WO2000055187A1 - Therapeutic and diagnostic applications of p400: a newly discovered beta-amyloid binding protein present in human biological fluids - Google Patents

Therapeutic and diagnostic applications of p400: a newly discovered beta-amyloid binding protein present in human biological fluids Download PDF

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WO2000055187A1
WO2000055187A1 PCT/US2000/006878 US0006878W WO0055187A1 WO 2000055187 A1 WO2000055187 A1 WO 2000055187A1 US 0006878 W US0006878 W US 0006878W WO 0055187 A1 WO0055187 A1 WO 0055187A1
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amyloid
disease
deπved
polypeptides
fragments
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PCT/US2000/006878
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French (fr)
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Gerardo Castillo
Alan D. Snow
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Proteotech, Inc.
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711Alzheimer's disease; Amyloid plaque core protein
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • the invention relates to the discover) , identification and use of P400 (a new ly discovered A ⁇ - binding protein present in human blood and cerebrospinal fluid), P400-de ⁇ ved protein fragments, and P400-de ⁇ ved polypeptides. as well as related peptides and antibodies, for the therapeutic intervention and diagnosis of Alzheimer ' s disease and other amyloidoses
  • Alzheimer's disease is characterized by the accumulation of a 39-43 amino acid peptide termed the beta-amyloid protein or A ⁇ in a fib ⁇ llar form, existing as extracellular amyloid plaques and as ai loid within the walls of cerebral blood vessels Fib ⁇ llar A ⁇ amv loid deposition m Alzheimer ' s disease is believed to be det ⁇ mental to the patient and eventually leads to toxicity and neuronal cell death, characteristic hallmarks of Alzheimer ' s disease. Accumulating evidence implicates amyloid as a major causative factor of Alzheimer ' s disease pathogenesis
  • a va ⁇ ety of other human diseases demonstrate amyloid deposition and usually involve systemic organs (i.e. organs or tissues lying outside the central nervous system), with the amyloid accumulation leading to organ dysfunction or failure.
  • systemic organs i.e. organs or tissues lying outside the central nervous system
  • amyloid accumulation leading to organ dysfunction or failure.
  • Alzheimer's disease and "systemic" amyloid diseases there is currently no cure or effective treatment, and the patient usually dies within 3 to 10 years from disease onset
  • New compounds or agents for therapeutic regimes to arrest or reverse amyloid formation, deposition, accumulation and/or persistence that occurs in Alzheimer's disease and other amyloidoses are therefore urgent sought.
  • Implicated proteins which may cont ⁇ bute to such A ⁇ solubility m biological fluids include apolipoproteins, albumin and laminin.
  • Implicated proteins which may cont ⁇ bute to such A ⁇ solubility m biological fluids include apolipoproteins, albumin and laminin.
  • P400 cerebrospinal fluid
  • Our data indicates that P400 in human serum and CSF binds tightly to A ⁇ and is decreased substantially in serum de ⁇ ved from Alzheimer's disease patients in compa ⁇ son to normal aged controls.
  • Implicated proteins which may contribute to such maintenance of A ⁇ solubility in biological fluids include apolipoproteins (apolipoproteins J and E)( Ghiso et al, Biochem. J. 293:27-30, 1993; Wisniewski et al, Biochem. Res. Commun 192:359-365, 1993; St ⁇ ttmatter et al, Proc. Natl. Acad. Sci. U. S. A. 90:8098-8102, 1993; LaDu et al. J. Biol. Chem.
  • a p ⁇ mary object of the present invention is to establish new therapeutic methods and diagnostic applications for the amyloid diseases.
  • the amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease and Down's syndrome (wherein the specific amyloid is referred to as beta-arm loid protein or A ⁇ ), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (wherein the specific ai loid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is refe ⁇ ed to as AL amyloid), the amyloid associated w ith t ⁇ pe II diabetes (wherein the specific amyloid is referred to as amyhn or islet amyloid), the amyloid associated with the pnon diseases including Creutzfeldt- Jakob disease, Gerstmann Straussler syndrome, kuru and
  • P400 refers to a newly discovered A ⁇ -binding protein present in human serum and CSF, and which has a molecular weight of approximately 350 to 450 kilodaltons as determined by standard SDS-PAGE (as descnbed herein)
  • a p ⁇ mary object of the present invention is to use P400, P400 protein fragments and/or P400- de ⁇ ved polypeptides as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer ' s disease and other amyloidoses
  • Yet another object of the present invention is to use conformational dependent proteins. polypeptides, or fragments thereof for the treatment of Alzheimer ' s disease and other amyloidoses.
  • conformational dependent proteins include, but are not limited to, P400. P400-de ⁇ ved fragments and/or portions thereof
  • Yet another aspect of the present invention is to use peptidomimetic compounds modeled from P400, P400-de ⁇ ved protein fragments and/or P400-de ⁇ ved polypeptides, and fragments thereof, as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses
  • Yet another object of the present invention is to mimic the 3-d ⁇ mens ⁇ onal A ⁇ -binding s ⁇ te(s) on P400, P400-de ⁇ ved protein fragments and/or P400-de ⁇ ved polypeptides and use these mimics as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses
  • Yet a further aspect of the present invention is to use anti-idiotypic antibodies to P400, P400- de ⁇ ved protein fragments and/or P400-de ⁇ ved polypeptides as potent inhibitors of amyloid formation, deposition accumulation and/or persistence in Alzheimer's disease and other amyloidoses
  • Another aspect of the invention is to provide new and novel polyclonal and/or monoclonal peptide antibodies which can be utilized in a number of in vitro assays to spec ⁇ f ⁇ call> detect A ⁇ - binding P400-de ⁇ ved protein fragments and/or A ⁇ -binding P400 derived polypeptides in human tissues and/or biological fluids
  • Polyclonal or monoclonal antibodies that are made specifically against a peptide portion or fragment of P400 hich interacts with A ⁇ can be utilized to detect and quantify amyloid disease specific P400 fragments in human tissues and/or biological fluids
  • These antibodies can be made b ⁇ administe ⁇ ng the peptides in antigemc form to a suitable host
  • Polyclonal or monoclonal antibodies may be prepared by standard techniques known to those skilled in the art
  • Another object of the present invention is to use P400.
  • Yet another aspect of the present invention is to use antibodies recognizing P400, any of the AB-binding P400 fragments, and/or P400-de ⁇ ved polypeptides, and fragments thereof, for m vivo labeling, for example with a radionucleotide. for radioimag g to be utilized for in vivo diagnosis and/or for in ⁇ itro diagnosis
  • Yet another aspect of the present invention is to make use of P400, P400-de ⁇ ved protein fragments, and P400-de ⁇ ved polypeptides. and fragments thereof, as potential therapeutics to inhibit the deposition, formation, and accumulation of fib ⁇ llar amyloid in Alzheimer's disease and other amyloidoses (desc ⁇ bed above), and to enhance the clearance and/or removal of preformed amyloid deposits in brain (for Alzheimer's disease and Down's syndrome amyloidosis) and in systemic organs (for systemic amyloidoses)
  • Another object of the present invention is to use A ⁇ -bmding P400-de ⁇ ved polypeptides or fragments thereof, in conjunction with polyclonal and/or monoclonal antibodies generated against these peptide fragments, using in vitro assays to detect amyloid disease specific autoantibodies in human biological fluids
  • Specific assay systems can be utilized to not only detect the presence of autoantibodies against A ⁇ -bindmg P400-de ⁇ ved protein fragments or polypeptides thereof in biological fluids, but also to monitor the progression of disease by following elevation or diminution of P400 protein fragments and/or P400-de ⁇ ved polypeptide autoantibody levels
  • Another aspect of the invention is to utilize P400. P400-de ⁇ ved protein fragments, and P400- de ⁇ ved antibodies and/or molecular biology probes for the detection of these P400 de ⁇ vatives in human tissues in the amyloid diseases
  • Yet another object of the present invention is to use the P400-de ⁇ ved protein fragments of the present invention in each of the various therapeutic and diagnostic applications desc ⁇ bed above
  • Specific P400-de ⁇ ved protein fragments or peptides as desc ⁇ bed above may be de ⁇ ved from any species including, but are not limited to, human, mu ⁇ ne, bovine, porcine, and/or equine species
  • Another object of the invention is to provide polyclonal and/or monoclonal peptide antibodies which can be utilized in a number of in vitro assays to specifically detect P400 protein fragments in human tissues and/or biological fluids
  • Polyclonal or monoclonal antibodies made specifically against a peptide portion or fragment of any of the P400 fragments desc ⁇ bed herein can be utilized to detect and quantify P400-de ⁇ ved protein fragments in human tissues and/or biological fluids
  • a prefe ⁇ ed embodiment is a polyclonal antibody made to P400 present in human serum and cerebrospinal fluid
  • These antibodies can be made by isolating and administe ⁇ ng P400, P400 fragments and/or P400 polypeptides in antigemc form to a suitable host.
  • Polyclonal or monoclonal antibodies may be prepared by standard techniques by one skilled in the art
  • Yet another object of the present invention is to use P400-de ⁇ ved antibodies as desc ⁇ bed herein as a specific indicator for the presence and extent of P400 breakdown in brain by monitoring biological fluids including, but not limited to. cerebrospinal fluid, blood, serum, u ⁇ ne, saliva, sputum, and stool
  • Yet another object of the present invention is to use P400-de ⁇ ved antibodies as desc ⁇ bed herein as a specific indicator for the presence, extent and/or progression of Alzheimer's disease and/or other brain amyloidoses by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, u ⁇ ne, saliva, sputum, and stool
  • biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, u ⁇ ne, saliva, sputum, and stool
  • Yet another object of the present invention is to use P400-de ⁇ ved antibodies as desc ⁇ bed herein as a specific indicator for the presence and extent of P400 breakdown in systemic organs by momto ⁇ ng biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, u ⁇ ne, saliva, sputum, and stool
  • Yet another object of the present invention is to use P400-de ⁇ ved antibodies as desc ⁇ bed herein as a specific indicator for the presence and extent of amyloidosis m type II diabetes by monito ⁇ ng biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, u ⁇ ne, saliva, sputum, and stool
  • biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, u ⁇ ne, saliva, sputum, and stool
  • Yet another object of the present invention is to use P400-de ⁇ ved antibodies as desc ⁇ bed herein as a specific indicator for the presence and extent of amyloidosis in other systemic amyloidoses by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool
  • Yet another object of the invention is to utilize specific P400-de ⁇ ved antibodies, as described herein, for the detection of P400 in human tissues in the amyloid diseases
  • Another object of the present invention is to use P400, P400-de ⁇ ved protein fragments, and P400-de ⁇ ved polypeptides. as descnbed herein, for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses
  • Another object of the present invention is to use pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, e xers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositones, ste ⁇ le injectable solutions, and ste ⁇ le packaged powders, which contain P400, P400-de ⁇ ved protein fragments, and P400-de ⁇ ved polypeptides, and fragments thereof, to treat patients with Alzheimer's disease and other amyloidoses
  • Yet another object of the present invention is to use P400, P400-de ⁇ ved protein fragments, and P400-de ⁇ ved polypeptides, and fragments thereof, as potent agents which inhibit amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, and/or cause a dissolution of preformed or pre deposited amyloid fib ⁇ ls in Alzheimer's disease, and other amyloidoses
  • Yet another object of the present invention is to provide the use of P400, P400-de ⁇ ved protein fragments, and P400-de ⁇ ved polypeptides, as described herein, for inhibition of amyloid formation, deposition, accumulation, and/or persistence, regardless of its clinical setting
  • Yet another object of the present invention is to provide compositions and methods involving administe ⁇ ng to a subject a therapeutic dose of P400, P400-de ⁇ ved protein fragments, and P400- de ⁇ ved polypeptides, which inhibit amyloid deposition, and fragments thereof. Accordingly, the compositions and methods of the invention are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs.
  • the proteins or polypeptides of the invention can be used therapeutically to treat amyloidosis or can be used prophylactically in a subject susceptible to amyloidosis.
  • the methods of the invention are based, at least in part, in directly inhibiting amyloid fib ⁇ l formation, and/or causing dissolution of preformed amyloid fibrils BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGURE 1 is a black and white photograph of a gand blot analysis of serum separated by non- reducing SDS-PAGE. transfe ⁇ ed to PVDF membrane, and probed with 5nM of b ⁇ ot ⁇ nylated-A ⁇ (1-40) Laminin and P400 are demonstrated to be major A ⁇ -binding proteins in human serum Low concentrations of A ⁇ (1-40) detected only those proteins that have dissociation constants in the nanomolar range A prominent and more focused -130 kDa A ⁇ -bmding protein (lower arrow) is believed to represent the E8 fragment of laminin (Begovac et al.
  • FIGURE 2 is a black and white photograph of a Western blot demonstrating that P400 is lmmunologically distinct from laminin This is a Western blot of human CSF (30 ⁇ l/lane, Lanes 1-6) and serum (10 ⁇ l/lane.
  • Lanes 7 - 14) probed with a laminin antibody See text for individual lane details Intact laminin (M r -850 kDa) (upper a ⁇ ow ) was present in human CSF (lanes 1 -6) but not as prominent in human serum (lanes 7- 14) No lmmunostainmg was observed in the region >205 kDa up to the gel interface, (compare to Fig 1) indicating that P400 is lmmunologically distinct from laminin
  • FIGURE 3 is a black and white photograph demonstrating isolation of P400 using Sephacryl S1000 gel filtration chromatography
  • This is an A ⁇ -hgand blot of va ⁇ ous fractions de ⁇ ved from Sephacryl S 1000 gel filtration chromatography, where a concentrated sample from 10 ml CSF was applied The blot was probed with 50 nM of biotinylated A ⁇ (1-40)
  • This blot shows that P400 is present in human CSF and can be pu ⁇ fied from other A ⁇ -binding proteins
  • Coomassie blue staining of similar blots indicate that very little low molecular weight proteins are present in fractions with K a , of 0 5 (not shown), indicating that this method is useful in initial pu ⁇ fication of P400
  • Amyloid is a gene ⁇ c term refer ⁇ ng to a group of diverse, but specific extracellular protein deposits which all have common morphological properties, staining characte ⁇ stics, and x-ray diffraction spectra. Regardless of the nature of the amyloid protein deposited all amyloids have the following characte ⁇ stics 1) an amorphous appearance at the light microscopic level and appear eosinophilic using hematoxvlin and eosm stains: 2) all stain with Congo red and demonstrate a red/green biref ⁇ ngence as viewed under pola ⁇ zed light (Puchtler et al., J Histochem Cytochem 10:355-364, 1962), 3) all contain a predominant beta-pleated sheet secondary structure, and 4) ultrastructurally amyloid usually consist of non-branching fib ⁇ ls of indefinite length and with a diameter of 7-10 nm
  • amyloid today is classified according to the specific amyloid protein deposited.
