GenBank Accession No. 1 ,NM_017212.2. Other native tau sequences may be readily determined by methods known in the art, e.g. in silico by employing publicly available databases, or empirically by isolating and sequencing the native tau of interest.
[0036] In other embodiments, the tau polypeptide comprises variant tau sequence. In other words, the tau of the subject compositions comprises a sequence having less than 100% sequence identity with a native wild type tau sequence over the length of the fragment, i.e. it is “variant tau”. Variant tau polypeptides include polypeptides wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the native sequence. In such instances, from about one to 20 amino acid residues may be deleted, and optionally substituted by one or more amino acid residues. Also encompassed as variant tau polypeptides are derivatives of the above native polypeptides, wherein an amino acid residue has been substituted or covalently modified so that the resulting product has a non-naturally occurring amino acid. Ordinarily, biologically active variants will have an amino acid sequence having 75% or more sequence identity, 80% or more sequence identity, 85% or more amino acid sequence identity, 90% or more amino acid sequence identity with a native sequence polypeptide, preferably 95% or more, in some instances at least 99% sequence identity. Various methods known in the art may be utilized in developing such variant tau.
[0037] As mentioned above, in addition to comprising amyloid peptide/polypeptide, the amyloid monomer subunits of the amyloid oligomers comprise an oligomerization domain. In some instances, the oligomerization domain comprises a ligand that complexes with a ligand binding partner, which, in turn, promotes the directed oligomerization of the amyloid peptide/polypeptide. By a “ligand” and a “ligand binding partner” it is meant a complementary set of molecules that demonstrate specific binding, generally of relatively high affinity (an affinity constant, Ka, of about 106 M-1), e.g., a biotin ligand and its binding partner streptavidin or avidin, a hapten and antibody, an enzyme and inhibitor, a lectin and carbohydrate, a ligand and its receptor. In such instances, the amyloid peptide/polypeptide is typically covalent bound to the ligand. In other instances, the oligomerization domain comprises an oligomerization moiety, i.e. one or more functional groups - e.g., peptide, polypeptide, small molecule or nucleic acid composition -- that oligomerizes in solution, thereby promoting the directed oligomerization of the amyloid peptide/polypeptide to which it is covalently bound. In such instances, the amyloid peptide/polypeptide is covalent bound to the oligomerization moiety. [0038] As mentioned above, in some embodiments, the amyloid monomer subunits comprise an amyloid peptide/polypeptide covalently bound to a ligand that, in turn, complexes with a ligand binding partner. As used herein, the phrase "ligand-ligand binding partner pair" refers to any ligand and its ligand binding partner that are capable of recognizing and binding to each other. The ligand(s) and binding partner can be any moieties that are capable of recognizing and binding to each other to form a complex. In some instances, the ligand and binding partner are non-covalently bound. In other instances, the ligand and binding partner form a complex via the binding of a third intermediary substance. The ligand and ligand binding partner can be naturally occurring or artificially produced, and optionally can be aggregated with other species of molecules. In some instances, the ligand binding partner is an oligomerizing ligand binding partner. In other words, the ligand binding partner oligomerizes in solution, thereby promoting the directed oligomerization of the ligand (and hence amyloid peptide/polypeptide) to which it is complexed, e.g. streptavidin. In some instances, the ligand binding partner is polyvalent. In other words, the ligand binding partner that has two or more, e.g. three or four, ligand binding sites, e.g. the F(ab’)2 of an antibody, thereby promoting the directed oligomerization of the ligand (and hence amyloid peptide/polypeptide) to which it is complexed. [0039] In some embodiments, the ligand binding partner dimerizes in solution, or is divalent, e.g., an antibody. In such instances, ligand binding partner promotes the dimerization of the amyloid peptide/polypeptide into homo- or hetero-dimers. By a “dimer”, it is meant that the oligomer comprises two polypeptides, each comprising an amyloid peptide/polypeptide. In other embodiments, the ligand binding partner trimerizes in solution, or is trivalent. In other words, it promotes the trimerization of the amyloid peptide/polypeptide into homo- or heterotrimers. By a “trimer”, it is meant that the oligomer comprises three polypeptides, each comprising an amyloid peptide/polypeptide. In other embodiments, the ligand binding partner tetramerizes in solution, e.g., streptavidin, or is tetravalent, e.g. a streptavidin complex. In other words, it promotes the tetramerization of the amyloid peptide/polypeptide into homo- or hetero-tetramers. By a “tetramer”, it is meant that the oligomer comprises four polypeptides, each comprising an amyloid peptide/polypeptide. By a /lomo-dimer, /lomo-trimer, homotetramer, and the like, it is meant that all polypeptides of the oligomer composition comprise the same amyloid peptide/polypeptide. In other words, the oligomer composition is homogeneous for the type of amyloid peptide/polypeptide comprised therein. By a heterodimer, hetero-trimer, hetero-tetramer, and the like, it is meant that the polypeptides of the oligomer composition comprise different amyloid peptide/polypeptide. In other words, the oligomer composition is heterogeneous for the type of amyloid peptide/polypeptide comprised therein.
[0040] Examples of ligands and ligand binding partners include, but are not limited to, biotin (i.e. biotin or biotin derivatives, e.g. Biocytin, NHS-Biotin, Sulfo-NHS-Biotin, TFP-PEG3-Biotin, BMCC-Biotin, lodoacetyl-LC-Biotin, HPDP-Biotin, Amine-PEG2-Biotin, Hydrazide-Biotin, Alkoxyamine-PEG4-Biotin, the photoactivatable derivate TFPA-PEG3-Biotin ), avidin, streptavidin, agonists and antagonists for cell membrane receptors, receptors, toxins and venoms, viral epitopes, hormones such as steroids, hormone receptors, peptides, enzymes and other catalytic polypeptides, enzyme substrates, cofactors, drugs including small organic molecules, opiates, opiate receptors, lectins, sugars, saccharides including polysaccharides, proteins, and antibodies including monoclonal antibodies and synthetic antibody fragments. The ligand may be a B cell epitope (e.g., antibody), either naturally-occurring or synthetic, such as for example, a polyoma epitope, a FLAG epitope, a hemagglutinin epitope for the 12CA5 monoclonal antibody, a polyhistidine tract, and the like. The ligand binding partner can be an antibody, such as, for example, an IgG, IgM, and the like, or an antigen binding fragment thereof. Examples of ligand-ligand binding partner complexes include the following: biotinstreptavidin; antibody-antigen; lectin-carbohydrate; peptide-cell membrane receptor; protein A-antibody; hapten-anti-hapten; digoxigenin-anti-digoxigenin; enzyme-cofactor; and enzymesubstrate. Optionally, ligand binding partners can be formed into polyvalent ligand binding partners according to methods known in the art.
[0041] The amyloid peptide/polypeptide may be covalently bound to the ligand by any convenient method. For example, the amyloid peptide/polypeptide and ligand may be encoded by the same nucleic acid, such that upon translation, the polypeptide that is produced is a fusion polypeptide comprising amyloid peptide/polypeptide covalently bound to the ligand. As another example, the ligand may become covalently bound to the amyloid peptide/polypeptide by modification of an amyloid peptide/polypeptide, e.g by use of a modifying enzyme. Suitable modifying enzymes include, for example, BirA, various glycosylases, famesyl transferase protein, and the like. Nonlimiting examples of ligand that may become covalently bound to an amyloid peptide/polypeptide or subunit thereof by a modifying enzyme include biotin, a sugar, a famesyl group, and the like. In an exemplary embodiment, an amyloid peptide/polypeptide, e.g an Ap peptide, includes a modification site (e.g., the BirA recognition sequence GGGLNDIFEAQKIEWH (Castro, C. et al. J Biol Chem 284, 31028 (2009)), which is used to covalently bind a biotin moiety to the Ap peptide. The biotinylated amyloid peptide/polypeptide, e.g. Ap peptide, is then complexed to streptavidin or avidin, which binds biotin with extremely high affinity. The amyloid multimers, e.g. “Ap-streptavidin tetramers”, or “Ap-SavT”, can then be stored until needed.
[0042] In some instances, the ligand binding partner is free in solution. In other instances, the ligand binding partner is attached to a solid support. Examples of suitable solid supports include beads (e.g., magnetic beads), membranes, microtiter plates, and the like. The support can be glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, PVDF, and the like. The use of a binding partner linked to a solid support can be useful for immobilization and/or isolation of cells that recognize the multimeric amyloid peptide/polypeptide, or the purification of receptor(s) that are bound by the multimeric amyloid peptide/polypeptide.
[0043] As mentioned above, in other embodiments, the amyloid monomer subunits that form the subject amyloid oligomer compositions comprise an amyloid peptide/polypeptide covalently bound to an oligomerization moiety. By an oligomerization moiety, it is meant a functional group - e.g., peptide, polypeptide, small molecule or nucleic acid composition -- that oligomerizes in solution, thereby promoting the directed oligomerization of the amyloid peptide/polypeptide to which it is fused. Any moiety that is known in the art to oligomerize in solution at physiological pH, salt concentration, and temperature may be used as an oligomerization moiety in the subject amyloid subunits. By oligomerizing in solution, it is meant that 50% or more of the moiety complexes with itself in solution, e.g. 60% or more, 65% or more, 70% or more, or 75% or more of the moiety binds with itself, in some instances, 80% or more, 85% or more, or 90% or more of the moiety binds with itself, in some cases, 95% or more, 97% or more, or virtually all of the moiety binds with itself.
[0044] In some embodiments, the oligomerization moiety is a dimerization moiety. In other words, it promotes the dimerization of the amyloid peptide/polypeptide into homo- or heterodimers. Non-limiting examples of polypeptides that dimerize in solution that may be used as oligomerization moieties in the subject compositions include Verde Fluorescent Protein (VFP) and VFP mutant dVFP (Hagan, RP et al. (2010) FEBS J. 277(8):1967-19780, Midoriishi Cyan (MBL International), Turbo Yellow Fluorescent Protein (TurboYFP; Evrogen), dimerizationdependent red fluorescent protein (ddRFP; Alford S.C. et al. (2012) Chem Biol. 19(3):353-60), HCRedl and dTomato (Shaner et al., Nat. Biotechnol., 2004, 22, 1524). In other embodiments, the oligomerization moiety is a trimerization moiety. In other words, it promotes the trimerization of the amyloid peptide/polypeptide into homo- or hetero-trimers. Non-limiting examples of polypeptides that form trimers in solution that may be used as oligomerization moieties in the subject compositions include the C-terminal domain of T4 fibritin (foldon) (Guthe, S., et al. (2004) J Mol Biol 337(4):905-15) and the trimerization domain of human Collagen XVIII (Boudko, S.P., et al. (2009) J. Mol. Biol. 392(3) :787-802). In other embodiments, the oligomerization moiety is a tetramerization moiety. In other words, it promotes the tetramerization of the amyloid peptide/polypeptide into homo- or heterotetramers. Non-limiting examples of polypeptides that form tetramers in solution that may be used as oligomerization moieties in the subject compositions include DsRed (US Application No. US2004/0137642), HcRed, ZsGreen, (Clontech), cOFP (Stratagene), cFP484, and streptavidin.
[0045] In some instances, the ligand or oligomerization moiety is covalently bound directly to the amyloid peptide/polypeptide. In other instances, the ligand or oligomerization moiety is covalently bound to the amyloid peptide/polypeptide indirectly, i.e. via a linker region. In other words, the ligand or oligomerization moiety is separated from the amyloid peptide/polypeptide by a linker region. By a “linker region” it is meant a sequence of amino acids, e.g. polar or amphipathic amino acids, that links the amyloid peptide/polypeptide to the ligand or oligomerization moiety. Linker regions are typically about 3 to about 25 amino acids in length, e.g. 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids or more in length, although longer linker regions may also be employed in the subject composition. Often, the linker region allows a flexible, unconstrained solution conformation, in which the geometry of the amyloid oligomer, or its subunits, is unrestricted relative to other domains. In other words, it is a “conformationally flexible linker region”. In some instances, a modification site, such as a BirA modification site, may be included in a linker region, e.g. for the conjugation of ligand to the linker region.
[0046] In some instances, the ligand or oligomerization moiety is covalently bound to the N- terminus of the amyloid peptide/polypeptide. In other words, it is covalently bound, either directly or indirectly, to the N-terminal amino acid of the peptide. In other instances, the ligand or oligomerization moiety is covalently bound to the C-terminus of the amyloid peptide/polypeptide. In other words, it is covalently bound, either directly or indirectly, to the C-terminal amino acid of the peptide. In other instances, the ligand is covalently bound to an amino acid of the amyloid peptide/polypeptide other than the N-terminal or C-terminal amino acid. In other words, it is covalently bound, either directly or indirectly, to an internal amino acid of the peptide.
[0047] In some instances, the ligand binding domain or oligomerization moiety is labeled with a detectable label. As used herein, the terms "label" or "labeled" refer to a molecule or groups of molecules which can provide a detectable signal when the label is incorporated into, or attached to, a polypeptide such as an oligomerization moiety or a ligand binding partner. For example, an oligomerization moiety or a ligand binding partner can be labeled with a radioactive molecule, a luminescent molecule, a fluorescent molecule, a chemi-luminescent molecule, an enzyme, biotinyl moieties, and the like. Methods of labeling polypeptides and binding partners are well known in the art. (See, e.g., Ausubel et al.; Sambrook et al.) Examples of detectable labels include, but are not limited to, radioisotopes (e.g., 3H, 14C, 32P, 35S, 125l, 131l, and the like); fluorescent molecules (e.g., Alexa 488, Alexa 598, Alexa 647, fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin (PE), phycocyanin, allophycocyanin, ortho-phthaldehyde, fluorescamine, peridinin-chlorophyll a (PerCP), Cy3 (indocarbocyanine), Cy5 (indodicarbocyanine), lanthanide phosphors, and the like); enzymes (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase); biotinyl groups (e.g. Biocytin, NHS-Biotin, Sulfo-NHS-Biotin, TFP-PEG3-Biotin, BMCC-Biotin, lodoacetyl-LC-Biotin, HPDP-Biotin, Amine-PEG2-Biotin, Hydrazide-Biotin, Alkoxyamine- PEG4-Biotin, the photoactivatable derivate TFPA-PEG3-Biotin); and the like. In some embodiments, detectable labels are attached by spacer arms or linkers of various lengths to reduce potential steric hindrance.
[0048] The subject amyloid oligomer compositions may be prepared following any convenient method for the preparation of polypeptides and polypeptide complexes. In one exemplary embodiment, amyloid peptide/polypeptide is synthesized in vitro or in vivo. The amyloid peptide/polypeptide is subsequently covalently bound to ligand, e.g. biotin, by chemical reaction to produce an amyloid peptide/polypeptide-ligand composition, e.g. biotinylated amyloid peptide/polypeptide. The amyloid peptide/polypeptide-ligand composition is incubated with binding partner, e.g. streptavidin, under conditions that promote the non- covalent binding of ligand with ligand binding partner and the assembly of amyloid monomer compositions into oligomers, e.g. tetramers. In another exemplary embodiment, amyloid peptide/polypeptide is synthesized in vitro or in vivo. The amyloid peptide/polypeptide is subsequently covalently bound to an oligomerization domain by chemical reaction to produce an amyloid monomer composition, which is allowed to assemble into oligomers. In another exemplary embodiment, amyloid peptide/polypeptide is synthesized in vitro or in vivo already covalently bound to a ligand, i.e., no modification of the amyloid peptide/polypeptide is required to produce the amyloid-ligand fusion polypeptide. The amyloid-ligand fusion polypeptide is incubated with binding partner and allowed to assemble into oligomers. In another exemplary embodiment, amyloid peptide/polypeptides are synthesized in vitro or in vivo covalently fused to an oligomerization moiety, i.e., no modification of the amyloid peptide/polypeptide is required to produce the amyloid-oligomerization moiety fusion polypeptide. The amyloid-oligomerization moiety fusion polypeptide are then allowed to assemble into oligomers at physiologic pH.
[0049] Amyloid peptide/polypeptides and amyloid fusion polypeptides may be produced by any convenient method. For example, amyloid peptide/polypeptides and amyloid fusion polypeptides may be chemically synthesized, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied BioSystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules, e.g. ligands, or to a surface, e.g. a solid support. Thus, for example, cysteines could be introduced to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, BirA recognition sequences for BirA-mediated modification of the peptide, and the like. [0050] As another example, amyloid peptide/polypeptides and amyloid fusion polypeptides may be synthesized in a cell, for example by natural or recombinant synthesis of the peptide or fusion polypeptide, and isolated and purified in accordance with conventional methods of recombinant synthesis. Likewise, amyloid peptide/polypeptide may be synthesized by synthesizing the precursor protein, e.g. APP, in a cell, and subsequently processing the precursor protein in vitro to produce the amyloid peptide/polypeptide. In such cases as when the synthesis is cell-based, a lysate may be prepared of an expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. In some instances, the expression host, i.e. the cell, will endogenously express the amyloid peptide/polypeptide or precursor thereof or amyloid fusion polypeptide. In other instances, the expression host will ectopically express the amyloid peptide/polypeptide or amyloid fusion polypeptide, e.g., the host will be transformed with a nucleic acid vector that comprises nucleic acid sequence that encodes for the amyloid peptide/polypeptide, precursor protein of the amyloid peptide/polypeptide, or amyloid fusion protein, and the host will ectopically express the amyloid peptide/polypeptide, amyloid precursor protein, or amyloid fusion protein from the vector. For the most part, the compositions which are used will comprise at least 50% by weight of the desired product, more usually at least about 75%, 80%, 85%, or 90% by weight, preferably at least about 95% by weight, and in some instances 97%, 98%, 99%, or 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.
[0051] The subject amyloid oligomer compositions of the present disclosure may be readily recognized by virtue of both their structure and function. For example, because the oligomerization of the subject amyloid oligomer compositions is directed, i.e., controlled, by their oligomerization domains, the subject compositions comprise a controlled amount of amyloid peptide/polypeptide per oligomer. As such, the amyloid oligomer compositions of the subject disclosure are typically a substantially homogeneous composition of oligomers comprising the same number of amyloid peptide/polypeptide per oligomer. By a “substantially homogeneous” composition of oligomers, it is meant that about 60% or more of the oligomers in the composition comprise the same number of amyloid monomer subunits, e.g. 65% or more, 70% or more, 75% or more, 80% or more, for example, 85% or more, 90% or more, 95% or more, in some instances 97% or more, comprise the same number of amyloid monomer subunits. For example, an amyloid tetramer composition created by the oligomerization of a tetramerization domain would be substantially homogenous for amyloid tetramers, i.e. oligomers comprising 4 amyloid monomer subunits; while an amyloid dimer composition created by the oligomerization of a dimerization domain would be substantially homogenous for amyloid dimers, i.e. oligomers comprising 2 amyloid monomer subunits. This is in contrast to current methods in the art, which rely upon a random, low-efficiency chemical process that creates a range of oligomerization states simultaneously, with no method to control the number of amyloid peptides per oligomer. The number of subunits in the oligomers of the subject oligomer compositions and the extent of homogeneity of the subject amyloid oligomer compositions may be detected using any convenient method for visualizing amyloid oligomer content in the sample. For example, oligomeric forms may be detected by gel shift and Coomassie gel staining or Western blotting with an antibody specific for the amyloid peptide or oligomerization domain, where oligomeric forms comprising the same number of amyloid subunits will appear as discrete bands, in contrast to the heterogenous smear patterns of bands seen in Western blots for amyloid oligomers generated using current methods in the art.