  • the amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease, Down ' s syndrome and Hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is referred to as beta-amyloid protein or A ⁇ ), the amyloid associated with chronic inflammation, va ⁇ ous forms of malignancy and Familial Medite ⁇ anean Fever (wherein the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is refe ⁇ ed to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is refe ⁇ ed to as amylin or islet amyloid), the amyloid associated with the p ⁇ on diseases including Creutzf el dt-
  • Gerstmann- Straussler syndrome, kuru and animal scrapie wherein the specific amyloid is refe ⁇ ed to as PrP amyloid
  • the amyloid associated with long-term hemodialysis and carpal tunnel syndrome wherein the specific amyloid is referred to as beta2-m ⁇ croglobul ⁇ n amyloid
  • the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy wherein the specific amyloid is refe ⁇ ed to as prealbumin or transthyretin amyloid
  • the amyloid associated with endoc ⁇ ne tumors such as medullary carcinoma of the thyroid
  • amyloid deposits in clinical conditions share common physical properties relating to the presence of a beta-pleated sheet conformation
  • many different chemical types exist and additional ones are likely to be desc ⁇ bed in the future.
  • a circulating precursor protein may result from overproduction of either intact or aberrant molecules (ex. plasma cell dyscrasias), reduced degradation or excretion (serum amyloid A in some secondary amyloid syndromes and beta2-rrucroglobuhn in long-term hemodialysis), or genetic abnormalities associated with variant proteins (ex.
  • Systemic amyloids which include the amyloid associated with chronic inflammation, va ⁇ ous forms of malignancy and Familial Medite ⁇ anean Fever (le AA amyloid or inflammation-associated amylo ⁇ dos ⁇ s)(Benson and Cohen, Arth Rheum. 22.36-42, 1979, Ka ei et al, Acta Path Jpn 32: 123-133, 1982; McAdam et al, Lancet 2 572-573, 1975; Metaxas, Kidnev Int.
  • amyloid associated with multiple myeloma and other B-cell dyscrasias (le AL amylo ⁇ d)(Harada et al, J_ Histochem Cytochem 19 1 15, 1971), as examples, are known to involve amyloid deposition in a va ⁇ ety of different organs and tissues generally lying outside the central nervous system Amyloid deposition in these diseases may occur, for example, in liver, heart, spleen, gastrointestinal tract, kidney, skin, and/or lungs (Johnson et al, N. Engl J Med 321:513-518, 1989). For most of these amyloidoses.
  • amyloid deposition in kidney may lead to renal failure
  • amyloid deposition in heart may lead to heart failure.
  • amyloid accumulation in systemic organs leads to eventual death generally w ithin 3-5 years
  • Other amyloidoses may affect a single organ or tissue such as observed with the A ⁇ amyloid deposits found in the brains of patients with Alzheimer ' s disease and Down's syndrome: the PrP amyloid deposits found in the brains of patients with Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, and kuru; the islet amyloid (amylin) deposits found in the islets of Langerhans in the pancreas of 907c of patients with type II diabetes (Johnson et al.
  • Alzheimer's disease is a leading cause of dementia in the elderly, affecting 5-109-- of the population over the age of 65 years (A Guide to Understanding Alzheimer's Disease and Related Disorders, edited by Jorm. New York University Press, New York, 1987).
  • Alzheimer's disease the parts of the brain essential for cognitive processes such as memory, attention, language, and reasoning degenerate, robbing victims of much that makes us human, including independence
  • onset is in middle age, but more commonly, symptoms appear from the m ⁇ d-60's onward Alzheimer's disease today affects 4-5 million Ame ⁇ cans, with slightly more than half of these people receiving care at home, while the others are in many different health care institutions
  • the prevalence of Alzheimer ' s disease and other dementias doubles every 5 years beyond the age of 65, and recent studies indicate that nearly 509c of all people age 85 and older have symptoms of Alzheimer's disease (1997 Progress Report on Alzheimer's Disease, National Institute on Aging/National Institute of Health). 13% (33 million people)
  • Alzheimer's disease also puts a heavy economic burden on society as well.
  • a recent study estimated that the cost of ca ⁇ ng for one Alzheimer's disease patient with severe cognitive impairments at home or in a nursing home, is more than $47,000 per year (A Guide to Understanding Alzheimer's Disease and Related Disorders, edited by Jorm, New York University Press, New York, 1987)
  • the overall cost of Alzheimer's disease to families and to society is stagge ⁇ ng
  • the annual economic toll of Alzheimer's disease in the United States in terms of health care expenses and lost wages of both patients and their caregivers is estimated at $80 to $100 billion (1997 Progress Report on Alzheimer's Disease, National Institute on Aging/National Institute of Health)
  • Tac ⁇ ne hydrochlo ⁇ de (“Cognex"), the first FDA approved drug for Alzheimer's disease is a acetylcholinesterase inhibitor (Cutler and Sramek, N. Engl. J. Med. 328:808 810, 1993). However, this drug has showed limited success in the cognitive improvement m Alzheimer's disease patients and initially had major side effects such as liver toxicity.
  • the second more recently FDA approved drug, donepezil also known as "A ⁇ cept”
  • a ⁇ cept also known as “A ⁇ cept”
  • tac ⁇ ne is more effective than tac ⁇ ne, by demonstrating slight cognitive improvement in Alzheimer's disease patients (Barner and Gray, Ann. Pharmacotherapy 32:70-77, 1998; Rogers and F ⁇ edhoff, Eur. Neuropsvch. 8:67-75, 1998), but is not believed to be a cure. Therefore, it is clear that there is a need for more effective treatments for Alzheimer's disease patients.
  • Amyloid as a Therapeutic Target for Alzheimer's Disease
  • Alzheimer ' s disease is characte ⁇ zed by the deposition and accumulation of a 39-43 amino acid peptide termed the beta-am loid protein, A ⁇ or ⁇ /A4 (Glenner and Wong, Biochem. Biophys Res Comm 120:885-890, 1984; Masters et al, Proc Natl Acad. Sci. U.S.A. 82:4245-4249, 1985; Husby et al, Bull WHO 71 :105-108, 1993 j A ⁇ is de ⁇ ved from larger precursor proteins termed beta-amyloid precursor proteins (or BPPs) of which there are several alternatively spliced va ⁇ ants.
  • beta-am loid precursor proteins or BPPs
  • the most abundant forms of the ⁇ PPs include proteins consisting of 695, 751 and 770 amino acids (Tanzi et al, Nature 331:528-530, 1988; Kitaguchi et al, Nature 331.530-532, 1988; Ponte et al, Nature 331:525-527, 1988)
  • the small A ⁇ peptide is a major component which makes up the amyloid deposits of "plaques" in the brains of patients with Alzheimer ' s disease. In addition.
  • Alzheimer's disease is characte ⁇ zed by the presence of numerous neurofib ⁇ llary "tangles " , consisting of paired helical filaments which abnormally accumulate in the neuronal cytoplasm (Grundke-Iqbal et al, Proc. Natl. Acad Sci U S.A. 83:4913-4917, 1986; Kosik et al, Proc. Natl. Acad Sci U S.A 83:4044-4048, 1986; Lee et al, Science 251:675-678, 1991).
  • the pathological hallmarks of Alzheimer's disease is therefore the presence of "plaques” and "tangles", with amyloid being deposited in the central core of plaques.
  • amyloid The other major type of lesion found in the Alzheimer ' s disease brain is the accumulation of amyloid in the walls of blood vessels, both within the brain parenchyma and in the walls of meningeal vessels which lie outside the brain.
  • the amyloid deposits localized to the walls of blood vessels are refe ⁇ ed to as cerebrovascular amyloid or congophihc ang ⁇ opath ⁇ (Mandybur, J Neuropath Exp Neurol 45.79-90, 1986, Pard ⁇ dge et al, J_ Neurochem 49' 1394-1401. 1987)
  • Alzheimer's A ⁇ protein in cell culture has been shown to cause degeneration of nerve cells within short pe ⁇ ods of time (Pike et al, Br. Res. 563:311-314, 1991: J.
  • Neurochem 64:253-265, 1995 Studies suggest that it is the fib ⁇ llar structure (consisting of a predominant ⁇ -pleated sheet secondary structure), characte ⁇ stic of all amyloids, that is responsible for the neurotoxic effects.
  • a ⁇ has also been found to be neurotoxic in slice cultures of hippocampus (Har ⁇ gan et al, Neurobiol. Aging 16:779-789, 1995) and induces nerve cell death in transgenic mice (Games et al, Nature 373:523-527. 1995; Hsiao et al, Science 274:99-102, 1996). Injection of the Alzheimer's A ⁇ into rat brain also causes memory impairment and neuronal dysfunction (Flood et al, Proc. Natl.
  • a ⁇ amyloid is directly involved in the pathogenesis of Alzheimer ' s disease comes from genetic studies. It has been discovered that the production of A ⁇ can result from mutations in the gene encoding, its precursor, beta amyloid precursor protein (Van Broeckhoven et al, Science 248: 1120-1122, 1990; Murrell et al, Science 254:97-99, 1991 , Haass et al, Nature Med 1 : 1291-1296.
  • a ⁇ is normally present in biological fluids including its presence in both cerebrospinal fluid (CSF)(Shoj ⁇ et al, Science 258: 126-129, 1992; Seubert et al, Nature 359 325-327, 1992, Vigo-Pelfrey et al, J. Neurochem 61 : 1965-1968, 1993) and plasma (Seubert et al, Nature 359.325-327, 1992). Seubert et al (Nature 359:325-327, 1992) found A ⁇ peptide in both CSF and plasma of humans, dogs, guinea pigs and rats.
  • CSF cerebrospinal fluid
  • Seubert et al found A ⁇ peptide in both CSF and plasma of humans, dogs, guinea pigs and rats.
  • a ⁇ in CSF In humans, the concentration of A ⁇ in CSF is approximately 2 5 ng/ml whereas in plasma the A ⁇ concentration is approximately 0.9 ng/ml (Seubert et al, Nature 359.325-327, 1992).
  • a ⁇ in CSF appears to consist of va ⁇ ous lengths, 70% of A ⁇ in CSF consists of A ⁇ 1-34 and A ⁇ 1-40 (Vigo-Pelfrev et al. J. Neurochem. 61 : 1965-1968. 1993). Since previous studies have demonstrated that synthetic peptides homologous to A ⁇ 1-40 and 1-42 spontaneously form amyloid-like fib ⁇ ls (Kirschner et al, Proc. Natl. Acad. Sci.
  • a prominent -130 kDa A ⁇ -binding band (lower arrow) was present in all patients serums and was believed to represent the E8 fragment of laminin (Yurchenco et al, J Biol Chem 268 17286-17299, 1993)
  • a prominent >205 kilodalton band (and designated as P400 due to an estimated approximate size of -350-450 k ⁇ lodaltons)(upper a ⁇ ow) is also present in human serum de ⁇ ved from normal (lanes 1 and 4) and type II diabetes (lanes 2, 5, 7 and 8) patients, and appears to be markedly decreased in the serum de ⁇ ved from 2 patients with moderate AD (lanes 3 and 6). All of these serums were similarly obtained from live patients so the decreased P400 in the serum of probable AD patients is not believed to represent "breakdown" products Evidence also indicates that P400 is also present in human CSF samples (see
  • a polyclonal antibody against laminin was used to probe (by Western blot) human CSF and serum samples to determine whether P400 was lmmunologically related to laminm.
  • human CSF (30 ⁇ l/lane, lanes 1-6) and serum (10 ⁇ l/lane, lanes 7-14) was probed with a polyclonal antibody against laminm (obtained from Sigma Chemical Co., St Louis. MO).
  • Figure 2 the following human biological fluid samples (i.e.
  • Lane 1 #5211- CSF from a 66 year old male with probable AD with a mini-mental score of 25
  • Lane 2 #5113- CSF from a 83 year old normal male with a mini-mental score of 27
  • Lane 3 #5112- CSF from a 78 year old normal female with a mini-mental score of 30
  • Lane 4 #5111- CSF from a 69 year old female with presymptomatic Alzheimer ' s disease with rrum-mental score of 28
  • Lane 5 #5110- CSF from a 67 year old normal male with a mini-mental score of 30
  • Lane 6 #5109- CSF from a 78 year old normal female with a mini-mental score of 30
  • the co ⁇ esponding serum from these same patients are in Lanes 7- 12, and in the same order as the CSF samples.
  • Lane 13 #5101- serum from a 73 year old normal male with a mini-mental score of 30
  • P400 is present in human CSF and can be isolated by Sephacryl S1000 fractionation (Fig. 3).
  • 10 ml of CSF were fractionated through an Sephacryl S1000 column, and the va ⁇ ous fractions were separated by non-reducing SDS-PAGE, transfe ⁇ ed to PVDF membrane and probed with 50 nM of b ⁇ ot ⁇ nylated-A ⁇ .
  • This concentration of b ⁇ ot ⁇ nylated-A ⁇ was chosen so as to detect most proteins in CSF which bind A ⁇ .
  • P400 (upper a ⁇ ow) is present in human CSF and can be ennched using Sephacryl S1000 fractionation.
  • the AB-ligand blot (Fig. 3).
  • the precipitate obtained is then dissolved in 2 ml of 50 mM T ⁇ s-HCl, 150 mM NaCl, pH 7 5 (TBS) and passed through an A ⁇ affinity column for further pu ⁇ fication
  • a ⁇ affinity column For preparation of the A ⁇ affinity column, 1 mg of A ⁇ 1-40 (Bachem Ine, To ⁇ ance, CA) in 0.5 ml of double distilled water is added to 1 ml of washed Affigel beads (Biorad) suspended in 1 volume of 0 2 M HEPES 160 mM CaCl (pH 7.5) The mixture is incubated overnight with gentle agitation at room temperature and stopped with lOO ⁇ l of 1 M ethanolamine at pH 8.0 for 1-2 hours at room temperature. The denvatized gel is washed in a minicolumn and the flow through is collected and assayed for protein content using a Buffalo black or Bradford reagent (Biorad) to determine the % of protein bound to the column.