[0052] As also demonstrated in the working examples of the present disclosure, the subject amyloid oligomer compositions of the present disclosure may also be recognized by their high affinity and specificity for their cognate binding partners, which can be directly attributed to the substantial homogeneity of the subject amyloid oligomer composition as well as the methods by which the subject amyloid compositions are made. This is in contrast to the affinity and specificity observed of amyloid oligomers prepared using current methods in the art, in which only a small amount of amyloid monomer is formed into the active oligomer, creating a reagent that is largely and variably inactive, and which rely on high temperature/high pH to induce aggregation, which renders polypeptides “sticky” and increases the chance of nonspecific interactions. By “affinity” as used herein, it is meant the binding strength between an amyloid oligomer and its binding partner, e.g. receptor. By “receptor specificity”, “specific binding,” “specifically bind,” and the like as used herein, it is meant the ability of the subject amyloid oligomer composition to preferentially bind directly to one protein relative to other molecules or moieties on the cell. In certain embodiments, the affinity between an amyloid oligomer and the protein to which it specifically binds when they are specifically bound to each other in a binding complex is characterized by a KD (dissociation constant) of less than 10'6 M, less than 107 M, less than 10-8 M, less than 10-9 M, less than 10-10 M, less than 10-11 M, less than 10-12 M, less than 10-13 M, less than 10-14 M, or less than 10-15 M. As demonstrated using the A|3 tetramers in figures 2 and 3 of the present disclosure, this high affinity and specificity may be observed as a significantly larger amount of receptor binding and downstream signaling per molar concentration of composition than an oligomer composition prepared using current methods in the art, i.e. without the guidance of an oligomerization domain.
[0053] Moreover, and perhaps in view of this increased affinity and specificity, the subject amyloid oligomer compositions have a physiological effect on cells that better recapitulates the physiological effect of A|3 oligomers that form in vivo than amyloid peptides that are allowed to oligomerize randomly. In other words, amyloid oligomers that are formed by directed oligomerization more accurately model amyloid oligomers that form naturally in vivo than amyloid oligomers that are allowed to freely aggregate.
[0054] Because of these characteristics of the subject amyloid oligomer compositions - substantial homogeneity, high affinity and specificity of the amyloid oligomers therein for their binding partner in or on cells -the subject amyloid oligomer compositions find many uses in research and drug development, as described in greater detail in, for example, the Utility section below.
Nucleic acids
[0055] Also encompassed by the present disclosure are nucleic acids that encode for amyloid monomer compositions of the present disclosure, e.g. nucleic acids that encode for amyloid peptide/polypeptide fused to a ligand, nucleic acids that encode for amyloid peptide/polypeptide fused to an oligomerization moiety; and nucleic acids that encode for peptides/polypeptides that may be used to generate the amyloid monomer compositions of the present disclosure, e.g. nucleic acids that encode for amyloid peptide/polypeptides, nucleic acids that encodes for precursor proteins of the amyloid peptide/polypeptide, etc. These nucleic acid compositions are described in greater detail below.
[0056] In some aspects of the invention, nucleic acids that encode for amyloid monomer compositions of the present disclosure or peptides/polypeptides that may be used to generate the amyloid monomer compositions of the present disclosure are provided. By a nucleic acid composition is meant a composition comprising a sequence of DNA, i.e. a DNA molecule, having an open reading frame, or coding sequence, that encodes for an amyloid monomer composition of the present disclosure or a peptide/polypeptide that may be used to generate the amyloid monomer compositions of the present disclosure. By a "DNA molecule" it is meant the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. Also encompassed in this term are DNA molecules that are homologous, substantially similar or identical to the nucleic acids of the present invention. By a "coding sequence" it is meant a DNA sequence which is transcribed and translated into a peptide or polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences, e.g. non-naturally occurring fusion polypeptides, e.g. an amyloid peptide/polypeptide-ligand fusion polypeptide or amyloid peptide/polypeptide- oligomerization moiety fusion polypeptide. By "isolated" is meant to describe a polynucleotide, a polypeptide, an antibody, or a host cell that is in an environment different from that in which the polynucleotide, the polypeptide, the antibody, or the host cell naturally occurs. In other words, the subject nucleic acids are present in other than their natural environment, e.g., they are isolated, present in enriched amounts, etc., from their naturally occurring environment, e.g., the organism from which they are obtained. Thus, the subject invention provides coding sequences encoding the amyloid monomer compositions of the present disclosure or peptides/polypeptides that may be used to generate the amyloid monomer compositions of the present disclosure, as well as homologs thereof.
[0057] In addition to the coding sequence, the subject nucleic acid compositions may comprise one or more DNA regulatory sequences. As used herein, “DNA regulatory sequences” are transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for and/or regulate expression of a coding sequence in a host cell. For example, the coding sequence may be associated with a polyadenylation signal and transcription termination sequence 3' to the coding sequence. As another example, the coding sequence may be operably linked to a promoter sequence 5’ to the coding sequence. By a "promoter sequence" it is meant a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters may be used to drive the various vectors of the present invention. For example, the promoter may be a constitutively active promoter, i.e. a promoter that is active in the absence externally applied agents, e.g. the CMV IE1 promoter, the SV40 promoter, GAPDH promoter, Actin promoter. The promoter may be an inducible promoter, i.e. a promoter whose activity is regulated upon the application of an agent to the cell, e.g. doxycycline, the tet-on or tet-off promoter, the estrogen receptor promoter, etc. The promoter may be a tissue-specific promoter, i.e. a promoter that is active on certain types of cells.
[0058] In some instances, the coding sequence is inserted into a vector. By a “vector”, it is meant a nucleic acid that is a replicon, such as plasmid, minicircle, phage, cosmid, etc, to which another DNA segment, i.e. an “insert”, e.g. a coding sequence, an expression cassette, etc., may be attached so as to bring about the replication of the attached segment. Thus, the subject invention also provides constructs comprising the subject nucleic acids inserted into a vector, where such constructs may be used for a number of different applications, including propagation, protein production, etc. Viral and non-viral vectors may be prepared and used, including plasmids. The choice of vector will depend on the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal or person. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially. To prepare the constructs, the partial or full-length polynucleotide is inserted into a vector typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination in vivo. Typically this is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence. Regions of homology are added by ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence, for example.
[0059] Also provided are expression cassettes or systems that find use in, among other applications, the synthesis of the subject proteins. By an “expression cassette” it is meant a DNA coding sequence operably linked to a promoter and/or other DNA regulatory sequences. For expression, the peptide or polypeptide encoded by a polynucleotide of the invention is expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. Suitable vectors and host cells are described in U.S. Patent No. 5,654,173. In the expression vector, a subject polynucleotide, for example, a polynucleotide encoding an amyloid monomer composition, e.g. an amyloid peptide/polypeptide fused to a ligand, an amyloid peptide/polypeptide fused to an oligomerization moiety, or a polynucleotide encoding a peptide/polypeptide that may be used to generate an amyloid monomer composition, is linked to a regulatory sequence as appropriate to obtain the desired expression properties. These regulatory sequences can include promoters (attached either at the 5' end of the sense strand or at the 3' end of the antisense strand), enhancers, terminators, operators, repressors, and inducers. The promoters can be regulated or constitutive. In some situations it may be desirable to use conditionally active promoters, such as tissue-specific or developmental stage-specific promoters. These are linked to the desired nucleotide sequence using the techniques described above for linkage to vectors. Any techniques known in the art can be used. In other words, the expression vector will provide a transcriptional and translational initiation region, which may be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. These control regions may be native to the subject species from which the subject nucleic acid is obtained, or may be derived from exogenous sources.
[0060] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Expression vectors may be used for, among other things, the production of fusion proteins, as described above.
[0061] Expression cassettes may be prepared comprising a transcription initiation region, the gene or fragment thereof, and a transcriptional termination region. Of particular interest is the use of sequences that allow for the expression of functional epitopes or domains, usually at least about 8 amino acids in length, more usually at least about 15 amino acids in length, to about 25 amino acids, and up to the complete open reading frame of the gene. After introduction of the DNA, the cells containing the construct may be selected by means of a selectable marker, the cells expanded and then used for expression.
[0062] The above described expression systems may be employed with prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. For large scale production of the protein, a unicellular organism, such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, e.g. COS 7 cells, HEK 293, CHO, Xenopus Oocytes, etc., may be used as the expression host cells. In some situations, it is desirable to express the gene in eukaryotic cells, where the expressed protein will benefit from native folding and post-translational modifications. Small peptides can also be synthesized in the laboratory. Polypeptides that are subsets of the complete protein sequence may be used to identify and investigate parts of the protein important for function.
[0063] Specific expression systems of interest include bacterial, yeast, insect cell and mammalian cell derived expression systems. Representative systems from each of these categories is are provided below:
[0064] Bacteria. Expression systems in bacteria include those described in Chang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979) 281 :544; Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0036,776; U.S. Patent No. 4,551 ,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA) (1983) 80:21 25; and Siebenlist et al., Cell (1980) 20:269.
[0065] Yeast. Expression systems in yeast include those described in Hinnen et al., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J. Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142; Kunze et al., J. Basic Microbiol. (1985) 25:141 ; Gleeson et al., J. Gen. Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986) 202:302; Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt et al., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology (1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141 ; Cregg et al., Mol. Cell. Biol. (1985) 5:3376; U.S. Patent Nos. 4,837,148 and 4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr. Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49; Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284289; Tilburn et al., Gene (1983) 26:205221 ; Yelton et al., Proc. Natl. Acad. Sci. (USA) (1984) 81 :1470 1474; Kelly and Hynes, EMBO J. (1985) 4:475479; EP 0 244,234; and WO 91/00357.
[0066] Insect Cells. Expression of heterologous polypeptides in insects is accomplished as described in U.S. Patent No. 4,745,051 ; Friesen et al., “The Regulation of Baculovirus Gene Expression”, in: The Molecular Biology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0 155,476; and Vlak et al., J. Gen. Virol. (1988) 69:765 776; Miller et al., Ann. Rev. Microbiol. (1988) 42:177; Carbonell et al., Gene (1988) 73:409; Maeda et al., Nature (1985) 315:592 594; Lebacq Verheyden et al., Mol. Cell. Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad. Sci. (USA) (1985) 82:8844; Miyajima et al., Gene (1987) 58:273; and Martin et al., DNA (1988) 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts are described in Luckow et al., Bio/Technology (1988) 6:47 55, Miller et al., Generic Engineering (1986) 8:277 279, and Maeda et al., Nature (1985) 315:592-594.
[0067] Mammalian Cells. Mammalian expression is accomplished as described in Dijkema et al., EMBO J. (1985) 4:761 , Gorman et al., Proc. Natl. Acad. Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41 :521 and U.S. Patent No. 4,399,216. Other features of mammalian expression are facilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44, Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.
[0068] When any of the above host cells, or other appropriate host cells or organisms, are used to replicate and/or express the polynucleotides or nucleic acids of the invention, the resulting replicated nucleic acid, RNA, expressed protein or polypeptide, is within the scope of the invention as a product of the host cell or organism. The product is recovered by any appropriate means known in the art.
[0069] Also provided are nucleic acids that hybridize to the above described nucleic acid under stringent conditions. An example of stringent hybridization conditions is hybridization at 50°C or higher and O.IxSSC (15 mM sodium chloride/1 .5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42eC in a solution: 50 % formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 |ig/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65eC. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least about 90% as stringent as the above specific stringent conditions. Other stringent hybridization conditions are known in the art and may also be employed to identify nucleic acids of this particular embodiment of the invention. [0070] The subject nucleic acid compositions may be introduced into cells by any convenient method. For example, nucleic acids may be introduced via electroporation, calcium chloride transfection, or lipofection. As another example, nucleic acids may be provided to cells via a virus. In other words, the cells are contacted with viral particles comprising the nucleic acid of interest. Retroviruses, for example, lentiviruses, and adenoviruses are particularly suitable to the method of the invention. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells. Such methods are well known in the art.
[0071] In some instances, the subject nucleic acid may be maintained episomally, e.g. as plasmids, minicircle DNAs, virus-derived vectors such cytomegalovirus, adenovirus, etc.. In other instances, the subject nucleic acids may be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. In contrast, when producing a transgenic animal, e.g. as described below, it will be desirable to have the subject nucleic acid integrate into the genome. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell 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 primary cell that is capable of stable growth in vitro for many generations.
Transgenics
[0072] The subject amyloid monomer nucleic acids can be used to generate transgenic, nonhuman animals or site specific gene modifications in cell lines. Transgenic cells of the subject invention include one or more amyloid oligomer nucleic acids according to the subject invention present as a transgene, where included within this definition are the parent cells transformed to include the transgene and the progeny thereof. Transgenic organisms of the subject invention include cells and multicellular organisms, e.g. animals, in which the subject composition is expressed in cells or tissues where amyloid peptide/polypeptide, e.g. A|3, are normally expressed and/or at levels normally present in such cells or tissues. Transgenic organisms of the subject invention also include cells and multicellular organisms, e.g. animals, in which the subject composition is expressed in cells or tissues where amyloid peptide/polypeptide, e.g. A|3, are not normally expressed and/or at levels not normally present in such cells or tissues.
[0073] Transgenic organisms of the subject invention may be made through any convenient method. For example, transgenic organisms may be made by homologous recombination, in which a particular locus, e.g. the locus from which the precursor protein of the amyloid peptide/polypeptide is produced (for example, for the A|3 peptide, the APP locus), is targeted by the subject nucleic acid composition using homologous recombination. In some such instances, the subject nucleic acid may replace the targeted gene, e.g. the APP gene, or a coding region thereof. In other such instances, the subject nucleic acid may be inserted into the targeted gene locus, e.g. the APP locus, such that APP gene expression is not disrupted but expression of the subject nucleic acid composition is also regulated by the endogenous target gene’s promoter. As another example, a subject nucleic acid composition may be randomly integrated into the genome, e.g. as a nucleic acid construct comprising the subject nucleic acid in operable linkage with a promoter, or as a nucleic acid composition not operably linked to a promoter . Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like.
[0074] DNA constructs for homologous recombination will comprise at least a portion of the APP-oligomer nucleic acid of the subject invention, wherein the DNA construct includes regions of homology to the target locus. DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection may be included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990), Meth. Enzymol. 185:527-537.
[0075] For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF). When ES or embryonic cells have been transformed, they may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting offspring screened for the construct. By providing for a different phenotype of the blastocyst and the genetically modified cells, chimeric progeny can be readily detected.
[0076] The chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vitro culture. The transgenic animals may be any non-human mammal, such as laboratory animals, domestic animals, etc. The transgenic animals may be used in functional studies, drug screening, etc. Representative examples of the use of transgenic animals include those described infra.
Utility
[0077] Because of the substantial homogeneity of the subject amyloid oligomer compositions, and the high affinity and specificity of the amyloid oligomers therein for their binding partner in or on cells, the subject compositions find many uses in research and drug discovery.
[0078] For example, amyloid oligomer compositions can be used to model amyloid binding to a cell or amyloid signaling in a cell that expresses an amyloid receptor, e.g. in a cell that expresses PirB/LilrB2, e.g. to study the effects of amyloid oligomer binding on intracellular signaling pathways, to screen for agents that inhibit amyloid oligomer signaling, etc. The subject amyloid oligomer compositions can also be used to identify novel amyloid oligomer binding partners, e.g. new receptors for amyloid oligomers. The subject amyloid oligomer compositions can be used to generate animal models of diseases and disorders associated with amyloid formation, e.g. Alzheimer’s disease, cerebral amyloid angiopathy, Down’s Syndrome, Diabetes mellitus type 2, Parkinson's disease, tauopathies, spongiform encephalopathies, Huntington's Disease, Medullary carcinoma of the thyroid, cardiac arrhythmias and isolated atrial amyloidosis, Atherosclerosis, Rheumatoid arthritis, Aortic medial amyloid, prolactinomas, Familial amyloid polyneuropathy, Hereditary non-neuropathic systemic amyloidosis, Dialysis related amyloidosis, Finnish amyloidosis, Lattice corneal dystrophy, systemic AL amyloidosis, and Sporadic Inclusion Body Myositis.
[0079] In some aspects of the invention, the subject amyloid oligomer compositions are used to promote amyloid peptide/polypeptide signaling in a cell. In such methods, a cell expressing an amyloid receptor is contacted with amyloid oligomer in a concentration, or amount, that is effective to promote amyloid oligomer receptor signaling, e.g. to increase signaling by 25%, 50%, 75%, 90%, 95%, or more, relative to the signaling in the absence of the amyloid oligomer. [0080] In some aspects of the invention, the subject amyloid oligomer compositions find use in screens to identify agents that modulate the effects of amyloid peptide/polypeptides on a cell. For example, it may be desirable to identify an agent that modulates the binding of Ap to Ap receptor, and/or that modulates intracellular signaling events promoted by the binding of Ap to Ap receptor. It is expected that a candidate agent that is identified as having activity in disrupting Ap binding to its receptor or disrupting Ap-mediated intracellular signaling will be an agent that finds use in the treatment of an Ap-associated disease or disorder, e.g., Alzheimer’s disease, Down’s Syndrome, etc. In such instances, the ability of the candidate agent to modulate, e.g. promote or disrupt, binding of Ap to Ap receptor and/or Ap-mediated intracellular signaling may be accurately determined by assessing the ability of the subject composition to bind receptor and/or promote intracellular signaling in the presence of candidate agent and comparing that ability to its ability in the absence of candidate agent.
[0081] For example, the ability of an agent to inhibit amyloid receptor binding may be assessed by employing the subject amyloid oligomer composition in a cell-free assay, e.g. using surface plasmon resonance (SPR). For example, an Ap receptor or domain thereof, e.g. the extracellular domain of PirB/LILRB2, the receptor amy3, etc., is immobilized on a sensor surface, the receptor is contacted with the Ap tetramer and the candidate agent, and the affinity of the Ap tetramer for the immobilized receptor is compared to the affinity of the Ap tetramer for the immobilized receptor in the absence of candidate agent. A reduced affinity of Ap tetramer for the receptor in the presence of candidate agent indicates that the candidate agent will disrupt, i.e. inhibit, reduce, or otherwise suppress, Ap binding to a cell expressing that receptor, e.g. PirB/LILRB2.
[0082] As another example, the ability of an agent to inhibit amyloid receptor binding can be assessed by employing the subject amyloid oligomer composition in a cell-based assay. For example, a cell expressing an Ap receptor or domain thereof, e.g. PirB/LILRB2, may be contacted with Ap tetramer and candidate agent, and the effect of the agent on the ability of the Ap tetramer to bind the cell and/or promoter intracellular signaling is assessed by assessing one or more output parameters.
[0083] Parameters are quantifiable components of cells, particularly components that can be accurately measured, desirably in a high throughput system. A parameter can be any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc. Characteristically a range of parameter readout values will be obtained for each parameter from a multiplicity of the same assays. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values. Thus, for example, one such method may comprise contacting a cell that expresses an A|3 receptor with the subject A|3 tetramer composition and a candidate agent; and comparing the parameter to the parameter in a cell that expresses the receptor and that was contacted with the APP oligomer composition but was not contacted with the candidate agent, wherein a difference in the parameter in the cell contacted with the candidate agent indicates that the candidate agent will modulate cell binding of A|3 or cell signaling mediated by A|3.