  • Affigel beads Biorad
  • the denvatized gel is washed in a minicolumn and the flow through is collected and assayed for protein content using a Buffalo black or Bradford reagent (Biorad) to determine the % of protein bound to
  • Tightly bound matenals are then be eluted with 10 ml of 50 mM Tns-HCl, 3M NaCl, 7 M Urea (pH 8.0). A quots of each of 2ml fractions are precipitated by 15-m ⁇ nute cent ⁇ fugation at 14,000 Xg in the presence of 4 volumes of 95 % ethanol with 1 % sodium acetate The pellets are then made up to 10 ⁇ l 1 x SDS sample buffer and are subjected to SDS-PAGE and transfe ⁇ ed to PVDF membrane (see below) to identify fractions containing P400 Identification of P400 is based on size and its ability to bind A ⁇ as determined by A ⁇ ligand blot analysis
  • protease digestions are performed to expose internal portions ot the protein which are anticipated to allow for N-terminal sequencing
  • P400 is either left undigested, or digested with V8, trypsin, or elastase (Sigma Chem Co St Louis, MO) p ⁇ or to SDS-PAGE More specifically 2 ⁇ g of trypsm, V8 protease, or elastase in 2 ⁇ l of 50 mM T ⁇ s-HCl buffer (pH 8 0) is added to either buffer only, or to 50 ⁇ l of P400 (50 ⁇ g) in the same buffer The mixtures are incubated at 37°C and ahquots (10 ⁇ l) taken at va ⁇ ous times are mixed ith equal volumes of 2X non-reducing SDS-sample buffer for SDS- PAGE The separated P400 fragments are then transfe ⁇ ed to PVDF membranes and
  • a more accurate molecular weight of P400 is also determined by slightly decreasing the acrylamide concentration (from 4% to 3 5%) and include larger molecular weight standards (i.e >400 kDa) obtained commerciallv
  • Example 5 Determination of the Strength of Binding of P400 to Beta-Amyloid Protein Rationale: The data indicates that P400 binds to A ⁇ with high affinity human biological fluids
  • solid phase binding assays are used to determine the dissociation constant of P400 to immobilized A ⁇ using b ⁇ ot ⁇ nylated-P400
  • polyclonal antibodies to P400 are generated in rabbits and used in ELISA assays to determine the dissociation constant of A ⁇ binding to P400 as previously descnbed (Castillo et al, J Neurochem 69:2452-2465, 1997)
  • Nunc plates (Maxisorb) containing immobilized A ⁇ is first prepared by incubating each well overnight with 2 ⁇ g of A ⁇ (Bachem Ine, To ⁇ ance, CA) in 40 ⁇ l of TBS containing lOOmM Tns-HCl, 50 mM NaCl, and 3 mM NaN3 (pH 7.4) The next day the wells are blocked with 300 ⁇ l of TBS containing 0.05% Tween-20 (TTBS) plus 2% bovine serum albumin (BSA) Wells without immobilized A ⁇ (i.e. blank wells) are also blocked with TTBS containing 2 % albumin as desc ⁇ bed above.
  • a ⁇ Bachem Ine, To ⁇ ance, CA
  • TBS Tween-20
  • BSA bovine serum albumin
  • Pu ⁇ fied and biotinylated P400 (1 mg/ml) is diluted in TTBS at dilutions of 1 10, 1 30, 1:90, 1.270, 1:810, 1:2430, 1.7290 (v/v) and blank. These dilutions are plated (250 ⁇ l) in t ⁇ phcate wells in the presence or absence of immobilized A ⁇ (1-40) and incubated overnight The next day, the wells are nnsed 3 times with TTBS and probed for 30 min with 100 ⁇ l of streptavidm-peroxidase (1:500 of 2 ⁇ g/ml) in TTBS containing 0 1 % BSA (i.e secondary probe) The wells are then nnsed 3 times with TTBS and 100 ⁇ l of substrate solution ( OPD-Sigma Fast from Sigma Chemical Co., St.
  • rabbit polyclonal antibodies are utilized to detect the relative amount of P400 bound to A ⁇ immobilized on rmcrotiter wells.
  • P400 at va ⁇ ous dilutions desc ⁇ bed above are plated (250 ⁇ l) in t ⁇ phcate wells in the presence or absence of immobilized A ⁇ (1-40) and incubated overnight The next da> the wells are nnsed 3 times with TTBS and probed for 1 hour with 100 ⁇ l of an ant ⁇ -P400 polyclonal antibody diluted in TTBS (dilution to be determined empincally).
  • TTBS tinylated goat anti-rabbit secondary antibody
  • streptavidin-peroxidase 1:500 of 2 ⁇ g/ml
  • the wells are then be nnsed 3 times with TTBS and 100 ⁇ l of the substrate solution ( OPD-Sigma Fast from Sigma Chemical Co., St. Louis, MO.) is then added to each well and allowed to develop for 10 rrunutes or until there is a significant color difference.
  • the reaction is stopped with 50 ⁇ l of 4 N Sulfu ⁇ c acid and read at 490 nm on an ELISA plate reader. The data is plotted and the K d is determined using the Ultrafit program (Biosoft, UK)
  • the binding data is analyzed assuming a thermodynarmc equilibrium for the formation of the complex BL, from the P400 ligand in solution, L, and the uncomplexed A ⁇ adsorbed to the rmcrotiter well, B, according to the equation.
  • K d [B] X [L]/[BL] (Castillo et al, J Neurochem 69:2452-2465, 1997).
  • the K d 's are determined by using an enzyme-linked immunoassay that gives a color signal that is proportional to the amount of P400 bound to A ⁇ (Engel and Schalch, Mol Immunol.
  • control wells without A ⁇ are included for each concentration of P400 used in each binding expenment.
  • Optical densities of the control wells usually never exceed 0.050 at all ligand concentrations based on previous studies (Castillo et al, J. Neurochem 69:2452-2465, 1997).
  • the derivatized gel is washed in a minicolumn and the flow-through is collected and assayed for protein content using the Bradford reagent according to manufacturer protocol (Biorad).
  • the column is equilibrated with TBS with 0.1 % TX-100 and 10 ml of serum diluted 1:4 in 100 mM Tris-HCl, 50 mM NaCl, pH 7.5 (TBS) is applied several times. Unbound materials are washed with 20 ml TBS. Bound antibodies are eluted with 1 % acetic acid/water and lyophilized.
  • Example 6 Effects of P400 on Inhibition/Dissolution of A ⁇ Fibril Formation Rationale: The initial data demonstrates that P400 binds to A ⁇ with high affinity in biological fluids. P400 is believed to be a potent inhibitor of A ⁇ fibril formation and likely dissolves pre-formed A ⁇ amyloid fibrils. For these studies, Thioflavin T fluorometry, Congo red staining assays and negative stain electron microscopy are employed to determine the effects of purified P400 on inhibition of A ⁇ fibril formation and whether P400 is capable of dissolving preformed A ⁇ amyloid fibrils.
  • Fibrillogenesis is determined by addition of 1.2 ml Thioflavin T solution (100 ⁇ M Thioflavin T in 50 mM Sodium phosphate buffer pH 6.0) to 50 ⁇ l aliquots, followed by reading on the fluorometer as previously described (Castillo et al, J. Neurochem. 69:2452-2465, 1997). The effect of various concentrations of P400 (5- lOOnM) on A ⁇ fibrillogenesis (25 ⁇ M) is also tested in a similar manner as described above.
  • Example 7 P400 as a Specific Diagnostic Marker For Alzheimer's Disease Rationale: The data suggests that P400 is reduced in the biological fluids in Alzheimer's disease and may therefore potentially serve as a specific biochemical marker. Studies utilizing A ⁇ -hgand blotting techniques followed by scanning densitometry can quantitate changes relative intensity P400 among Alzheimer's disease versus normal aged-matched control patients.
  • sandwich or competitive ELISAs are used to determine whether P400 is useful as a specific marker protein for Alzheimer's disease and/or its progression Methodology: A ⁇ -hgand blotting techniques are used to assess levels of P400 in biological fluids of Alzheimer's disease versus normal aged-matched controls Biotinylation of A ⁇ 1-40, and assessment of serum and CSF samples by A ⁇ -hgand blotting is as described in example 5. Following transfer of serum and CSF proteins to PVDF membranes as desc ⁇ bed in example 5, membrane blots are probed for 2 hrs with 50 nM biotinylated AB (1-40) in TTBS which is heated to 90°C for 5 minutes pnor to use.
  • the membranes are then nnsed three times (5 seconds each) with TTBS, probed for 30 minutes with streptavidin alkaline phosphatase conjugate (Vectastain), nnsed again (as desc ⁇ bed above), followed by the addition of an alkaline phosphatase substrate solution (Vectastain). Following color development, flushing the membranes with distilled deiomzed water stops the reaction.
  • the relative intensity of P400 bands is determined by scanning the blots and quatifying using a Biorad scanning densitometer, as previously descnbed (Maresh et al, Soc Neurosci. Abst. 23.2221, 1997)
  • TTBS Tween-20
  • BSA bovine serum albumin
  • Serum samples diluted 1 100 are applied in tnphcate wells in the presence or absence of immobilized A ⁇ (1-40), and then incubated overnight.
  • Known amounts of punfied P400 are also applied to serve as an internal quantitative standard.
  • the wells are nnsed 3 times with TTBS and probed for 30 min with 100 ⁇ l of a secondary probe consisting of peroxidase conjugated anti-rabbit antibody diluted 1:500 (out of 2 ⁇ g/ml )(Jackson immunoresearch, Westgrove, PA) in TTBS containing 0.1 % BSA.
  • the wells are then nnsed 3 times with TTBS, and 100 ⁇ l of substrate solution (OPD-Sigma Fast from Sigma Chemical Co., St. Louis. MO.) is added to each well and allowed to develop for 10 minutes or until there is a significant color difference.
  • substrate solution OPD-Sigma Fast from Sigma Chemical Co., St. Louis. MO.
  • the reaction is stopped with 50 ⁇ l of 4 N Sulfu ⁇ c acid and read at 490 nm on an ELISA plate reader.
  • the optical density of vanous concentrations of standard P400 is plotted, and the concentrations of P400 in CSF and serum samples is determined based on the standard curve. If polyclonal antibodies against P400 are not available, levels of P400 in biological fluids are quantitated by a competitive ELISA format.
  • the competitive ELISA is performed based on the competition for binding of biotinylated P400 versus nonbiotinylated P400 (in serum or CSF) to immobilized A ⁇ .
  • the amounts of biotinylated P400 bound to immobilized A ⁇ is inversely proportional to the amounts of P400 in the biological sample. More specifically, Nunc plates (Maxisorb) containing immobilized A ⁇ (1-40 or 1-42; Bachem Inc., To ⁇ ance, CA) is prepared by incubating each well overnight with 2 ⁇ g of A ⁇ in 40 ⁇ l Tris-buffered saline (TBS; 100 mM Tris-HCl, 50 mM NaCl, and 3 mM NaN3, pH 7.4) and blocking the next day with 300 ⁇ l TBS containing 0.05% Tween-20 (TTBS) plus 2% bovine serum albumin (BSA).
  • TBS Tris-buffered saline
  • Wells without immobilized A ⁇ are also blocked with TTBS containing 2 % albumin as described above (i.e. blank wells).
  • TTBS containing 2 % albumin as described above
  • Various known standard concentrations of nonbiotinylated P400 and serum or CSF samples with unknown concentrations of P400 are mixed with fixed concentration of biotinylated-P400.
  • the fixed concentration of biotinylated-P400 is chosen so that it is close to saturating all of the immobilized A ⁇ sites in each well. These solutions are applied in triplicate wells in the presence or absence of immobilized A ⁇ (1-40) and incubated overnight.
  • the wells are rinsed 3 times with TTBS and probed for 30 min with 100 ⁇ l of secondary probe made up with streptavidin-peroxidase (1 :500 of 2 ⁇ g/ml) in TTBS containing 0.1 % BSA.
  • the wells are then rinsed 3 times with TTBS and 100 ⁇ l of the substrate solution ( OPD-Sigma Fast from Sigma Chemical Co., St. Louis, MO.) is added to each well and allowed to develop for 10 minutes or until there is a significant color differences.
  • the reaction is stopped with 50 ⁇ l of 4 N Sulfu ⁇ c acid and read at 490 nm.
  • a standard curve is then plotted from the optical density results of the standard concentrations of P400, which is an inverse relationship. The concentrations of P400 in CSF and serum is determined based on the standard curve.
  • One therapeutic application of the present invention is to use P400, P400 protein fragments which bind A ⁇ or other amyloid proteins, and/or P400 polypeptides derived from amino acid sequencing of the P400 fragments which bind A ⁇ or other amyloid proteins, as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses.
  • amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease and Down's syndrome (wherein the specific amyloid is refe ⁇ ed to as beta-amyloid protein or A ⁇ ), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Medite ⁇ anean Fever (wherein the specific amyloid is refe ⁇ ed to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is refe ⁇ ed to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is refe ⁇ ed to as amylin or islet amyloid), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is refe ⁇ ed to as PrP amyloid
  • the polypeptides refe ⁇ ed to above may be a natural polypeptide, a synthetic polypeptide or a recombinant polypeptide.
  • the fragments, derivatives or analogs of the polypeptides to any P400 fragment refe ⁇ ed to herein may be a) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be encoded by the genetic code, or b) one in which one or more of the amino acid residues includes a substituent group, or c) one in which the mature polypeptide is fused with another compound, such as a compound used to increase the half-life of the polypeptide (for example, polylysine), or d) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • the tertiary structure of proteins refers to the overall 3-dimensional architecture of a polypeptide chain.
  • the complexity of 3-dimensional structure arises from the intrinsic ability of single covalent bonds to be rotated. Rotation about several such bonds in a linear molecule will produce different nonsuperimpossable 3-dimensional a ⁇ angements of the atoms that are generally described as confo ⁇ nations.
  • Protein conformation is an essential component of protein-protein, protein-substrate, protein- agonist, protein-antagonist interactions. Changes in the component amino acids of protein sequences can result in changes that have little or no effect on the resultant protein conformation. Conversely, changes in the peptide sequences can have effects on the protein conformation resulting in reduced or increased protein-protein, etc. interactions. Such changes and their effects are generally disclosed in Proteins: Structures and Molecular Properties by Thomas Creightonm W.H Freeman and Company, New York, 1984 which is hereby inco ⁇ orated by reference
  • Conformation and “conformation simila ⁇ ty” when used in this specification and claims refers to a polypeptide's ability (or any other organic or inorganic molecule) to assume a given shape, through folding and the like, so that the shape, or conformation, of the molecule becomes an essential part of it ' s functionality, sometimes to the exclusion of its chemical makeup It is generally known that m biological processes two conformational similar molecules may be interchangeable in the process, even the chemically different "Conformational simila ⁇ ty” refers to the latter mterchangeability or substitutabi ty
  • P400 and P400-de ⁇ ved protein fragments are among the subjects of the invention because they have been shown to bind the A ⁇ protein and render it inactive in fib ⁇ l fo ⁇ nation; it is contemplated that other molecules that are conformationally similar to P400, or any claimed P400 fragment or polypeptide, may be substituted in the claimed method to similarly render the A ⁇ inactive in fibnllogenesis and other amyloid processes
  • polypeptides of the present invention include the polypeptides or fragments of P400 and fragments thereof, as well as polypeptides which have at least 70% similanty (preferably 70 % identity) and more preferably a 90% similanty (more preferably a 90% identity) to the polypeptides of P400.
  • Fragments or portions of the polypeptides or fragments of P400 of the present invention may be employed for producing the co ⁇ esponding full-length polypeptides by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full length polypeptides.
  • polypeptides of the present invention may be a naturally punfied product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bactenal, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant procedure, the polypeptides of the present invention may be glycosylated or may be non glycosylated Polypeptides of the invention may also include an initial methiomne amino acid residue
  • Standard reference works seting forth the general pnnciples of recombinant DNA technology include Watson, Molecular Biology of the Gene, Volumes I and II, The Benjamin/Cummings Publishing Company Ine , publisher, Menlo Park, Calif 1987, Ausubel et al, eds , Cu ⁇ ent Protocols in Molecular Biology, Wiley Interscience, publisher, New York, N Y 1987, 1992, and Sambrook et al, Molecular Cloning A Laboratory Manual, Second Edition, Cold Sp ⁇ ng Harbor Laboratory, publisher, Cold Sp ⁇ ng Harbor, N.Y 1989, the entire contents of which references are herein inco ⁇ orated by reference
  • polypeptides of the present invention may also be utilized as research reagents and mate ⁇ als for discovery of treatments and diagnostics for human diseases
  • Antibodies generated against the polypeptides co ⁇ esponding to specific sequences recognizing the P400 fragments of the present invention hich bind A ⁇ or other amyloid proteins can be obtained by direct injection of the polypeptides into an animal or by administe ⁇ ng the polypeptides to an animal, preferably a nonhuman The antibody so obtained will then bind the polypeptides itself In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides Such antibodies can then be used to isolate the polypeptides from tissue expressing that polypeptide
  • antibody is meant to include polyclonal antibodies, monoclonal antibodies, chime ⁇ c antibodies, anti-idiotypic antibodies to antibodies specific for P400 de ⁇ ved protein fragments or polypeptides of the present invention
  • Polyclonal antibodies are heterogeneous populations of antibody molecules denved from the sera of animals immunized with an antigen
  • a monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybndo a technique (Kohler and Milstem, Nature 256:495-497, 1975), the t ⁇ oma technique, the human B-cell hybndoma technique (Kozbor et al. Immunology Today 4 72, 1983), and the EBV-h b ⁇ doma technique to produce human monoclonal antibodies (Cole et al, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp 77-96, 1985)
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE. IgA, GILD and any subclass thereof
  • Chimenc antibodies are molecules different portions of which are de ⁇ ved from different animal species, such as those having vanable region denved from a munne monoclonal antibody and a human immunoglobulin constant region, which are p ⁇ ma ⁇ ly used to reduce immunogenicity in application and to increase yields in production Chime ⁇ c antibodies and methods for their production are known in the art (ex. Cabilly et al. Proc Natl Acad Sci U S A 81 :3273-32 " 77, 1984, Harlow and Lane Antibodies' A Laboratory Manual. Cold Sp ⁇ ng Harbor Laboratory 1988)
  • An anti-idiotypic antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An anti-iodiotypic antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the monoclonal antibody with the monoclonal antibody to which an anti-iodiotypic antibody is being prepared. The immunized animal will recognize and respond to the ldiotypic determinants of the immunizing antibody by producing an antibody to these ldiotypic determinants (the anti-idiotypic antibody). See, for example. U S Patent No 4,699.880. which is herein inco ⁇ orated by reference
  • antibody is also meant to include both intact molecules as well as fragments thereof, such as. for example. Fab and F(ab')2, which are capable of binding antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less nonspecific tissue binding than an intact antibody (Wahl et al, J Nucl Med. 24-316-325. 1983)
  • the antibodies or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect P400 or P400-de ⁇ ved fragments in a sample or to detect presence of cells which express a P400 polypeptide of the present invention.This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytomet ⁇ c or fluoromet ⁇ c detection
  • a P400 antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme lmmunoassay (EIA).
  • EIA enzyme lmmunoassay
  • This enzyme when later exposed to an approp ⁇ ate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometnc, fluoromet ⁇ c, or by visual means.
  • Enzymes which can be used detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-stero ⁇ d isomerase, yeast alcohol dehydrogenase, alpha glycerophosphate dehydrogenase, t ⁇ ose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparagmase, glucose oxidase. beta-galactosidase. ⁇ bonuclease, urease, catalase, glucose-6-phosphate dehydrogenase.
  • glucoamylase and acetylcholinesterase The detection can be accomplished by colomet ⁇ c methods which employ a chromogenic substrate for the enzyme Detection can be accomplished by colometnc methods which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual companson of the extent of enzymatic reaction of a substrate with similarly prepared standards (see Harlow and Lane, Antibodies. A Laboratory Manual. Cold Spnng Harbor Laboratory 1988; Ausubel et al, eds., Cu ⁇ ent Protocols in Molecular Biology. Wiley Interscience, N.Y. 1987, 1992)
  • Detection may be accomplished using any of a vanety of other immunoassays.
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a gamma-counter, a scintillation counter or by autoradiography.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythnn, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. commercially available, e.g , from Molecular Probes, Ine (Eugene, Oregon, U S A )
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152EU, or other of the lanthanide senes. These metals can be attached to the antibody using such metal groups as diethylenetnamine pentaacetic acid (EDTA)
  • EDTA diethylenetnamine pentaacetic acid
  • the antibody can also be detectably labeled by coupling it to a chemiluminescent compound
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that anses du ⁇ ng the course of a chemical reaction
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acndinium ester, lmidazole, ac ⁇ dinium salt, and oxalate ester
  • bioluminescent compound may be used to label the antibody of the present invention
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for pu ⁇ oses of labeling are lucifenn, luciferase and aequonn.
  • the antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of a P400 fragment of the present invention
  • In situ detection may be accomplished by removing a histological specimen from a patient, and providing the labeled antibody of the present invention to such a specimen
  • the antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample.
  • antibodies against P400, P400 fragments and/or P400-de ⁇ ved polypeptides which interact with A ⁇ or other amyloid proteins, or de ⁇ vatives thereof can be used for a number of important diagnostic and/or therapeutic applications as desc ⁇ bed herein.
  • polyclonal and/or monoclonal antibodies made against P400, P400 fragments and/or P400-de ⁇ ved polypeptides which bind A ⁇ or other amyloid proteins may be utilized for Western blot analysis (using standard Western blotting techniques knowledgeable to those skilled in the art) to detect the presence of amyloid protein-binding P400 fragments or amyloid protein-binding P400 polypeptides in human tissues and in tissues of other species.
  • Western blot analysis can also be used to determine the apparent size of each amyloid protein-binding P400 fragment
  • Western blotting following by scanning densitometrv can be used to quantitate and compare levels of each of the P400 fragments in tissue samples.
  • Bio fluids or biopsies obtained from individuals with specific diseases (such as the amyloid diseases) in comparison to tissue samples, biological fluids or biopsies obtained from normal individuals or controls.
  • Biological fluids include, but are not limited to, blood, plasma, serum, cerebrospinal fluid, sputum, saliva, u ⁇ ne and stool
  • polyclonal and/or monoclonal antibodies made against P400, P400 fragments and or P400-de ⁇ ved peptides which bind A ⁇ or other amyloid proteins can be utilized for immunoprecipitation studies (using standard immunoprecipitation techniques known to one skilled in the art) to detect P400, P400 fragments and/or P400-denved peptides which bind A ⁇ or other amyloid proteins, in tissues, cells and/or biological fluids.
  • P400, P400 fragments and/or P400- denved peptide antibodies for immunoprecipitation studies can also be quantitated to determine relative levels of P400, P400 fragments and/or P400-de ⁇ ved peptides which interact with A ⁇ or other amyloid proteins, in tissues, cells and/or biological fluids.
  • Quantitative immunoprecipitation can be used to compare levels of P400, P400 fragments and/or P400 amyloid protem-bmding peptides in tissue samples, biological fluids or biopsies obtained from individuals with specific diseases (such as the amyloid diseases) in comparison to tissue samples, biological fluids or biopsies obtained from normal individuals or controls.
  • Yet another aspect of the present invention is to make use of P400, P400 fragments and/or P400- derived polypeptides as amyloid inhibitory therapeutic agents.
  • the P400-derived peptide sequences or fragments can be synthesized utilizing standard techniques (ie. using an automated synthesizer).
  • P400, P400 fragments and/or P400-derived peptides which bind A ⁇ or other amyloid proteins can be used as potential blocking therapeutics for the interaction of P400 in a number of biological processes and diseases (such as in the amyloid diseases described above).
  • specific peptides made against P400 or fragments thereof may be used to aid in the inhibition of amyloid formation, deposition, accumulation, and /or persistence in a given patient.
  • anti-idiotypic antibodies made against P400, P400 fragments and/or P400-derived peptides may be given to a human patient as potential blocking antibodies to disrupt continued amyloid formation, deposition, accumulation and/or persistence in the given patient.
  • Preparations of P400-derived polypeptides for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain axillary agents or excipients which are known in the art.
  • Pharmaceutical compositions such as tablets, pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositories, sterile injectable solutions, sterile packaged powders, can be prepared according to routine methods and are known in the art.
  • P400, P400 fragments and/or P400-derived peptides may be used as an effective therapy to block amyloid formation, deposition, accumulation and/or persistence as observed in the amyloid diseases.
  • the invention includes a pharmaceutical composition for use in the treatment of amyloidoses comprising a pharmaceutically effective amount of a P400, P400 fragments and/or P400-derived peptide anti-idiotypic antibody and a pharmaceutically acceptable carrier.
  • the compositions may contain P400, P400 fragments and/or P400-derived peptide anti-idiotypic antibody, either unmodified, conjugated to a potentially therapeutic compound, conjugated to a second protein or protein portion or in a recombinant form (ie.
  • compositions may additionally include other antibodies or conjugates.
  • the antibody compositions of the invention can be administered using conventional modes of administration including, but not limited to, topical, intravenous, intra-arterial, intraperitoneal, oral, intralymphatic, intramuscular or intralumbar. Intravenous administration is prefe ⁇ ed.
  • the compositions of the invention can be a variety of dosage forms, with the prefe ⁇ ed form depending upon the mode of administration and the therapeutic application. Optimal dosage and modes of administration for an individual patient can readily be determined by conventional protocols.
  • P400, P400 fragments and/or P400-derived peptides, or antibodies of the present invention may be administered by any means that achieve their intended pu ⁇ ose, for example, to treat P400 involved pathologies, such as Alzheimer's disease and other amyloid diseases, or other related pathologies, using a P400-derived polypeptide described herein, in the form of a pharmaceutical composition.
  • administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal or buccal routes.
  • parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal or buccal routes.
  • administration may be by the oral route.
  • Parenteral administration can be by bolus injection or by gradual perfusion over time.
  • a prefe ⁇ ed mode of using a P400-derived polypeptide, or antibody pharmaceutical composition of the present invention is by oral administration or intravenous application.
  • a typical regimen for preventing, suppressing or treating P400-involved pathologies, such as Alzheimer's disease amyloidosis comprises administration of an effective amount of P400-derived polypeptides, administered over a period of one or several days, up to and including between one week and about 24 months.
  • the dosage of the P400-derived polypeptides of the present invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concu ⁇ ent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the most prefe ⁇ ed dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.
  • the total dose required for each treatment may be administered by multiple doses or in a single dose.
  • a P400-derived polypeptide may be administered alone or in conjunction with other therapeutics directed to P400-involved pathologies, such as Alzheimer's disease or amyloid diseases, as described herein.
  • Effective amounts of a P400-derived polypeptide or composition, which may also include a P400- fragment derived antibody, are about O.Ol ⁇ g to about lOOmg/kg body weight, and preferably from about 10 ⁇ g to about 50 mg/kg body weight, such as 0.05, 0.07, 0.09, 0.1, 0.5, 0.7, 0.9. , 1, 2, 5, 10, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/kg.
  • Preparations for parenteral administration include ste ⁇ le aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain axillary agents or excipients which are known in the art
  • Pharmaceutical compositions comp ⁇ sing at least one P400 denved polypeptide, such as 1-10 or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 P400-denved polypeptides, of the present invention may include all compositions wherein the P400- de ⁇ ved polypeptide is contained in an amount effective to achieve its intended pu ⁇ ose
  • a pharmaceutical composition may contain suitable pha naceutically acceptable earners, such as excipients, earners and/or axillanes which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • compositions comp ⁇ sing at least one P400-de ⁇ ved polypeptide or antibody may also include suitable solutions for administration intravenously, subcutaneously, dermally, orallv , mucosally, rectally or may by injection or orally, and contain from about 0.01 to 99 percent, preferably about 20 to 75 percent of active component (i.e. polypeptide or antibody) together with the excipient.
  • suitable solutions for administration include pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, and syrups.
  • the P400, P400 fragments and or P400-denved peptides for Alzheimer's disease and other central nervous system amyloidoses may be optimized to cross the blood-brain banner.
  • Methods of introductions include but are not limited to systemic administration, parenteral administration i.e., via an intrape ⁇ toneal, intravenous, penoral. subcutaneous, intramuscular, mtraartal, intradermal, intramuscular, mtranasal, epidural and oral routes.
  • P400, P400 fragments and/or P400-denved peptides may be directly administered to the cerebrospinal fluid by intravent ⁇ cular injection.
  • P400, P400 fragments and/or P400-denved peptides may be delivered in a controlled release system, such as an osmotic pump.
  • a controlled release system can be placed in proximity to the therapeutic target, ie. the brain, thus requmng only a fraction of the systemic dose.
  • peptidomimetic compounds modelled from P400, P400 fragments and/or P400-denved peptides identified as binding A ⁇ or other amyloid proteins may serve as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses Peptidomimetic modelling is implemented by standard procedures known to those skilled in the art
  • compounds that mimic the 3 dimensional A ⁇ binding site on P400 using computer modelling may serve as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses. Design and production of such compounds using computer modeling technologies is implemented by standard procedures known to those skilled in the art
  • Recombinant DNA technology has direct applicability to the P400 and their fragments, of this invention
  • One skilled in the art can take the peptide sequences and create co ⁇ esponding nucleotide sequences that code for the co ⁇ esponding peptide sequences. These sequences can be cloned into vectors such as retroviral vectors, and the like. These vectors can, in turn, be transfected into human cells such as hepatocytes or fibroblasts, and the like. Such transfected cells can be introduced into humans to treat amyloid diseases. Alternatively, the genes can be introduced into the patients directly.
  • Another aspect of the invention is to provide polyclonal and or monoclonal antibodies against P400, P400 fragments and/or P400-denved peptides which bind A ⁇ or other amyloid proteins, which would be utilized to specifically detect P400.
  • P400 fragments and/or P400-denved peptides in human tissues and/or biological fluids are preferred.
  • polyclonal or monoclonal antibodies made against a peptide portion or fragment of P400. can be used to detect and quantify P400, P400 fragments and/or P400-denved peptides in human tissues and/or biological fluids.
  • Polyclonal and/or monoclonal peptide antibodies can also be utilized to specifically detect P400 fragments and/or P400-denved polypeptides in human tissues and/or biological fluids.
  • a polyclonal or monoclonal antibody made specifically against a peptide portion of P400 which binds A ⁇ can be used to detect and quantify this P400 fragment in human tissues and/or biological fluids.
  • a polyclonal or monoclonal antibody made specifically against a peptide portion or fragment of P400 which is present in human biological fluids and binds A ⁇ can be used to detect and quantify this P400 fragment in human tissues and/or biological fluids
  • polyclonal and/or monoclonal antibodies can be utilized using standard lmmunohistochemical and lmmunocytochemical techniques, known to one skilled in the art.
  • vanous types of ELISA assays can be utilized, known to one skilled in the art.