[0084] One example of an output parameter, that may be quantified when screening for agents that modulate the effects of A|3 on a cell, is A|3 tetramer binding to the cell. A change in the ability of an A|3 tetramer to bind to a cell can be readily visualized by, for example, flow cytometry, immunohistochemistry, fluorescence resonance energy transfer (FRET), and the like. Another example of an output parameter is an increase in cytosolic cAMP and Ca(2+), or the activation of one or more of the signal transduction mediators protein kinase A, MAPK, Akt, and cFos. Other output parameters could include those that are reflective of the function of the cells in the culture. For example, neurons contacted with candidate agent may be assessed for the number of spines, the number of synapses shared with neighboring neurons, the plasticity of the neurons, and the like, where an increase in the number of spines, number of synapses, and plasticity of the neuron in the presence of candidate agent indicates that the agent disrupts the effect of A|3 on a neuron. As another example, cell viability following exposure to the subject A|3 composition, e.g. a 24-48h exposure at low micromolar concentrations, may be used as an output parameter, where a reduction in cell death in the presence of candidate agent indicates that the agent disrupts the effect of A|3 on a cell. In some instances, one parameter is measured. In some instances, multiple parameters are measured.
[0085] Cells useful for screening include any cell that endogenously or ectopically expresses a receptor for the subject amyloid oligomer composition, e.g., for A|3 tetramers, PirB/LILRB2 receptor, amylin receptor 3, etc. Cells may be from any mammalian species, e.g. murine, rodent, canine, feline, equine, bovine, ovine, primate, human, etc. Cells may be from established cell lines, e.g. HEK293 cells, 293T cells, NG108 cells, PC-12 cells, etc., or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary cell lines of the present invention are maintained for fewer than 10 passages in vitro.
[0086] If the cells are primary cells, they may be harvested from an individual by any convenient method. For example, cells may be harvested by biopsy, or by harvesting a tissue in its entirety. An appropriate solution may be used for dispersion or suspension of the harvested cells. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, DMEM-F12, etc., conveniently supplemented with a synthetic supplement, e.g. B27, NGS, N2, etc., or with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. The cells may be used immediately, or they may be stored, frozen, for long periods of time, being thawed and capable of being reused. In such cases, the cells will usually be frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells.
[0087] Candidate agents of interest for screening include known and unknown compounds that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. An important aspect of the invention is to evaluate candidate drugs, including toxicity testing; and the like.
[0088] Candidate agents include organic molecules comprising functional groups necessary for structural interactions, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Included are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, hormones or hormone antagonists, etc. Exemplary of pharmaceutical agents suitable for this invention are those described in, "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition. Also included are toxins, and biological and chemical warfare agents, for example see Somani, S. M. (Ed.), "Chemical Warfare Agents," Academic Press, New York, 1992).
[0089] Candidate agents of interest for screening also include nucleic acids, for example, nucleic acids that encode siRNA, shRNA, antisense molecules, or miRNA, or nucleic acids that encode polypeptides. Many vectors useful for transferring nucleic acids into target cells are available. The vectors may be maintained episomally, e.g. as plasmids, minicircle DNAs, virus-derived vectors such cytomegalovirus, adenovirus, etc., or they may be integrated into the target cell genome, through homologous recombination or random integration, e.g. retrovirus derived vectors such as MMLV, HIV-1 , ALV, etc. Vectors may be provided directly to the subject cells. In other words, the pluripotent cells are contacted with vectors comprising the nucleic acid of interest such that the vectors are taken up by the cells.
[0090] Methods for contacting cells with nucleic acid vectors, such as electroporation, calcium chloride transfection, and lipofection, are well known in the art. Alternatively, the nucleic acid of interest may be provided to the subject cells via a virus. In other words, the pluripotent cells are contacted with viral particles comprising the nucleic acid of interest. Retroviruses, for example, lentiviruses, are particularly suitable to the method of the invention. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells. Envelope proteins are of at least three types, ecotropic, amphotropic and xenotropic. Retroviruses packaged with ecotropic envelope protein, e.g. MMLV, are capable of infecting most murine and rat cell types, and are generated by using ecotropic packaging cell lines such as BOSC23 (Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses bearing amphotropic envelope protein, e.g. 4070A (Danos et al, supra.), are capable of infecting most mammalian cell types, including human, dog and mouse, and are generated by using amphotropic packaging cell lines such as PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431 -437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with xenotropic envelope protein, e.g. AKR env, are capable of infecting most mammalian cell types, except murine cells. The appropriate packaging cell line may be used to ensure that the subject CD33+ differentiated somatic cells are targeted by the packaged viral particles. Methods of introducing the retroviral vectors comprising the nucleic acid encoding the reprogramming factors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art.
[0091] Vectors used for providing nucleic acid of interest to the subject cells will typically comprise suitable promoters for driving the expression, that is, transcriptional activation, of the nucleic acid of interest. This may include ubiquitously acting promoters, for example, the CMV- b-actin promoter, or inducible promoters, such as promoters that are active in particular cell populations or that respond to the presence of drugs such as tetracycline. By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by at least about 10 fold, by at least about 100 fold, more usually by at least about 1000 fold. In addition, vectors used for providing reprogramming factors to the subject cells may include genes that must later be removed, e.g. using a recombinase system such as Cre/Lox, or the cells that express them destroyed, e.g. by including genes that allow selective toxicity such as herpesvirus TK, bcl-xs, etc.
[0092] Candidate agents of interest for screening also include polypeptides. Such polypeptides may optionally be fused to a polypeptide domain that increases solubility of the product. The domain may be linked to the polypeptide through a defined protease cleavage site, e.g. a TEV sequence, which is cleaved by TEV protease. The linker may also include one or more flexible sequences, e.g. from 1 to 10 glycine residues. In some embodiments, the cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, e.g. in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and/or polynucleotides that increase solubility, and the like. Domains of interest include endosomolytic domains, e.g. influenza HA domain; and other polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE domain, and the like.
[0093] If the candidate polypeptide agent is being assayed for its ability to inhibit signaling intracellularly, the polypeptide may comprise the polypeptide sequences of interest fused to a polypeptide permeant domain. A number of permeant domains are known in the art and may be used in the non-integrating polypeptides of the present invention, including peptides, peptidomimetics, and non-peptide carriers. For example, a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK. As another example, the permeant peptide comprises the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein. Other permeant domains include poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine, octa-arginine, and the like. (See, for example, Futaki et al. (2003) Curr Protein Pept Sci. 2003 Apr; 4(2): 87-96; and Wender et al. (2000) Proc. Natl. Acad. Sci. U.S.A 2000 Nov. 21 ; 97(24):13003-8; published U.S. Patent applications 20030220334; 20030083256; 20030032593; and 20030022831 , herein specifically incorporated by reference for the teachings of translocation peptides and peptoids). The nona-arginine (R9) sequence is one of the more efficient PTDs that have been characterized (Wender et al. 2000; Uemura et al. 2002).
[0094] If the candidate polypeptide agent is being assayed for its ability to inhibit signaling extracellularly, e.g. to disrupt A|3 tetramer binding to a cell, the polypeptide may be formulated for improved stability. For example, the peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream. The polypeptide may be fused to another polypeptide to provide for added functionality, e.g. to increase the in vivo stability. Generally such fusion partners are a stable plasma protein, which may, for example, extend the in vivo plasma half-life of the polypeptide when present as a fusion, in particular wherein such a stable plasma protein is an immunoglobulin constant domain. In most cases where the stable plasma protein is normally found in a multimeric form, e.g., immunoglobulins or lipoproteins, in which the same or different polypeptide chains are normally disulfide and/or noncovalently bound to form an assembled multichain polypeptide, the fusions herein containing the polypeptide also will be produced and employed as a multimer having substantially the same structure as the stable plasma protein precursor. These multimers will be homogeneous with respect to the polypeptide agent they comprise, or they may contain more than one polypeptide agent.
[0095] The candidate polypeptide agent may be produced by eukaryotic cells or by prokaryotic cells, it may be further processed by unfolding, e.g. heat denaturation, DTT reduction, etc. and may be further refolded, using methods known in the art. Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine. The polypeptides may have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non- naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.
[0096] The candidate polypeptide agent may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. Alternatively, the candidate polypeptide agent may be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.
[0097] In some cases, the candidate polypeptide agents to be screened are antibodies. The term “antibody” or “antibody moiety” is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The specific or selective fit of a given structure and its specific epitope is sometimes referred to as a “lock and key” fit. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammal, chicken, other avians, etc., are considered to be “antibodies.” Antibodies utilized in the present invention may be either polyclonal antibodies or monoclonal antibodies. Antibodies are typically provided in the media in which the cells are cultured.
[0098] Candidate agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
[0099] Candidate agents are screened for biological activity by adding the agent to at least one and usually a plurality of cell samples, usually in conjunction with cells not contacted with the agent. The change in parameters in response to the agent is measured, and the result evaluated by comparison to reference cultures, e.g. in the presence and absence of the agent, obtained with other agents, etc.
[00100] The agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.
[00101 ] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.
[00102] Various methods can be utilized for quantifying the selected parameters. For example, flow cytometry, immunohistochemistry, or FRET may be employed to measure A|3 tetramer binding to the cell. Western blotting and kinase assays may be used to measure phosphorylation of PKA, MAPK, Akt, and cFos. The fluorescent indicators Fura-2 and BCECF may be employed to measure changes in cytosolic Ca(+2) Radioimmunoassays or FRET may be employed to measure changes in cytosolic cAMP. Such methods would be well known to one of ordinary skill in the art.
[00103] In some aspects of the invention, the subject amyloid oligomer compositions find use in experiments to identify signaling pathways that are activated by binding of an amyloid oligomer of interest to its receptor. For example, cells expressing an amyloid receptor may be contacted with a subject amyloid oligomer composition, and the gene expression profile of the contacted cells compared to the gene expression profile of cells not contacted with the subject composition, e.g. by isolating RNA or protein from contacted cells and cells not contacted with the subject composition, and analyzing the RNA or protein by, e.g., RNA or protein microarray, qRT-PCR, Northern blotting, Western blotting, and the like. As another example, cells expressing an amyloid receptor may be contacted with a subject amyloid oligomer composition, and the activation state (e.g. phosphorylation state, ubiquitination state, acetylation state, proteolytic cleavage state, etc.) of one or more proteins in the contacted cells may be compared to the activation state of one or more proteins in cells not contacted with the subject composition, e.g. by Western blotting or flow cytometry using antibodies that are specific for the activation state of the protein(s) of interest. Such experiments may be used to identify changes in gene expression or protein activity in the presence of the amyloid oligomer, which may in turn be used to identify which signaling pathways have become more or less active in the cells as a result of contacting with the amyloid oligomer.
[00104] In some aspects of the invention, the subject amyloid oligomer compositions find use in experiments to identify a novel receptor for an amyloid oligomer of interest. For example, the amyloid oligomer composition may be immobilized on a solid support (e.g. polystyrene bead, microplate, etc), and a cell lysate passed over the immobilized oligomer composition. Bound receptor may be identified by, e.g. 2D-electrophoresis, MALDI-TOF, mass spectrometry, etc. The specificity of the protein that is identified for the amyloid oligomer may be confirmed by, for example, co-immunoprecipitation of labeled amyloid oligomer and receptor, e.g. ectopically expressed by cells, or endogenously expressed in vivo, e.g. using receptor-specific antibodies or antibodies specific for a receptor tag.
[00105] In some aspects of the invention, the subject amyloid oligomer compositions find use in creating an animal model of an amyloid-associated disease. For example, an animal model of an amyloid-associated disease may be created by ectopically expressing a subject amyloid monomer composition comprising a disease-associated amyloid peptide/polypeptide from the genome in the animal, e.g. as a transgene in the animal, e.g. as described above. As another example, the animal model may be created by administering to the animal a subject amyloid oligomer composition comprising the disease-associated amyloid peptide/polypeptide in an amount effective to induce the disease. The administration of the subject composition can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. The subject composition may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation. The subject composition may be formulated for immediate activity or it may be formulated for sustained release. For some conditions, particularly central nervous system conditions, it may be necessary to formulate the subject composition to cross the blood-brain barrier (BBB). One strategy for drug delivery through the blood-brain barrier (BBB) entails disruption of the BBB, either by osmotic means such as mannitol or leukotrienes, or biochemically by the use of vasoactive substances such as bradykinin. The potential for using BBB opening to target specific agents to brain tumors is also an option. A BBB disrupting agent can be co-administered with the subject composition when the composition is administered by intravascular injection. Other strategies to go through the BBB may entail the use of endogenous transport systems, including Caveolin-1 mediated transcytosis, carrier- mediated transporters such as glucose and amino acid carriers, receptor-mediated transcytosis for insulin or transferrin, and active efflux transporters such as p-glycoprotein. Active transport moieties may also be conjugated to the therapeutic compounds for use in the invention to facilitate transport across the endothelial wall of the blood vessel. Alternatively, drug delivery of the subject composition behind the BBB may be by local delivery, for example by intrathecal delivery, e.g. through an Ommaya reservoir (see e.g. US Patent Nos. 5,222,982 and 5385582, incorporated herein by reference); by bolus injection, e.g. by a syringe, e.g. intravitreally or intracranially; by continuous infusion, e.g. by cannulation, e.g. with convection (see e.g. US Application No. 20070254842, incorporated here by reference); or by implanting a device upon which the subject composition has been reversably affixed (see e.g. US Application Nos. 20080081064 and 20090196903, incorporated herein by reference).
REAGENTS AND KITS
[00106] Also provided are reagents, devices and kits thereof for practicing one or more of the above-described methods. The subject reagents and kits thereof may vary greatly. For example, reagents and kits may include one or more of the following: amyloid peptide/polypeptide, ligand, ligand binding protein, reagents for the conjugation of ligand to amyloid peptide/polypeptide, oligomerization moiety, reagents for the conjugation of oligomerization moiety to amyloid peptide/polypeptide, amyloid monomers, amyloid oligomers, reagents to test the purity of an amyloid monomer or oligomer composition, and the like.
[00107] In addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
EXAMPLES
[00108] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
[00109] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.
Ap-streptavidin tetramers for quantitative study of Ap oligomer binding and signaling
[00110] Soluble beta-amyloid (Ap) oligomers are highly heterogeneous among different preparations, limiting their use in quantitative cell biological and biochemical studies. Here we report a homogeneous, chemically defined version of tetrameric Ap conjugated by site- specif ically labeled streptavidin. Like Ap oligomers, Ap-streptavidin tetramers (Ap-SAvT) bind to known Ap receptors such as PirB and LilrB2 with nanomolar affinity and alter downstream signaling pathways implicated in Ap oligomer-induced synapse loss. Results suggest that Ap- SAvT is a reproducible Ap mimetic that can act as a potent, biologically active ligand for PirB/LilrB2, and can be used for Alzheimer's disease research and drug development.
[00111] Alzheimer’s disease (AD) is the major cause of dementia in the elderly and is characterized by progressive memory loss and cognitive decline. Many recent studies have demonstrated that soluble oligomeric aggregates of p-amyloid (Ap) are key mediators of synaptic and cognitive dysfunction in AD and that these effects can be initiated by Ap oligomer binding to receptors. One such receptor expressed in neurons and associated with synapses is PirB (Paired Immunoglobulin-like Receptor B); its homologue LilrB2 (Leukocyte Immunoblobulin-like Receptor B2) is also present in human brain. Ap oligomer binding to PirB/LilrB2 perturbs downstream signaling cascades implicated in neuronal actin organization (e.g. cofilin hyperactivation), leading to synaptic and cognitive dysfunction in AD models in vivo and in vitro. However, because Ap oligomer preparations consist of heterogeneous mixes of various aggregates and monomers of Ap peptides, it has been difficult to generate reproducible, synaptotoxic species of Ap oligomers, and to study how they interact with receptors and engage downstream pathways. To address this shortcoming of Ap oligomers, we created a chemically defined Ap tetramer amenable to quantitative cell biological and biochemical studies by using streptavidin (SAv) that is site-specifically labeled with a fluorophore (i.e. Alexa Fluor 647 or Fluorescein) (Fig. 1a).
[00112] N-terminally biotinylated Api -42 (Ap42) or Api -40 (Ap40) peptides were conjugated to SAv, generating a highly reproducible form of tetrameric Ap: Ap42- or Ap40-SAv tetramers (Ap42-SAvT; Ap40-SAvT) (Fig. 1b). The Ap42-SAv conjugates also contain small amounts of 8-mer and 16-mer forms as detected by Western blot for Ap as discrete bands; this pattern is distinct from the broad, heterogeneous distribution of bands seen in Western blots for A|342 oligomers generated by standard methods (Fig. 1 b). To determine if A|342-SAvT can recapitulate the behavior of A|3 oligomers for receptor binding, we stained human embryonic kidney (HEK) 293 cells expressing PirB, a high-affinity receptor for A|3 oligomers, with either A|342-SAvT or a variety of controls (Fig. 1c, d). The cell surface pattern of binding of A|342- SAvT to PirB expressing HEK293 cells is comparable to that of A|342 oligomers (Fig. 1c and Fig. 4a). In contrast, monomeric A|342, scrambled A|342 peptide conjugated SAvT (Scr-SAvT) or control SAvT do not bind to PirB expressed on HEK293 cells (Fig. 1c and Fig. 4a, b). In addition, there is no significant binding of A|342-SAvT to HEK293 cells transfected with vector alone, or to PirB-related receptors PirA1 and PirA4 compared to PirB-expressing cells (Fig. 1 d, e), matching what was previously observed for A|342 oligomers. A|342-SAvT also exhibits prominent binding to LilrB2-expressing HEK293 cells, in comparison to barely detectable binding to LilrBI - or LilrB3-expressing cells (Fig. 5), again consistent with A|342 oligomers and with the high homology between the D1 D2 domains of LilrB2 and PirB. These observations indicate that A|342-SAvT, like A|342 oligomers, selectively binds to PirB and LilrB2.
[00113] To see if A|340, which is more abundantly produced in Alzheimer's-affected brain tissue, can also bind to PirB when it is tetramerized, we tested A|340 tetramers. A|340- SAvT (Fig. 1b), but not monomeric A|340, binds to PirB expressing cells (Fig. 1f), suggesting that both A|340 and A|342 can bind to PirB by increasing the avidity and apparent affinity of the interaction, and share a common PirB binding epitope. The observed interactions are saturable, with an apparent dissociation constant ( d) of 33 nM for A|342-SAvT, a binding affinity about 6 times greater than that of A|342 oligomers (i.e. 180 nM6), and with a Kti of 65 nM for A|340-SAvT (Fig. 1g, h). Together, these results show that conjugation of A|3 peptides onto streptavidin- either A|340 or 42- enables reproducible A|3 tetramerization, leading to enhanced binding to PirB/LilrB2 compared to A|3 oligomers (Fig. 4c).