  • An antibody molecule of the present invention may be adapted for utilization in an immunometnc assay, also known as a "two-site” or “sandwich” assay.
  • a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or earner, and a quantity of detectable labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid phase antibody, antigen, and labeled antibody
  • a "sandwich" type of ELISA can be used.
  • a pilot study is first implemented to determine the quantity of binding of each P400-fragment monoclonal antibody to microtiter wells. Once this is determined, aliquots (usually m 40 ⁇ l of TBS; pH 7.4) of the specific P400-fragment antibody are allowed to bind overnight to microtiter wells (Maxisorb C plate from Nunc) at 4oC. A series of blank wells not containing any P400-fragment specific monoclonal antibody are also utilized as controls. The next day, non-bound monoclonal antibody is shaken off the microtiter wells.
  • All of the microtiter wells are then blocked by incubating for 2 hours with 300 ⁇ l of Tns-buffered saline containing 0.05% Tween-20 (TTBS) plus 2% bovine serum albumin, followed by 5 ⁇ nses with TTBS 200 ⁇ l of cerebrospinal fluid, blood, plasma, serum, unne, sputum, and/or stool and or any other type of biological sample is then diluted (to be determined empincally) in TTBS containing 2% bovine serum albumin and placed in wells (in t ⁇ phcate) containing bound P400 fragment antibody (or blank) and incubated for 2 hours at room temperature The wells are then washed 5 times with TTBS.
  • TBS Tween-20
  • a second biotinylated-monoclonal antibody against the same P400-denved fragment (but which is against a different epitope) is then added to each well (usually in 40 ⁇ l of TBS; pH 7.4) and allowed to bind for 2 hours at room temperature to any P400-fragment captured by the first antibody.
  • the wells are washed 5 times with TTBS.
  • Bound mate ⁇ als are then detected by incubating with 100 ⁇ l of peroxidase-avidm complex (1.250 dilution in TTBS with 0.1% BSA) for 1 hour on a rotary shaker. After 5 washes with TTBS, a substrate solution (100 ⁇ l, OPD-Sigma Fast from Sigma Chemical Co., St.
  • a competition assay may also be employed wherein antibodies specific to P400, P400 fragments and/or P400-denved peptides are attached to a solid support and labeled P400, P400 fragments and/or P400-de ⁇ ved peptides and a sample de ⁇ ved from a host are passed over the solid support and the amount of label detected attached to the solid support can be co ⁇ elated to the quantity of P400, P400 fragments and/or P400-denved peptides in the sample
  • This standard technique is known to one skilled in the art
  • Another object of the present invention is to use P400, P400 fragments and/or P400-denved peptides, in conjunction with P400, P400 fragment and/or P400 denved peptide antibodies, in an ELISA assay to detect potential P400, P400 fragment and/or P400-denved peptide autoantibodies in human biological fluids.
  • Such a diagnostic assay may be produced in a kit form
  • peptides containing the sequences of P400, P400 fragments and/or P400-denved peptides, as well as polypeptides which have at least 70% similanty (preferably 70 % identity) and more preferably a 90% similanty (more preferably a 90% identity) to the polypeptides descnbed above will be used to initially bind to microtiter wells in an ELISA plate
  • a pilot study is first implemented to determine the quantity of binding of each P400 fragment polypeptide to microtiter wells Once this is determined, aliquots (usually l-2 ⁇ g in 40 ⁇ l of TBS, pH 7 4) of specific P400 fragment polypeptides (as descnbed herein) are allowed to bind overnight to microtiter wells (Maxisorb C plate from Nunc) at 4°C All the microtiter wells (including blank wells without the P400 fragment polypeptid
  • TTBS 100 ⁇ l of biotinylated polyclonal goat anti-human IgGs (Sigma Chemical company, St. Louis, MO, USA), diluted 1:500 in TTBS with 0.1% bovine serum albumin, is then ahquoted into each well. Bound matenals are detected by incubating with 100 ⁇ l of peroxidase-avidin complex (1.250 dilution in TTBS with 0.1% bovine serum albumin) for 1 hour on a rotary shaker Following 5 washes with TTBS, substrate solution (100 ⁇ l, OPD Sigma Fast from Sigma Chemical Company, St. Louis, MO, USA) is added and allowed to develop significant color (usually 8-10 minutes).
  • the reaction is stopped with 50 ⁇ l of 4N sulfuric acid added to each well and read on a standard spectrophotometer at 490 nm.
  • This assay system can be utilized to not only detect the presence of autoantibodies against P400 fragments in biological fluids, but also to monitor the progression of disease by following elevation or diminution of P400 fragment autoantibody levels. It is believed that patients demonstrating excessive P400 fragment formation, deposition, accumulation and/or persistence as may be observed in the amyloid diseases will also ca ⁇ y autoantibodies against the P400 fragments in their biological fluids.
  • ELISA assay systems knowledgeable to those skilled in the art, can be used to accurately monitor the degree of P400 fragments in biological fluids as a potential diagnostic indicator and prognostic marker for patients during the progression of disease (i.e. monitoring of an amyloid disease for example).
  • Such assays can be provided in a kit form.
  • quantitative changes in P400 fragment autoantibody levels can also serve as a prognostic indicator monitoring how a live patient will respond to treatment which targets a given amyloid disease.
  • diagnostic methods utilizing the invention include diagnostic assays for measuring altered levels of P400, P400 fragments and/or P400-derived peptides in various tissues compared to normal control tissue samples.
  • Assays used to detect levels of P400, P400 fragments and/or P400-derived peptides in a sample derived from a host are well-known to those skilled in the art and included radioimmunoassays, competitive-binding assays, Western blot analysis and preferably ELISA assays (as described above).
  • Yet another aspect of the present invention is to use the antibodies recognizing P400, P400 fragments and/or P400-derived peptides for labellings, for example, with a radionucleotide, for radioimaging or radioguided surgery, for in vivo diagnosis, and/or for in vitro diagnosis.
  • radiolabelled peptides or antibodies made (by one skilled in the art) against P400, P400 fragments and/or P400-derived peptides may be used as minimally invasive techniques to locate P400, P400 fragments and/or P400-derived peptides, and concu ⁇ ent amyloid deposits in a living patient. These same imaging techniques could then be used at regular intervals (i.e. every 6 months) to monitor the progression of the amyloid disease by following the specific levels of P400, P400 fragments and/or P400- derived peptides.
  • Yet another aspect of the present invention is to provide a method which can evaluate a compound's ability to alter (diminish or eliminate) the affinity of a given amyloid protein (as described herein) or amyloid precursor protein, to P400, P400 fragments and/or P400-derived peptides.
  • a given amyloid protein as described herein
  • amyloid precursor protein as described herein
  • the present invention is also useful in identifying compounds which can prevent or impair such binding interaction.
  • compounds can be identified which specifically affect an event linked with the amyloid formation, amyloid deposition, and/or amyloid persistence condition associated with Alzheimer's disease and other amyloid diseases as described herein.
  • amyloid or P400 fragments are immobilized, and the other of the two is maintained as a free entity.
  • the free entity is contacted with the immobilized entity in the presence of a test compound for a period of time sufficient to allow binding of the free entity to the immobilized entity, after which the unbound free entity is removed.
  • the amount of free entity bound to immobilized entity can be measured.
  • the effectiveness of the test compound to allow binding of free entity to immobilized entity can be determined and a quantitative determination of the effect of the test compound on the affinity of free entity to immobilized entity can be made.
  • the ability of the test compound to modulate the binding can be determined.
  • the amyloid is immobilized, it is contacted with free P400 derived fragments or polypeptides, in the presence of a series of concentrations of test compound.
  • immobilized amyloid is contacted with free P400-derived polypeptides, or fragments thereof in the absence of the test compound.
  • the dissociation constant (K d ) or other indicators of binding affinity of amyloid-P400 fragment binding can be determined.
  • the unbound P400 polypeptides are removed.
  • P400-derived fragment antibodies as described in the invention, to detect the amount of specific P400 fragments bound to the amyloid or the amount of free P400 fragments remaining in solution. This information is used to determine first qualitatively whether or not the test compound can allow continued binding between P400- derived fragments and amyloid.
  • the data collected from assays performed using a series of test compounds at various concentrations can be used to measure quantitatively the binding affinity of the P400 fragment-amyloid complex and thereby determine the effect of the test compound on the affinity between P400 fragments and amyloid.
  • compounds can be identified which do not modulate the binding of specific P400 fragments to amyloid and thereby allow the P400 fragments to reduce the amyloid formation, deposition, accumulation and/or persistence, and the subsequent development and persistence of amyloidosis.
  • kit for practicing a method for identifying compounds useful which do not alter P400, P400 fragments and/or P400-derived peptides to an immobilized amyloid protein comprising a) a first container having amyloid protein immobilized upon the inner surface, b) a second container which contains P400, P400 fragments and/or P400-derived peptides dissolved in solution, c) a third container which contains antibodies specific for said P400, P400 fragments and/or P400-derived peptides, said antibodies dissolved in solution, and d) a fourth container which contains labeled antibodies specific for P400, P400 fragments and/or P400-derived peptides, said antibodies dissolved in solution.

Abstract

Pharmaceutical composition and methods of use of an amyloid-beta binding protein from serum and cerebral spinal fluid of Alzheimer's disease and normal aged patients with an estimated size of 350-400 kilodaltons, designated P400. P400 is decreased in serum derived from Alzheimer's disease patients in comparison to normal aged controls. P400 is more abundant than apolipoproteins and laminin in biological fluids and binds Amyloid-beta with a higher affinity.

Description

Therapeutic and Diagnostic Applications of P400: A Newly Discovered Beta-Amyloid Binding Protein Present in Human Biological Fluids
TECHNICAL FLIELD
The invention relates to the discover) , identification and use of P400 (a new ly discovered Aβ- binding protein present in human blood and cerebrospinal fluid), P400-deπved protein fragments, and P400-deπved polypeptides. as well as related peptides and antibodies, for the therapeutic intervention and diagnosis of Alzheimer's disease and other amyloidoses
BACKGROUND OF THE INVENTION
Alzheimer's disease is characterized by the accumulation of a 39-43 amino acid peptide termed the beta-amyloid protein or Aβ in a fibπllar form, existing as extracellular amyloid plaques and as ai loid within the walls of cerebral blood vessels Fibπllar Aβ amv loid deposition m Alzheimer's disease is believed to be detπmental to the patient and eventually leads to toxicity and neuronal cell death, characteristic hallmarks of Alzheimer's disease. Accumulating evidence implicates amyloid as a major causative factor of Alzheimer's disease pathogenesis
A vaπety of other human diseases also demonstrate amyloid deposition and usually involve systemic organs (i.e. organs or tissues lying outside the central nervous system), with the amyloid accumulation leading to organ dysfunction or failure. In Alzheimer's disease and "systemic" amyloid diseases, there is currently no cure or effective treatment, and the patient usually dies within 3 to 10 years from disease onset
New compounds or agents for therapeutic regimes to arrest or reverse amyloid formation, deposition, accumulation and/or persistence that occurs in Alzheimer's disease and other amyloidoses are therefore desperately sought.
Recent studies indicate that the Aβ of Alzheimer's disease is maintained in biological fluids in a soluble state by an unknown mechanism Implicated proteins which may contπbute to such Aβ solubility m biological fluids include apolipoproteins, albumin and laminin. In the present invention, we have encountered a new Aβ-binding protein in serum and cerebrospinal fluid (CSF) of Alzheimer's disease and normal aged patients (designated as P400). Our data indicates that P400 in human serum and CSF binds tightly to Aβ and is decreased substantially in serum deπved from Alzheimer's disease patients in compaπson to normal aged controls. These studies suggest the identification of a new Aβ- binding protein w hich may be responsible for the maintenance of Aβ solubility in biological fluids. Identification of a new protein which may be a specific indicator for Alzheimer's disease and/or its progression will allow for both novel therapeutic and diagnostic approaches in the future.
SUMMARY OF THE INVENTION
It is known that the Aβ of Alzheimer's disease is maintained in biological fluids (i.e. blood and cerebrospinal fluid) in a soluble state by an unknown mechanism. Implicated proteins which may contribute to such maintenance of Aβ solubility in biological fluids include apolipoproteins (apolipoproteins J and E)( Ghiso et al, Biochem. J. 293:27-30, 1993; Wisniewski et al, Biochem. Res. Commun 192:359-365, 1993; Stπttmatter et al, Proc. Natl. Acad. Sci. U. S. A. 90:8098-8102, 1993; LaDu et al. J. Biol. Chem. 269:23403-23406. 1994: Naslund et al. Neuron 15:219-228, 1995; Chan et al, Biochemistry 35:7123-7130, 1996), albumin (Biere et al, J. Biol Chem. 271 : 32916-32922. 1996), and laminin (and/or laminin fragments)(Castιllo et al, Soc. Neurosci Abst. 23: 1882, 1997). During the course of our studies involving analysis of human biological fluids, we have encountered a new Aβ- binding protein in serum and CSF of Alzheimer's disease and normal aged patients, with a size. >205 kilodaltons (and designated P400 due to an approximate estimated size of -350-450 kilodaltons), which does not appear to be related to any of the previously descπbed proteins above. Our data indicates that P400 binds tightly to Aβ (I e. at subnanomolar concentrations) and is decreased in serum deπved from Alzheimer's disease patients in compaπson to normal aged controls. In addition, our data suggests that P400 is more abundant than other AB-binding proteins present in biological fluids including apolipoproteins, and laminin (and/or laminin fragments), and appears to bind Aβ with a higher affinity. These studies indicate that we have identified a new Aβ-b dmg protein in biological fluids, which may have diagnostic and therapeutic potential.