[00114] To further determine whether A|3-SAvT recapitulates the receptor binding characteristics of A|3 oligomers, we next examined if A|3-SAvT binding to PirB is competitive with that of known blocking reagents or competitors for A|3 oligomer-PirB interactions. First, A|342-SAvT binding is effectively prevented by co-incubation with an anti-Ap antibody, 4G8 (Fig. 2a). Second, A|342-SAvT binding is blocked by the anti-PirB antibody (6C1 ) (Fig. 2b). This antibody recognizes the D1 D2 domain of PirB, where A|3 oligomers are known to bind, and also prevents A|3 oligomer binding to PirB (Fig. 6a). In a competition experiment, A|342- SAvT binding is significantly reduced when cells are co-treated with A|342 oligomers (Fig. 3c), indicating that A|342-SAvT competes with A|342 oligomers for PirB binding. Similarly, binding of A|342-SAvT to LilrB2 is inhibited by anti-Ap (4G8) or anti-LilrB2 (AF2078) antibodies (Fig. 6b and 7). Together results suggest that A|342-SAvT binding to PirB/LilrB2 is mediated by Ap peptides, and that A|342-SAvT mimics A|342 oligomers for PirB/LilrB2 binding. [00115] It is known that A|3 oligomers can be pulled down using Fc reagents such as soluble PirB (D1 D6)-Fc and LilrB2 (D1 D4)-Fc, which lack the transmembrane and intracellular domains. These reagents, but not a control A|3 oligomer binding domain deficient PirB (D5D6)- Fc, can immunoprecipitate A|342-SAvT, as well as A|340-SAvT efficiently (Fig. 2d-f). We also examined if A|3-SAvT can pull down other known receptors for A|3 oligomers, notably EphB2. Indeed, an EphB2-Fc pulls down A|3-SAvT modestly but significantly (-30% of that detected with PirB (D1 D6)-Fc) (Fig. 2d-f), supporting the notion that A|3-SAvT broadly recapitulates A|3 oligomers for receptor binding.
[00116] Next we examined the effects of A|342-SAvT on neurons by preparing cultures of WT cortical neurons, which are known to express PirB. A punctate pattern of A|342- SAvT binding is seen along EGFP-expressing neuronal dendrites, but not with Scr-SAvT (Fig. 3a). Enriched tetramer binding zones are also visible along MAP-2 positive dendrites, and are greatly reduced in neurons from PirB /_ cortex (Fig. 3b). These observations suggest that A|342-SAvT binds to neuronal PirB. In addition, the remaining low level of staining could be due to other receptors expressed in cortical neurons known to bind A|3 oligomers.
[00117] A|342 oligomers are known to alter neuronal signaling pathways. To examine if A|342-
SAvT can affect neuronal signaling upon binding to PirB, cortical neurons cultured in vitro were studied. Cofilin activation and PSD-95 loss are induced following application of A|342-SAvT to WT, but not PirB /_, cortical neurons (Fig. 3c, d), as was observed when A|342 oligomers were applied, and in human Alzheimer’s brains. Together, these data show that homogeneous and chemically defined A|3- SAvT functions as a potent, biologically active ligand for PirB/LilrB2, and can also engage signaling pathways known to be perturbed in Alzheimer’s disease. Amyloid-|3 streptavidin tetramers can thus be used to study signaling pathways associated with Amyloid-|3.
[00118] The heterogeneous nature of A|3 oligomers has been a major hurdle in investigating the deleterious effects of A|3 oligomers at the molecular level. Here, we have used streptavidin (SAv) to generate a reproducible and homogenous version of A|3 oligomers. The major advantage of this approach is that streptavidin allows for a chemically defined, well-behaved method of tetramerization for A|3 peptide without any detectable non-specific effects. This point is well borne out through studies conducted over the past 15 years of other interactions in which streptavidin-mediated tetramers have been crucial for determining the binding specificities of low affinity monomeric interactions. In those studies, perhaps most notably those of T Cell Receptors (TCRs), binding is seen because the SAv tetramers increase the avidity and therefore the apparent affinity of ligand for receptors, rather than by an alteration of the “character” of the molecular interaction induced by tetramerization itself. In the case of A|3-SAvT, this increase in avidity permits the binding of A|3 peptides to PirB/LilrB2 with high apparent affinity and may also cluster PirB/LilrB2, initiating downstream signaling. The fact that addition of A|342-SAvT to cortical neurons engages cofilin signaling is strong support for the contention that these tetramers indeed recapitulate oligomeric forms of A|342. In contrast, when Ap peptides are in a monomeric state, it is likely that their inherent affinity for PirB/LilrB2 is too weak either to bind appreciably or to cluster receptors to engage downstream signaling. [00119] Results here indicate that Ap-SAv tetramers are homogeneous and chemically defined forms that mimic oligomeric Ap. Ap-SAv tetramers may be useful as potent reagents to elucidate the function and mechanism of action of Ap oligomers, as well as to identify and develop new therapeutics for Ap-related diseases. This new reagent also provides an experimental approach more firmly rooted in biophysical principles than current protocols, which create an indefinable mixture of Ap peptide species.
[00120] Amyloid-p streptavidin tetramers can be used in assays to screen small molecules or to discover drugs that block Ap binding to target receptors/proteins. Since Ap-SAv tetramers bind reproducibly to PirB/LilrB2 and recapitulate Ap oligomer binding (i.e. Ap42-SAvT competes with Ap42 oligomers for PirB binding), this interaction can be used to screen for blocking compounds including those from large compound libraries, antibodies etc.
Methods
[001 1] Reagents. Reagents were obtained from the following sources: antibodies to amyloid- P 17-24 (4G8; #SIG39220) and neuronal class III tubulin (Tuj1 ; #PRB-435P) are from Covance (Emerville, GA); blocking antibody to LilrB2 (#AF2078), as well as LilrB2-Fc (#2078- T4) and EphB2-Fc (#467-B2-200) from R&D Systems (Minneapolis, MN); antibodies to cofilin (#3312, #5175) and pCofilin (#3313) from Cell Signaling Technology (Danvers, MA); blocking antibody to PirB (6C1 ; #550348) from BD Biosciences (San Jose, CA); antibody to MAP2 (#AB15452) from Millipore (Temecula, CA); antibody to PSD-95 (#MA1 -045) and Fluorescein maleimide (Pierce #62245) from Thermo Scientific (Rockford, IL); HRP-conjugated secondary antibodies to rabbit, mouse, goat and human IgG from Jackson ImmunoResearch (West Grove, PA); Secondary antibodies with Alexa 488, 596 and 647 conjugates are from Invitrogen (Carlsbad, CA) and Jackson ImmunoResearch. DMEM, Neurobasal, B-27, fetal bovine serum, horse serum,
[00122] Lipofectamine and Alexa Fluor 647 C2-maleimide (#A-20347) are from Invitrogen (Grand Island, NY). Protease inhibitor cocktail (#P8340), phosphatase inhibitor cocktails (#P5726, #P0044) and all other chemicals are obtained from Sigma (St. Louis, MO).
[00123] Plasmids encoding Myc-tagged human LilrBI (accession #: NM_001081637.1 ), LilrB2 (accession #: NM_001080978.1 ) and LilrBS (accession #: NM_006864.2), as well as murine PirA1 (accession #: NM_011087.1 ), PirA4 (accession #: NM_011091.1) are obtained from OriGene Technologies (Rockville, MD); PirB 8, PirB-Fc and LilrB2-Fc constructs 6 were generated as described previously.
[00124] Ap preparation. Biotinylated (N-terminal) synthetic human Api-40, Api-42 and scrambled peptides for human Api -42 are obtained from Anaspec (Fremont, GA), and prepared as described with minor modifications. Briefly, Dissolved peptides were sonicated for 30 seconds and diluted in 1X phosphate buffered saline (PBS; 1 .06 mM KH2PO4, 155.17 mM NaCI, 2.97 mM Na2HPO4-7H2O, pH7.4; Invitrogen; #10010) to the final concentration of 100 pM to make freshly prepared Ap or scrambled peptides (mono). The A|342 peptides were then oligomerized as previously described (oligo). In each experiment, the uniformity of oligomerization was confirmed by Western blot analysis.
[001 5] Ap-SAvT generation. To generate tetrameric Ap streptavidin conjugates (Ap-SAvT), a custom-engineered streptavidin containing a short linker with the C-terminal Cysteine was expressed in E.coli, refolded in the presence of reducing agent as previously described, and site specifically coupled to Alexa Fluor 647 or fluorescein maleimide. The resulting labeled streptavidin was then mixed with monomeric N-terminal biotinylated A|340, A|342 or scrambled A|342 peptides (1 :4 molar ratio) in PBS and incubated on ice for 10 min to produce A|340- SAvT, A|342-SAvT, or Scr-SAvT. Quantitative coupling of Ap peptide with SAv was verified by gel shift assay as well as Western blot analysis for Ap in each experiment (e.g. Fig. 1 b and Fig. 4b).
[00126] Ap-SAvT I oligo-Ap binding assays. Human embryonic kidney (HEK) 293 cells maintained in DMEM containing 10% fetal bovine serum, supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM glutamine were transiently transfected with expression vectors encoding PirB, PirA1 , PirA4, LilrBI , LilrB2, LilrB3, or control plasmids, and plated onto 8 chamber slides (Thermo Scientific). Two days after transfection, cells were treated with monomeric or oligomeric Ap, or tetrameric Ap-SAvT or control-SAvT (SAvT; Scr- SAvT) for 1 h at 37°C, washed twice, and fixed with 2% paraformaldehyde (PFA) in 1X PBS for 20 min; protein expressions of transfected constructs were verified by Western blotting (i.e. Fig. 1e, inset) or immunostaining with appropriate antibodies. The bound mono-Ap, oligo- Ap, or Ap-SAvT was visualized with streptavidin-Alexa fluorophore (Alexa 488, 598 or 647; wavelength choice depending on experiments) or SAv-conjugated Alexa 647/FITC; DAPI was used to counterstain cell nuclei. Fluorescent images were captured with fixed illumination and exposure time using a Nikon 20X objective of numerical aperture 0.75, and then fluorescence intensity was quantified using Imaged software (NIH). In each experiment, values from control vector transfected cells were used as background, and subtracted from those of experimental cells.
[00127] For blocking or competition experiments, i) Ap42-SAvT (10 nM) were preincubated with anti-Ap (4G8) or isotype control IgG (50 nM each) in media for 10 min at 37°C- CO2 incubator, then treated to PirB or LilrB2-expressing HEK293 cells; II) PirB or LilrB2-expressing cells were pre-treated with anti-PirB (6C1 ), anti-LilrB2 (AF2078) or control antibodies (50 nM each) for 10 min at 37°C prior to the A|342-SAvT (10 nM) treatment (1 hr, 37°C); iii) PirB- expressing cells were co-treated with A|342-SAvT (10 nM) in the presence or absence of oligo- A|342 or control scrambled peptides (nonbiotinylated; 200 or 500 nM) for 1 hr. Cells were then processed and imaged as descried above.
[00128] For quantitative analysis of A|340/42-SAvT binding to PirB and binding affinity calculation, HEK293 cells transfected with PirB were treated with varying concentrations of A|340/42-SAvT, and the bound A|3-SAvT was visualized and quantified as described above. Saturation curves and scatchard plots were generated using Prism software (GraphPad, La Jolla, CA); half-saturation points (dissociation constant; Kti) were also determined as previously described.
[00129] In utero electroporation and neuronal A|3-SAvT binding. In utero electroporation was performed at E14.5-15.5 as described earlier. Tweezertype circular electrodes (0.5 cm in diameter) were custom-made using a platinum wire (PTP201 , World Precision Instruments Inc., FL) and electric pulses were delivered with a square-wave electroporation generator (ECM 830, BTX, Harvard Apparatus, MA). Mice were anesthetized with a constant flow of 1% - 2.5% isoflurane. After cleaning the abdomen with 70% ethanol, midline laparotomy was performed, and the uterine horns were exposed. For DNA injection, glass capillary tubes were pulled using a micropipette puller P-97 (Sutter Instrument, CA). 1 -2 pl of pCA-EGFP (1 pg/pl) (a gift from Dr. Susan K McConnell) was injected through the uterine wall into the lateral ventricles. Electrodes were positioned to the cortex and electric pulses were applied 5 times (50 ms pulse of 45 V with 950 ms intervals).
[00130] Mouse cortical cultures were prepared using HBSS supplemented with 41.55 mM glucose and antibiotics two days after in utero electroporation. Cortical neurons were isolated using Trypsin (0.05%) and plated onto poly-D-lysine coated coverslips (8,000 cells / coverslip) in Neurobasal (NB) media containing B27 and GlutaMAX with additional NaCI (37.5 mM) and glucose (0.3 %).
[00131] To visualize A|342-SAvT binding to neurons, primary cortical cultures from EGFP in utero electroporated WT, or non-electroporated WT vs PirB-/- mice were treated with A|342- SAvT (25 nM) in fresh culture media (Neurobasal + B27) for 2h at 37°C, washed extensively, and A|342-SAvT binding was detected by visualizing Alexa 647 conjugated to SAv using a Nikon 60X objective of numerical aperture 1 .40. Neuronal dendrites were visualized by EGFP expression or counterstaining with MAP2 antibodies.
[00132] All studies using primary cultures were conducted with approval of the Stanford IRB and Animal Care and Use Committees and in compliance with NIH guidelines for the use of experimental animals. All experiments and data analysis was performed blind to genotype. [00133] Neuronal signaling. Cortical neurons isolated from WT or PirB-/- mice were treated at DIV21 with A|342- SAvT or control SAvT (25 nM each) in fresh culture media (Neurobasal + B27) for 1 h (i.e. cofilin signaling) or for 24h (i.e. for PSD-95 levels) at 37°C, washed twice with 1X PBS, and harvested in ice cold lysis buffer containing 1% Triton-X 100, 150 mM NaCI, 50 mM Tris (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, protease inhibitor and phosphatase inhibitor cocktails. Samples were then subjected to Western blot analysis; for quantitative analysis of cofilin phosphorylation (pCofilin I total Cofilin), the signals for phospho-cofilin were quantified with Imaged and normalized by the signal intensities quantified from total cofilin bands. Detection of PSD-95 levels was determined and normalized by Tuj-1 using the same method.
[00134] A|3-SAvT immunoprecipitation with Fc-fusion proteins. To determine the levels of A|340- or A|342-SAvT binding to PirB, LilrB2 and EphB2, the same amount of A|340- or A|342- SAvT were immunoprecipitated with 1 pg each of PirB-Fc, LilrB2-Fc (R&D Systems) 6 or EphB2-Fc proteins (R&D Systems), followed by Western blot analysis. Truncated PirB-Fc lacking the binding domain for A|3 oligomers, PirB (D5D6)-Fc were used as controls. The A|3 signals were quantified by Imaged and normalized with the levels of Fc proteins.
[00135] Statistical analyses. Statistical significance was typically determined by two-tailed Student's t-test after testing for normal distribution. For values collected from discrete Western blot analyses with independent normalizations, Rvalues were calculated from non-parametric Mann- Whitney D-test using Prism software (GraphPad). Saturation binding curves, Kti and Scatchard plots were generated using Prism software. All analyses were performed blind to genotype.
[00136] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

THAT WHICH IS CLAIMED IS:
1. An amyloid oligomer composition, wherein each amyloid oligomer of the composition comprises two or more amyloid monomers, wherein each amyloid monomer comprises: an amyloid peptide/polypeptide and an oligomerization domain.
2. The amyloid oligomer composition according to claim 1 , wherein the oligomerization domain comprises a ligand and a ligand binding partner, wherein the amyloid peptide/polypeptide is covalently bound to the ligand.
3. The amyloid oligomer composition according to claim 1 or claim 2, wherein the ligand binding partner is detectably labeled.
4. The amyloid oligomer composition according to any of claims 1-3, wherein the detectable label is a fluorophor.
5. The amyloid oligomer composition according to any of claims 1-4, wherein the detectable label is a radioisotope.
6. The amyloid oligomer composition according to any of claims 1-5, wherein the ligand binding partner is free in solution.
7. The amyloid oligomer composition according to any of claims 1-5, wherein the ligand binding partner is attached to a solid support.
8. The amyloid oligomer composition according to any of claims 1-5, wherein the ligand is biotin and the ligand binding partner is streptavidin.
9. The amyloid oligomer composition according to any of claims 1-8, wherein the oligomerization domain comprises an oligomerization moiety, wherein the amyloid peptide/polypeptide is covalently bound to the oligomerization moiety.
10. The amyloid oligomer composition according to claim 9, wherein the oligomerization moiety is detectably labeled.
45
11. The amyloid oligomer composition according to claim 10, wherein the detectable label is a fluorophor.
12. The amyloid oligomer composition according to claim 10, wherein the detectable label is a radioisotope.
The amyloid oligomer composition according to claim 9, wherein the oligomerization moiety is free in solution.
14. The amyloid oligomer composition according to claim 9, wherein the oligomerization moiety is attached to a solid support.
15. The amyloid oligomer composition according to claim 9, wherein the oligomerization moiety is DsRed.
16. The amyloid oligomer composition according to any of claims 1 -15, wherein the peptide is Ap.
17. The amyloid oligomer composition according to any of claims 1 -15, wherein the composition is a substantially homogenous composition of amyloid oligomers.
18. An Ap-streptavidin tetramer (Ap-SavT) composition comprising: Ap peptide covalently bound to biotin, and streptavidin.
19. The composition according to claim 18, wherein the streptavidin is labeled with a fluorophor.
20. The composition according to claim 18 or 19, wherein the streptavidin is labeled with a radioisotope.
21. The composition according to any of claims 18-20, wherein the Ap peptide is covalently bound to the biotin on the N-terminus of the Ap peptide.
22. The composition according to any of claims 18-21 , wherein the biotin is separated from the Ap peptide by a linker.
46
23. The composition according to any of claims 18-22, wherein the composition is substantially homogeneous for Ap tetramers.
24. A method of screening a candidate agent for activity in inhibiting Ap binding to a cell, comprising: immobilizing PirB/LILRB2 or the extracellular domain (ECD) thereof to a sensor surface, comparing the binding of the Ap-streptavidin tetramer to immobilized PirB/LILRB2 in the present of candidate agent to the binding of the Ap-streptavidin tetramer to immobilized PirB/LILRB2 in the absence of candidate agent, wherein reduced binding of Ap-streptavidin tetramer in the presence of candidate agent indicates that the candidate agent will have activity in disrupting Ap binding to a cell.
25. The method according to claim 24, wherein the cell is a PirB/LilrB2-expressing cell.
26. A method for screening a candidate agent for activity in inhibiting Ap- associated synapse loss or cognitive impairment associated with Alzheimer’s disease or Down’s Syndrome, the method comprising: contacting a cell expressing PirB/LILRB2 with an Ap-streptavidin tetramer and the candidate agent, and comparing spine number, synapse number, or synaptic plasticity to spine number, synapse number, or synaptic plasticity in cells not contacted with candidate agent, wherein an improvement in spine number, synapse number, or synaptic plasticity indicates that the agent will have activity in inhibiting synaptic loss or cognitive impairment associated with Alzheimer’s disease or Down’s Syndrome.
47
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010900A2 (en) * 1999-08-04 2001-02-15 University Of Southern California Globular assembly of amyloid beta protein and uses thereof
US20080044356A1 (en) * 2004-10-22 2008-02-21 Regents Of The University Of Minnesota Assemblies of Oligomeric Amyloid Beta Protein and Uses Thereof
US20160090406A1 (en) * 2003-01-31 2016-03-31 AbbVie Deutschland GmbH & Co. KG Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof
US20210080463A1 (en) * 2017-06-08 2021-03-18 Carcidiag Biotechnologies Method of isolating and detecting cancer stem cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010900A2 (en) * 1999-08-04 2001-02-15 University Of Southern California Globular assembly of amyloid beta protein and uses thereof
US20160090406A1 (en) * 2003-01-31 2016-03-31 AbbVie Deutschland GmbH & Co. KG Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof
US20080044356A1 (en) * 2004-10-22 2008-02-21 Regents Of The University Of Minnesota Assemblies of Oligomeric Amyloid Beta Protein and Uses Thereof
US20210080463A1 (en) * 2017-06-08 2021-03-18 Carcidiag Biotechnologies Method of isolating and detecting cancer stem cells

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