FEATURES OFTHE INVENTION
A pπmary object of the present invention is to establish new therapeutic methods and diagnostic applications for the amyloid diseases. The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease and Down's syndrome (wherein the specific amyloid is referred to as beta-arm loid protein or Aβ), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (wherein the specific ai loid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is refeπed to as AL amyloid), the amyloid associated w ith t\pe II diabetes (wherein the specific amyloid is referred to as amyhn or islet amyloid), the amyloid associated with the pnon diseases including Creutzfeldt- Jakob disease, Gerstmann Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is referred to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-mιcroglobuhn amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is referred to as transthyretin or prealbumin), and the amyloid associated with endocπne tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as variants of procalcitomn)
As disclosed herein. "P400" refers to a newly discovered Aβ-binding protein present in human serum and CSF, and which has a molecular weight of approximately 350 to 450 kilodaltons as determined by standard SDS-PAGE (as descnbed herein)
A pπmary object of the present invention is to use P400, P400 protein fragments and/or P400- deπved polypeptides as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses
Yet another object of the present invention is to use conformational dependent proteins. polypeptides, or fragments thereof for the treatment of Alzheimer's disease and other amyloidoses. Such conformational dependent proteins include, but are not limited to, P400. P400-deπved fragments and/or portions thereof
Yet another aspect of the present invention is to use peptidomimetic compounds modeled from P400, P400-deπved protein fragments and/or P400-deπved polypeptides, and fragments thereof, as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses
Yet another object of the present invention is to mimic the 3-dιmensιonal Aβ-binding sιte(s) on P400, P400-deπved protein fragments and/or P400-deπved polypeptides and use these mimics as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses Yet a further aspect of the present invention is to use anti-idiotypic antibodies to P400, P400- deπved protein fragments and/or P400-deπved polypeptides as potent inhibitors of amyloid formation, deposition accumulation and/or persistence in Alzheimer's disease and other amyloidoses
Another aspect of the invention is to provide new and novel polyclonal and/or monoclonal peptide antibodies which can be utilized in a number of in vitro assays to specιfιcall> detect Aβ- binding P400-deπved protein fragments and/or Aβ-binding P400 derived polypeptides in human tissues and/or biological fluids Polyclonal or monoclonal antibodies that are made specifically against a peptide portion or fragment of P400 hich interacts with Aβ can be utilized to detect and quantify amyloid disease specific P400 fragments in human tissues and/or biological fluids These antibodies can be made b\ administeπng the peptides in antigemc form to a suitable host Polyclonal or monoclonal antibodies may be prepared by standard techniques known to those skilled in the art
Another object of the present invention is to use P400. the Aβ-binding P400 fragments and/or P400-deπved polypeptides referred to above, for the detection and specific localization of P400 peptides important in the amyloid diseases in human tissues, cells, and/or cell culture using standard lmmunohistochemical techniques
Yet another aspect of the present invention is to use antibodies recognizing P400, any of the AB-binding P400 fragments, and/or P400-deπved polypeptides, and fragments thereof, for m vivo labeling, for example with a radionucleotide. for radioimag g to be utilized for in vivo diagnosis and/or for in \ itro diagnosis
Yet another aspect of the present invention is to make use of P400, P400-deπved protein fragments, and P400-deπved polypeptides. and fragments thereof, as potential therapeutics to inhibit the deposition, formation, and accumulation of fibπllar amyloid in Alzheimer's disease and other amyloidoses (descπbed above), and to enhance the clearance and/or removal of preformed amyloid deposits in brain (for Alzheimer's disease and Down's syndrome amyloidosis) and in systemic organs (for systemic amyloidoses)
Another object of the present invention is to use Aβ-bmding P400-deπved polypeptides or fragments thereof, in conjunction with polyclonal and/or monoclonal antibodies generated against these peptide fragments, using in vitro assays to detect amyloid disease specific autoantibodies in human biological fluids Specific assay systems can be utilized to not only detect the presence of autoantibodies against Aβ-bindmg P400-deπved protein fragments or polypeptides thereof in biological fluids, but also to monitor the progression of disease by following elevation or diminution of P400 protein fragments and/or P400-deπved polypeptide autoantibody levels Another aspect of the invention is to utilize P400. P400-deπved protein fragments, and P400- deπved antibodies and/or molecular biology probes for the detection of these P400 deπvatives in human tissues in the amyloid diseases
Yet another object of the present invention is to use the P400-deπved protein fragments of the present invention in each of the various therapeutic and diagnostic applications descπbed above Specific P400-deπved protein fragments or peptides as descπbed above may be deπved from any species including, but are not limited to, human, muπne, bovine, porcine, and/or equine species
Another object of the invention is to provide polyclonal and/or monoclonal peptide antibodies which can be utilized in a number of in vitro assays to specifically detect P400 protein fragments in human tissues and/or biological fluids Polyclonal or monoclonal antibodies made specifically against a peptide portion or fragment of any of the P400 fragments descπbed herein can be utilized to detect and quantify P400-deπved protein fragments in human tissues and/or biological fluids A prefeπed embodiment is a polyclonal antibody made to P400 present in human serum and cerebrospinal fluid These antibodies can be made by isolating and administeπng P400, P400 fragments and/or P400 polypeptides in antigemc form to a suitable host. Polyclonal or monoclonal antibodies may be prepared by standard techniques by one skilled in the art
Yet another object of the present invention is to use P400-deπved antibodies as descπbed herein as a specific indicator for the presence and extent of P400 breakdown in brain by monitoring biological fluids including, but not limited to. cerebrospinal fluid, blood, serum, uπne, saliva, sputum, and stool
Yet another object of the present invention is to use P400-deπved antibodies as descπbed herein as a specific indicator for the presence, extent and/or progression of Alzheimer's disease and/or other brain amyloidoses by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, uπne, saliva, sputum, and stool
Yet another object of the present invention is to use P400-deπved antibodies as descπbed herein as a specific indicator for the presence and extent of P400 breakdown in systemic organs by momtoπng biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, uπne, saliva, sputum, and stool
Yet another object of the present invention is to use P400-deπved antibodies as descπbed herein as a specific indicator for the presence and extent of amyloidosis m type II diabetes by monitoπng biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, uπne, saliva, sputum, and stool Yet another object of the present invention is to use P400-deπved antibodies as descπbed herein as a specific indicator for the presence and extent of amyloidosis in other systemic amyloidoses by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool
Yet another object of the invention is to utilize specific P400-deπved antibodies, as described herein, for the detection of P400 in human tissues in the amyloid diseases
Another object of the present invention is to use P400, P400-deπved protein fragments, and P400-deπved polypeptides. as descnbed herein, for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses
Another object of the present invention is to use pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, e xers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositones, steπle injectable solutions, and steπle packaged powders, which contain P400, P400-deπved protein fragments, and P400-deπved polypeptides, and fragments thereof, to treat patients with Alzheimer's disease and other amyloidoses
Yet another object of the present invention is to use P400, P400-deπved protein fragments, and P400-deπved polypeptides, and fragments thereof, as potent agents which inhibit amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, and/or cause a dissolution of preformed or pre deposited amyloid fibπls in Alzheimer's disease, and other amyloidoses
Yet another object of the present invention is to provide the use of P400, P400-deπved protein fragments, and P400-deπved polypeptides, as described herein, for inhibition of amyloid formation, deposition, accumulation, and/or persistence, regardless of its clinical setting
Yet another object of the present invention is to provide compositions and methods involving administeπng to a subject a therapeutic dose of P400, P400-deπved protein fragments, and P400- deπved polypeptides, which inhibit amyloid deposition, and fragments thereof. Accordingly, the compositions and methods of the invention are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs The proteins or polypeptides of the invention can be used therapeutically to treat amyloidosis or can be used prophylactically in a subject susceptible to amyloidosis. The methods of the invention are based, at least in part, in directly inhibiting amyloid fibπl formation, and/or causing dissolution of preformed amyloid fibrils BRIEF DESCRIPTION OF THE DRAWINGS
The following dra ings are illustrative of the invention and are not meant to limit the scope of the invention
FIGURE 1 is a black and white photograph of a gand blot analysis of serum separated by non- reducing SDS-PAGE. transfeπed to PVDF membrane, and probed with 5nM of bιotιnylated-Aβ (1-40) Laminin and P400 are demonstrated to be major Aβ-binding proteins in human serum Low concentrations of Aβ (1-40) detected only those proteins that have dissociation constants in the nanomolar range A prominent and more focused -130 kDa Aβ-bmding protein (lower arrow) is believed to represent the E8 fragment of laminin (Begovac et al. J Cell Biol 113 637-644, 1991 ,Nurchenco and Cheng, J Biol Chem 268 17286-17299. 1993), whereas a prominent >205 kDa Aβ-bindmg protein (upper arrow) represents P400 which w as confirmed not to be lmmunologically related to laminin (see Fig 2) Lanes 1 and 4 (normal), lanes 3 and 6 (Alzheimer's disease), lanes 2,5, 7-8 (type II diabetes) Note the reduction of P400 in serum of Alzheimer's disease patients
FIGURE 2 is a black and white photograph of a Western blot demonstrating that P400 is lmmunologically distinct from laminin This is a Western blot of human CSF (30 μl/lane, Lanes 1-6) and serum (10 μl/lane. Lanes 7- 14) probed with a laminin antibody See text for individual lane details Intact laminin (Mr -850 kDa) (upper aπow ) was present in human CSF (lanes 1 -6) but not as prominent in human serum (lanes 7- 14) No lmmunostainmg was observed in the region >205 kDa up to the gel interface, (compare to Fig 1) indicating that P400 is lmmunologically distinct from laminin
FIGURE 3 is a black and white photograph demonstrating isolation of P400 using Sephacryl S1000 gel filtration chromatography This is an Aβ-hgand blot of vaπous fractions deπved from Sephacryl S 1000 gel filtration chromatography, where a concentrated sample from 10 ml CSF was applied The blot was probed with 50 nM of biotinylated Aβ (1-40) This demonstrates that the Aβ- bindmg P400 (upper aπow) can be ennched relative to the smaller laminin fragments (lower arrow) This blot shows that P400 is present in human CSF and can be puπfied from other Aβ-binding proteins Coomassie blue staining of similar blots indicate that very little low molecular weight proteins are present in fractions with Ka, of 0 5 (not shown), indicating that this method is useful in initial puπfication of P400
BEST MODE OF CARRYING OUT THE INVENTION
Turning now to the drawings, the invention will be descπbed in a preferred embodiment by reference to the numerals of the drawing figures wherein like numbers indicate like parts Amyloid and Amyloidosis
Amyloid is a geneπc term referπng to a group of diverse, but specific extracellular protein deposits which all have common morphological properties, staining characteπstics, and x-ray diffraction spectra. Regardless of the nature of the amyloid protein deposited all amyloids have the following characteπstics 1) an amorphous appearance at the light microscopic level and appear eosinophilic using hematoxvlin and eosm stains: 2) all stain with Congo red and demonstrate a red/green birefπngence as viewed under polaπzed light (Puchtler et al., J Histochem Cytochem 10:355-364, 1962), 3) all contain a predominant beta-pleated sheet secondary structure, and 4) ultrastructurally amyloid usually consist of non-branching fibπls of indefinite length and with a diameter of 7-10 nm
Amyloid today is classified according to the specific amyloid protein deposited. The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease, Down's syndrome and Hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is referred to as beta-amyloid protein or Aβ), the amyloid associated with chronic inflammation, vaπous forms of malignancy and Familial Mediteπanean Fever (wherein the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is refeπed to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is refeπed to as amylin or islet amyloid), the amyloid associated with the pπon diseases including Creutzf el dt- Jakob disease. Gerstmann- Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is refeπed to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-mιcroglobulιn amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is refeπed to as prealbumin or transthyretin amyloid), and the amyloid associated with endocπne tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as vaπants of procalcitomn).
Although amyloid deposits in clinical conditions share common physical properties relating to the presence of a beta-pleated sheet conformation, it is now clear that many different chemical types exist and additional ones are likely to be descπbed in the future. It is currently thought that there are several common pathogenetic mechanisms that may be operating in amyloidosis in general. In many cases, a circulating precursor protein may result from overproduction of either intact or aberrant molecules (ex. plasma cell dyscrasias), reduced degradation or excretion (serum amyloid A in some secondary amyloid syndromes and beta2-rrucroglobuhn in long-term hemodialysis), or genetic abnormalities associated with variant proteins (ex. familial amyloidotic polyneuropathy) Proteolysis of a larger protein precursor molecule occurs in many types of amyloidosis. resulting in the production of lower molecular weight fragments that polymeπze and assume a beta-pleated sheet conformation as tissue deposits, usually in an extracellular location. What are the precise mechanisms involved, and the aberrant causes leading to changes in proteolytic processing and/or translational modifications is not known in most amyloids
Systemic amyloids which include the amyloid associated with chronic inflammation, vaπous forms of malignancy and Familial Mediteπanean Fever (le AA amyloid or inflammation-associated amyloιdosιs)(Benson and Cohen, Arth Rheum. 22.36-42, 1979, Ka ei et al, Acta Path Jpn 32: 123-133, 1982; McAdam et al, Lancet 2 572-573, 1975; Metaxas, Kidnev Int. 20:676-685, 1981), and the amyloid associated with multiple myeloma and other B-cell dyscrasias (le AL amyloιd)(Harada et al, J_ Histochem Cytochem 19 1 15, 1971), as examples, are known to involve amyloid deposition in a vaπety of different organs and tissues generally lying outside the central nervous system Amyloid deposition in these diseases may occur, for example, in liver, heart, spleen, gastrointestinal tract, kidney, skin, and/or lungs (Johnson et al, N. Engl J Med 321:513-518, 1989). For most of these amyloidoses. there is no apparent cure or effective treatment and the consequences of amyloid deposition can be detπmental to the patient. For example, amyloid deposition in kidney may lead to renal failure, whereas amyloid deposition in heart may lead to heart failure. For these patients, amyloid accumulation in systemic organs leads to eventual death generally w ithin 3-5 years Other amyloidoses may affect a single organ or tissue such as observed with the Aβ amyloid deposits found in the brains of patients with Alzheimer's disease and Down's syndrome: the PrP amyloid deposits found in the brains of patients with Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, and kuru; the islet amyloid (amylin) deposits found in the islets of Langerhans in the pancreas of 907c of patients with type II diabetes (Johnson et al. N Engl. J Med 321 :513-518. 1989; Lab Invest. 66 522 535, 1992); the beta2-mιcroglobuhn amyloid deposits in the medial nerve leading to carpal tunnel syndrome as observed in patients undergoing long-term hemodialysis (Geyjo et al, Biochem. Biophys. Res. Comm. 129:701-706, 1985; Kidnev Int. 30:385-390, 1986); the prealbumin/ transthyretin amyloid observed in the hearts of patients with senile cardiac amyloid; and the prealbumin/ transthyretin amyloid observed in peπpheral nerves of patients who have Familial Amyloidotic Polyneuropathy (Skinner and Cohen, Biochem. Biophvs. Res Comm. 99.1326- 1332, 1981 , Saraiva et al, J Lab Chn Med 102:590-603, 1983, J. Clin. Invest. 74: 104-1 19, 1984, Tawara et al. J. Lab. Clin Med. 98:811-822. 1989) Alzheimer's Disease and the Aging Population
Alzheimer's disease is a leading cause of dementia in the elderly, affecting 5-109-- of the population over the age of 65 years (A Guide to Understanding Alzheimer's Disease and Related Disorders, edited by Jorm. New York University Press, New York, 1987). In Alzheimer's disease, the parts of the brain essential for cognitive processes such as memory, attention, language, and reasoning degenerate, robbing victims of much that makes us human, including independence In some inheπted forms of Alzheimer's disease, onset is in middle age, but more commonly, symptoms appear from the mιd-60's onward Alzheimer's disease today affects 4-5 million Ameπcans, with slightly more than half of these people receiving care at home, while the others are in many different health care institutions The prevalence of Alzheimer's disease and other dementias doubles every 5 years beyond the age of 65, and recent studies indicate that nearly 509c of all people age 85 and older have symptoms of Alzheimer's disease (1997 Progress Report on Alzheimer's Disease, National Institute on Aging/National Institute of Health). 13% (33 million people) of the total population of the United States are age 65 and older, and this % will climb to 20% by the year 2025 (1997 Progress Report on Alzheimer's Disease, National Institute on Aging/National Institute of Health)
Alzheimer's disease also puts a heavy economic burden on society as well. A recent study estimated that the cost of caπng for one Alzheimer's disease patient with severe cognitive impairments at home or in a nursing home, is more than $47,000 per year (A Guide to Understanding Alzheimer's Disease and Related Disorders, edited by Jorm, New York University Press, New York, 1987) For a disease that can span from 2 to 20 years, the overall cost of Alzheimer's disease to families and to society is staggeπng The annual economic toll of Alzheimer's disease in the United States in terms of health care expenses and lost wages of both patients and their caregivers is estimated at $80 to $100 billion (1997 Progress Report on Alzheimer's Disease, National Institute on Aging/National Institute of Health)
Tacπne hydrochloπde ("Cognex"), the first FDA approved drug for Alzheimer's disease is a acetylcholinesterase inhibitor (Cutler and Sramek, N. Engl. J. Med. 328:808 810, 1993). However, this drug has showed limited success in the cognitive improvement m Alzheimer's disease patients and initially had major side effects such as liver toxicity. The second more recently FDA approved drug, donepezil (also known as "Aπcept"), which is also an acetylcholinesterase inhibitor, is more effective than tacπne, by demonstrating slight cognitive improvement in Alzheimer's disease patients (Barner and Gray, Ann. Pharmacotherapy 32:70-77, 1998; Rogers and Fπedhoff, Eur. Neuropsvch. 8:67-75, 1998), but is not believed to be a cure. Therefore, it is clear that there is a need for more effective treatments for Alzheimer's disease patients. Amyloid as a Therapeutic Target for Alzheimer's Disease
Alzheimer's disease is characteπzed by the deposition and accumulation of a 39-43 amino acid peptide termed the beta-am loid protein, Aβ or β/A4 (Glenner and Wong, Biochem. Biophys Res Comm 120:885-890, 1984; Masters et al, Proc Natl Acad. Sci. U.S.A. 82:4245-4249, 1985; Husby et al, Bull WHO 71 :105-108, 1993 j Aβ is deπved from larger precursor proteins termed beta-amyloid precursor proteins (or BPPs) of which there are several alternatively spliced vaπants. The most abundant forms of the βPPs include proteins consisting of 695, 751 and 770 amino acids (Tanzi et al, Nature 331:528-530, 1988; Kitaguchi et al, Nature 331.530-532, 1988; Ponte et al, Nature 331:525-527, 1988) The small Aβ peptide is a major component which makes up the amyloid deposits of "plaques" in the brains of patients with Alzheimer's disease. In addition. Alzheimer's disease is characteπzed by the presence of numerous neurofibπllary "tangles", consisting of paired helical filaments which abnormally accumulate in the neuronal cytoplasm (Grundke-Iqbal et al, Proc. Natl. Acad Sci U S.A. 83:4913-4917, 1986; Kosik et al, Proc. Natl. Acad Sci U S.A 83:4044-4048, 1986; Lee et al, Science 251:675-678, 1991). The pathological hallmarks of Alzheimer's disease is therefore the presence of "plaques" and "tangles", with amyloid being deposited in the central core of plaques. The other major type of lesion found in the Alzheimer's disease brain is the accumulation of amyloid in the walls of blood vessels, both within the brain parenchyma and in the walls of meningeal vessels which lie outside the brain. The amyloid deposits localized to the walls of blood vessels are refeπed to as cerebrovascular amyloid or congophihc angιopath\ (Mandybur, J Neuropath Exp Neurol 45.79-90, 1986, Pardπdge et al, J_ Neurochem 49' 1394-1401. 1987)
For many years there has been an ongoing scientific debate as to the importance of "amyloid" in Alzheimer's disease and whether the "plaques" and "tangles" characteπstic of this disease, were a cause or merely the consequences of the disease Within the last few years, studies now indicate that amyloid ts indeed a causative factor for Alzheimer's disease and should not be regarded as merely an innocent bystander. The Alzheimer's Aβ protein in cell culture has been shown to cause degeneration of nerve cells within short peπods of time (Pike et al, Br. Res. 563:311-314, 1991: J. Neurochem 64:253-265, 1995) Studies suggest that it is the fibπllar structure (consisting of a predominant β-pleated sheet secondary structure), characteπstic of all amyloids, that is responsible for the neurotoxic effects. Aβ has also been found to be neurotoxic in slice cultures of hippocampus (Harπgan et al, Neurobiol. Aging 16:779-789, 1995) and induces nerve cell death in transgenic mice (Games et al, Nature 373:523-527. 1995; Hsiao et al, Science 274:99-102, 1996). Injection of the Alzheimer's Aβ into rat brain also causes memory impairment and neuronal dysfunction (Flood et al, Proc. Natl. Acad. Sci. U.S.A. 88:3363-3366, 1991; Br Res 663:271-276. 1994). Probably, the most convincing evidence that Aβ amyloid is directly involved in the pathogenesis of Alzheimer's disease comes from genetic studies. It has been discovered that the production of Aβ can result from mutations in the gene encoding, its precursor, beta amyloid precursor protein (Van Broeckhoven et al, Science 248: 1120-1122, 1990; Murrell et al, Science 254:97-99, 1991 , Haass et al, Nature Med 1 : 1291-1296. 1995) The identification of mutations in the beta-amyloid precursor protein gene which causes early onset familial Alzheimer's disease is the strongest argument that amyloid is central to the pathogenetic process underlying this disease Four reported disease-causing mutations have now been discovered which demonstrate the importance of Aβ in causing familial Alzheimer's disease (reviewed in Hardy, Nature Genet. 1:233-234, 1992). All of these studies suggest that providing a drug to reduce, eliminate or prevent fibπllar Aβ formation, deposition, accumulation and/or persistence in the brains of human patients is believed to serve as an effective therapeutic
Soluble Beta-Amyloid Protein in Biological Fluids
A number of different studies have now confirmed that Aβ is normally present in biological fluids including its presence in both cerebrospinal fluid (CSF)(Shojι et al, Science 258: 126-129, 1992; Seubert et al, Nature 359 325-327, 1992, Vigo-Pelfrey et al, J. Neurochem 61 : 1965-1968, 1993) and plasma (Seubert et al, Nature 359.325-327, 1992). Seubert et al (Nature 359:325-327, 1992) found Aβ peptide in both CSF and plasma of humans, dogs, guinea pigs and rats. In humans, the concentration of Aβ in CSF is approximately 2 5 ng/ml whereas in plasma the Aβ concentration is approximately 0.9 ng/ml (Seubert et al, Nature 359.325-327, 1992). Although Aβ in CSF appears to consist of vaπous lengths, 70% of Aβ in CSF consists of Aβ 1-34 and Aβ 1-40 (Vigo-Pelfrev et al. J. Neurochem. 61 : 1965-1968. 1993). Since previous studies have demonstrated that synthetic peptides homologous to Aβ 1-40 and 1-42 spontaneously form amyloid-like fibπls (Kirschner et al, Proc. Natl. Acad. Sci. U.S.A 84:6953-6957, 1987; Baπow et al, J. Mol. Biol. 225.1075-10793, 1992), the question aπses as to why Aβ in biological fluids is maintained in a soluble state? It is cuπently believed that CSF and plasma serum contain additional factors that maintain Aβ in a soluble state preventing its potential fibrillogenesis.
Previous studies (Wisniewski et al, Biochem. Biophys. Res. Commun. 192:359-365, 1993) indicated that some components of CSF are able to inhibit fibnl formation and aggregation of Aβ peptide such that it remains soluble. Apolipoproteins E and J (in blood and CSF with approximate SDS-PAGE mobility of 80 kDa or less) (Ghiso et al, Biochem. J. 293:27-30, 1993; Wisniewski et al, Biochem. Biophvs. Res. Commun 192:359-365, 1993; Stπttmatter et al, Proc. Natl. Acad. Sci. U. S A. 90:8098- 8102, 1993; LaDu et al, J. Biol. Chem. 269:23403-23406, 1994; Naslund et al, Neuron 15:219-228, 1995, Chan et al, Biochemistry 35:7123-7130, 1996), and albumin (in blood) (Biere et al, J. Biol. Chem. 271 32916-32922 1996) are proteins previously implicated in keeping Aβ in a soluble state in biological fluids Previous studies hav e also demonstrated that apoproteins bind Aβ w ithin the μM range (Stπttmatter et al, Proc Natl Acad Sci U S A 90 8098-8102, 1993. LaDu et al, J Biol Chem 269 23403-23406 1994) and their ability to bind Aβ is further attenuated when complexed with lipids (Biere et al, J Biol Chem 271 32916-32922. 1996) In our previous studies (Castillo and Snow, unpublished data) using a solid phase binding assav we determined that albumin binds Aβ with a dissociation constant of 0.05 mM, which is suggestive of rather weak binding However, due to the normally high concentrations of albumin in blood (1 mM), this binding appears significant if one increases the concentration of Aβ, by adding exogenous Aβ to the blood samples as was employed in a previous study (Biere et al. J Biol Chem 271 32916-32922, 1996) In the CSF however, the albumin concentration is only 3 μM, which makes the binding of albumin to Aβ with a Kd of 0 05 mM, insignificant in this not very tight biological fluid compartment Our previous studies have demonstrated that laminin in serum and CSF binds Aβ at low nanomolar concentrations (with a Kd = 2 7 x 10-9 M), suggesting that laminin (and/or laminin fragments) in blood and CSF can also contπbute to Aβ solubility (Castillo et al, Soc Neurosci Abst 23 1882, 1997) However, each of the descπbed proteins above may only be partially responsible for maintenance of Aβ in a soluble state in biological fluids Identification of other proteins which are also present in biological fluids and which bind Aβ very tightly, may also serve to maintain Aβ in a soluble state Data presented in this invention indicates that we have detected a new unidentified protein in serum and CSF which binds Aβ much tighter that any of the previous proteins implicated in biological fluids to maintain Aβ in a soluble state (l e lipoprotems, albumin and laminin)
Examples
The following examples are put forth so as to provide those with ordinary skill m the art with the disclosure and descπption of the discovery of P400 as a new Aβ-binding protein in serum and CSF, and its use as a diagnostic marker for Alzheimer's disease and/or its progression Example 1
Identification of a -350-450 Kilodalton Protein in Human Serum which Binds Aβ and is Reduced in Alzheimer's Disease
AB ligand blotting techniques were utilized to identify proteins present in human serum which bind Aβ In this initial studv , human serum was obtained from normal aged and Alzheimer's disease living patients who mostly hav e had coπesponding mini-mental state examinations (where a score of 30 is normal, a score of 15 suggests moderate dementia, and a score <10 suggests severe dementia), or from patients with confirmed type II diabetes For ligand blot analysis, lμl of human serum was separated b> non-reducing SDS-PAGE, transfeπed to PVDF membrane and probed with 5 nM of biotinylated Aβ 1-40 The probing with low concentrations of Aβ 1-40 will detect only those proteins which had a dissociation constant in the nanomolar range In Fig 1, the following human serums were obtained and analyzed as part of this studv Lane 1 patient #9, a normal 67 yr old female with a mini-mental score of 30, Lane 2 patient B- a 63 year old male with confirmed type II diabetes, Lane 3. patient #5223- a 68 year old female with probable Alzheimer's disease who had a mini-mental score of 22, Lane 4- patient #22- an 83 yr. old normal aged female who had a mini-mental score of 30; Lane 5: patient #E- a 54 year old male with confirmed type II diabetes, Lane 6 patient #5230- a 72 year old female with probable moderate Alzheimer's disease who had a mini-mental score of 19; Lane 7: patient #E-a 54 year old male with confirmed type II diabetes, and Lane 8 patient #F- a 69 year old male with confirmed type II diabetes
A prominent -130 kDa Aβ-binding band (lower arrow) was present in all patients serums and was believed to represent the E8 fragment of laminin (Yurchenco et al, J Biol Chem 268 17286-17299, 1993) A prominent >205 kilodalton band (and designated as P400 due to an estimated approximate size of -350-450 kιlodaltons)(upper aπow) is also present in human serum deπved from normal (lanes 1 and 4) and type II diabetes (lanes 2, 5, 7 and 8) patients, and appears to be markedly decreased in the serum deπved from 2 patients with moderate AD (lanes 3 and 6). All of these serums were similarly obtained from live patients so the decreased P400 in the serum of probable AD patients is not believed to represent "breakdown" products Evidence also indicates that P400 is also present in human CSF samples (see
Example 2 P400 is Not lmmunologically Related to Laminin In the next study, a polyclonal antibody against laminin was used to probe (by Western blot) human CSF and serum samples to determine whether P400 was lmmunologically related to laminm. In this study, human CSF (30 μl/lane, lanes 1-6) and serum (10 μl/lane, lanes 7-14) was probed with a polyclonal antibody against laminm (obtained from Sigma Chemical Co., St Louis. MO). In Figure 2, the following human biological fluid samples (i.e. CSF and serum) were obtained and analyzed as part of this study: Lane 1: #5211- CSF from a 66 year old male with probable AD with a mini-mental score of 25, Lane 2: #5113- CSF from a 83 year old normal male with a mini-mental score of 27, Lane 3: #5112- CSF from a 78 year old normal female with a mini-mental score of 30; Lane 4: #5111- CSF from a 69 year old female with presymptomatic Alzheimer's disease with rrum-mental score of 28, Lane 5: #5110- CSF from a 67 year old normal male with a mini-mental score of 30, Lane 6: #5109- CSF from a 78 year old normal female with a mini-mental score of 30 The coπesponding serum from these same patients are in Lanes 7- 12, and in the same order as the CSF samples. Lane 13: #5101- serum from a 73 year old normal male with a mini-mental score of 30; Lane 14- #5216- serum from a 74 year old female with confirmed Alzheimer's disease who had a mini-mental score of 14
As shown in Fig. 2, intact laminm (Mr -850 kιlodaltons)( which is observed at the gel interface and does not enter the gel due to its large size) (upper aπow) was present in human CSF (lanes 1-6) and to a lessor extent in human serum (lanes 7-14). The broad band observed between the 116 and 205 kDa markers is believed to represent the E8 fragment of laminin (Begovac et al, Cell Biol. 113:637-644, 1991; Yurchenco et al, J. Biol Chem. 268.17286-17299, 1993). The diffuseness of this band (in compaπson to Fig. 1) is believed to be due to the use of a polyclonal antibody against laminin which will recognize multiple epitopes The faintness of this band is believed to be due to the presence of the E8 fragment of laminin at low levels in CSF and serum. No lmmunostaining for laminin was observed the region >205 kilodaltons (up to the gel interface) suggesting that P400 was likely not lmmunologically related to laminin (compare the region >205 kilodaltons up to the gel interface in Fig. 1 versus Fig 2) We have employed other antibodies against laminin and have observed identical results (not shown), suggesting that P400 is not immunologicallv related to laminm
Example 3 Isolation and Purification of P400 from Human Cerebrospinal Fluid
Data also indicated that P400 is present in human CSF and can be isolated by Sephacryl S1000 fractionation (Fig. 3). For these studies 10 ml of CSF were fractionated through an Sephacryl S1000 column, and the vaπous fractions were separated by non-reducing SDS-PAGE, transfeπed to PVDF membrane and probed with 50 nM of bιotιnylated-Aβ. This concentration of bιotιnylated-Aβ was chosen so as to detect most proteins in CSF which bind Aβ. As shown in Fig. 3, P400 (upper aπow) is present in human CSF and can be ennched using Sephacryl S1000 fractionation. The AB-ligand blot (Fig. 3) demonstrated that P400 is present in human CSF and can be punfied from other Aβ-bmding proteins. Coomassie blue staining of similar blots indicate that very little low molecular weight proteins were present in fractions with Kα, of 0 5 (not shown), indicating that this method is useful in initial puπfication of P400
Example 4 Methodology to Determine the Identity of P400 Rationale: Our initial evidence suggests that P400 is present in both serum and CSF in humans, binds Aβ. and may be a potential marker for AD Gel filtration chromatography and Aβ-immunoaffinity chromatography are methods used to isolate P400 in sufficient quantities for microsequencmg Compaπsons are then be made to proteins in known data bases to identify P400
Methodology: Approximately 30 mis of serum from each of pooled Alzheimer's disease patients and normal aged controls are used. For this study, 30 mis of serum (from each of Alzheimer's disease and normal controls) is lyophilized separately and fractionated on a 40 ml Sephacryl S-1000 equilibrated with 50 mM Tπs-HCl, 150 mM NaCl, 1 M urea (pH 8.0), followed by elution using the same buffer. Our preliminary analysis (Fig 3) indicates that P400 is enπched in the fractions with a Kav = 0.45-0.55. Therefore, for this purification of P400, fractions with K,, = 0.45-0.55 are collected and precipitated by centπfugation at 14,000 Xg in the presence of 4 volumes of 95 % ethanol with 1 % sodium acetate The precipitate obtained is then dissolved in 2 ml of 50 mM Tπs-HCl, 150 mM NaCl, pH 7 5 (TBS) and passed through an Aβ affinity column for further puπfication
For preparation of the Aβ affinity column, 1 mg of Aβ 1-40 (Bachem Ine, Toπance, CA) in 0.5 ml of double distilled water is added to 1 ml of washed Affigel beads (Biorad) suspended in 1 volume of 0 2 M HEPES 160 mM CaCl (pH 7.5) The mixture is incubated overnight with gentle agitation at room temperature and stopped with lOOμl of 1 M ethanolamine at pH 8.0 for 1-2 hours at room temperature. The denvatized gel is washed in a minicolumn and the flow through is collected and assayed for protein content using a Buffalo black or Bradford reagent (Biorad) to determine the % of protein bound to the column. Previous studies (Snow et al, Arch Biochem. Biophys. 320:84-95, 1995) indicated that about 95 % of Aβ can be coupled to the column using this protocol. The column is first be equilibrated with TBS containing 0.1 % Tnton X-100 Then, P400 (isolated as descπbed above) is dissolved in TBS and is repeatedly applied to the column four times Unbound mateπals are washed with 10 ml TBS containing 0.1 % Tnton X-100. The bound mateπals are eluted with a 40 ml-hnear gradient of NaCl (0.15-1.0 M) in TBS containing 0.1 % Tnton X-100 at pH 7 5. Tightly bound matenals are then be eluted with 10 ml of 50 mM Tns-HCl, 3M NaCl, 7 M Urea (pH 8.0). A quots of each of 2ml fractions are precipitated by 15-mιnute centπfugation at 14,000 Xg in the presence of 4 volumes of 95 % ethanol with 1 % sodium acetate The pellets are then made up to 10 μl 1 x SDS sample buffer and are subjected to SDS-PAGE and transfeπed to PVDF membrane (see below) to identify fractions containing P400 Identification of P400 is based on size and its ability to bind Aβ as determined by Aβ ligand blot analysis
SDS-PAGE (4-15 % Tns-Glycine precast gels from BioRad, Hercules, CA) is performed under non-reducing conditions according to the method of Laemmh (Nature 227 680-685, 1970) using a Mini- Protean II electrophoresis system (BioRad) All samples are in 1 X SDS sample buffer and heated for 5 minutes in a boiling water bath before electrophoresis at 200V for 45 minutes along with pre-stained molecular weight protein standards The separated proteins are transfeπed from the gel to PVDF membrane (Immobilon-P for Western and ligand blot or Immobilon-PSQ for sequencing, Millipore Bedford, MA) using a Mini-transblot electrophoresis transfer cell according to manufacturers protocol (BioRad) Electrotransfer is performed at 100 V for 2 hours Following transfer, membranes are πnsed with methanol and dπed
For Coomassie blue staining, PVDF membranes are immersed with 0 2 % Coomassie bπlhant blue (w/v) in 50 % methanol, 10 % acetic acid, and 40% distilled water for 2 minutes The membranes are then nnsed with 50% methanol, 10% acetic acid, and 40% distilled water until the bands are visible and no background staining is remaining The coπesponding Aβ-binding P400 band (deteπmned by ligand blotting of adjacent lanes as descπbed below) is sent out for ammo-acid sequencing Amino acid sequencing is conducted using a Porton 2090 Gas-Phase Microsequencer ith on-line analysis of phenylthiohydantoin deπvatives (Porton Instruments Tarzana CA) as previously descnbed (Castillo and Templeton, FEBS Lett. 318 292-296, 1993) If the protein has a blocked N-terminus, protease digestions are performed to expose internal portions ot the protein which are anticipated to allow for N-terminal sequencing For these protease digestion studies, P400 is either left undigested, or digested with V8, trypsin, or elastase (Sigma Chem Co St Louis, MO) pπor to SDS-PAGE More specifically 2 μg of trypsm, V8 protease, or elastase in 2 μl of 50 mM Tπs-HCl buffer (pH 8 0) is added to either buffer only, or to 50 μl of P400 (50 μg) in the same buffer The mixtures are incubated at 37°C and ahquots (10 μl) taken at vaπous times are mixed ith equal volumes of 2X non-reducing SDS-sample buffer for SDS- PAGE The separated P400 fragments are then transfeπed to PVDF membranes and visualized by staining with Coomassie blue as descπbed above The most abundant P400 fragments as judged by Coomassie blue staining intensity are also be sent for microsequencing The partial P400 sequences obtained by microsequencing is then be identified by compaπson to known protein sequence hbraπes retneved from the National Center for Biotechnology Information, Bethesda, MD Initial studies indicate that 20-40 ml of serum gives a final yield of 04-0.8 mg of P400. which is more than sufficient for incorporation into all of the studies descπbed
A more accurate molecular weight of P400 is also determined by slightly decreasing the acrylamide concentration (from 4% to 3 5%) and include larger molecular weight standards (i.e >400 kDa) obtained commerciallv
Example 5 Determination of the Strength of Binding of P400 to Beta-Amyloid Protein Rationale: The data indicates that P400 binds to Aβ with high affinity human biological fluids In this study, solid phase binding assays are used to determine the dissociation constant of P400 to immobilized Aβ using bιotιnylated-P400 As an alternative assay to P400-bιotιnylatιon, polyclonal antibodies to P400 are generated in rabbits and used in ELISA assays to determine the dissociation constant of Aβ binding to P400 as previously descnbed (Castillo et al, J Neurochem 69:2452-2465, 1997)
Methodology: To biot ylate P400, 0 5 mg of puπfied P400 is diluted into 100 μl PBS, transfeπed into a tube containing 0 10 mg Sulfo-NHS-LC-Biotin, and incubated for 45 minutes at room temperature Bιotιnylated-P400 is then precipitated by addition of 95 % ethanol containing 1 % sodium acetate (w/v) followed by centnfugation at 14,000 Xg for 20 minutes The supernatant is decanted and P400 is nnsed twice by re-precipitation as above Biotin incorporation is determined per molecule of P400 using [2-(4'- hydroxyazobenzene)benzoιc acid] (HABA) according to the manufacturer's protocol (Pierce, Rockford,
U)
For solid phase binding assays, Nunc plates (Maxisorb) containing immobilized Aβ is first prepared by incubating each well overnight with 2 μg of Aβ (Bachem Ine, Toπance, CA) in 40 μl of TBS containing lOOmM Tns-HCl, 50 mM NaCl, and 3 mM NaN3 (pH 7.4) The next day the wells are blocked with 300 μl of TBS containing 0.05% Tween-20 (TTBS) plus 2% bovine serum albumin (BSA) Wells without immobilized Aβ (i.e. blank wells) are also blocked with TTBS containing 2 % albumin as descπbed above. Puπfied and biotinylated P400 (1 mg/ml) is diluted in TTBS at dilutions of 1 10, 1 30, 1:90, 1.270, 1:810, 1:2430, 1.7290 (v/v) and blank. These dilutions are plated (250 μl) in tπphcate wells in the presence or absence of immobilized Aβ (1-40) and incubated overnight The next day, the wells are nnsed 3 times with TTBS and probed for 30 min with 100 μl of streptavidm-peroxidase (1:500 of 2 μg/ml) in TTBS containing 0 1 % BSA (i.e secondary probe) The wells are then nnsed 3 times with TTBS and 100 μl of substrate solution ( OPD-Sigma Fast from Sigma Chemical Co., St. Louis, MO.) is added to each well and allowed to develop for 10 minutes or until there is a significant color difference The reaction is stopped w ith 50 μl of 4 N sulfuπc acid and read at 490 nm on a Model 450 ELISA plate reader (Biorad) The data is plotted and the Kd is determined using the Ultrafit program (Biosoft, UK) If there is a problem with proper biotinylation of P400 (for example, biotinylation of P400 may block the binding site on P400 which normally associates with Aβ, this would be determined by compaπng Kd's obtained using ELISA techniques to the descπbed ligand studies), then an alternative method as descπbed below can be used. For this method, rabbit polyclonal antibodies are utilized to detect the relative amount of P400 bound to Aβ immobilized on rmcrotiter wells. Bπefly, P400 at vaπous dilutions descπbed above are plated (250 μl) in tπphcate wells in the presence or absence of immobilized Aβ (1-40) and incubated overnight The next da> the wells are nnsed 3 times with TTBS and probed for 1 hour with 100 μl of an antι-P400 polyclonal antibody diluted in TTBS (dilution to be determined empincally). After 3 πnses with TTBS, the wells are incubated for 30 mm with 100 μl of biotinylated goat anti-rabbit secondary antibody (1 : 1000), and streptavidin-peroxidase (1:500 of 2 μg/ml) in TTBS containing 0.1 % BSA. The wells are then be nnsed 3 times with TTBS and 100 μl of the substrate solution ( OPD-Sigma Fast from Sigma Chemical Co., St. Louis, MO.) is then added to each well and allowed to develop for 10 rrunutes or until there is a significant color difference. The reaction is stopped with 50 μl of 4 N Sulfuπc acid and read at 490 nm on an ELISA plate reader. The data is plotted and the Kd is determined using the Ultrafit program (Biosoft, UK)
The binding data is analyzed assuming a thermodynarmc equilibrium for the formation of the complex BL, from the P400 ligand in solution, L, and the uncomplexed Aβ adsorbed to the rmcrotiter well, B, according to the equation. Kd = [B] X [L]/[BL] (Castillo et al, J Neurochem 69:2452-2465, 1997). The Kd's are determined by using an enzyme-linked immunoassay that gives a color signal that is proportional to the amount of P400 bound to Aβ (Engel and Schalch, Mol Immunol. 17:675-680, 1980, Mann et al, Eur J Biochem 178.71-80, 1988, Fox et al, EMBO J. 10.3137-3146, 1991; Battagha et al, Eur. J. Biochem. 208:359-366, 1992) To account for potential non-specific binding, control wells without Aβ (in tπphcate) are included for each concentration of P400 used in each binding expenment. Optical densities of the control wells usually never exceed 0.050 at all ligand concentrations based on previous studies (Castillo et al, J. Neurochem 69:2452-2465, 1997). The optical densities of the control wells are subtracted from the optical densities of the Aβ-containing wells that received similar P400 concentrations. Non-specific absorbance obtained from Aβ containing wells that did not receive P400 are also subtracted from all data points Thus, the equation in the form of: OD.xp=OD0 + (S x [P400]) + (ODmdX x [P400]/([P400] + Kd) where (S x [P400]) represents non-specific binding (control wells) and OD0 is the non-specific absorbance, becomes ODexp = ODmax x [P400]/([P400] + Kd). Therefore, at 50 % saturation OD_xp = 0.50 ODmax and Kd = [P400]. Determination of [P400] at 50% saturation is performed by nonlinear least square program (Ultrafit from Biosoft, UK) using a one-site model.
For preparation of polyclonal antibodies against P400, Zymed Laboratories are used. Once pure P400 is isolated, 100-200 μg are sent out to Zymed labs for the preparation of polyclonal antibodies. If necessary, affinity purification of P400 antibodies is performed, once the initial polyclonal antibodies have arrived. For such a procedure, 0.5 mg of P400 is incubated with 1 ml of a washed Affigel bead (Biorad) suspension in 0.5 ml 0.2 M HEPES 160 mM CaCl ( pH 7). The reaction is stopped with lOOμl of 1 M ethanolamine at pH 8.0 for 1-2 hours at room temperature. The derivatized gel is washed in a minicolumn and the flow-through is collected and assayed for protein content using the Bradford reagent according to manufacturer protocol (Biorad). The column is equilibrated with TBS with 0.1 % TX-100 and 10 ml of serum diluted 1:4 in 100 mM Tris-HCl, 50 mM NaCl, pH 7.5 (TBS) is applied several times. Unbound materials are washed with 20 ml TBS. Bound antibodies are eluted with 1 % acetic acid/water and lyophilized.
Example 6 Effects of P400 on Inhibition/Dissolution of Aβ Fibril Formation Rationale: The initial data demonstrates that P400 binds to Aβ with high affinity in biological fluids. P400 is believed to be a potent inhibitor of Aβ fibril formation and likely dissolves pre-formed Aβ amyloid fibrils. For these studies, Thioflavin T fluorometry, Congo red staining assays and negative stain electron microscopy are employed to determine the effects of purified P400 on inhibition of Aβ fibril formation and whether P400 is capable of dissolving preformed Aβ amyloid fibrils.
Methodology: To assess the potential inhibitory effects of P400 on Aβ amyloid fibril formation, fluorometry assays will be used as previously described (Castillo et al, J. Neurochem. 69:2452-2465, 1997). Briefly, 25 μM of Aβ (1-40) (Bachem Inc., Toπance, CA; Lot # WM 365) in TBS solution (pH 7.0) is incubated at 37°C (in triplicate) in the presence or absence of 100 nM of P400. The mixtures are incubated for 1 week at 37°C with aliquots analyzed at lh, 1 day, 3 days and 1 week. Fibrillogenesis is determined by addition of 1.2 ml Thioflavin T solution (100 μM Thioflavin T in 50 mM Sodium phosphate buffer pH 6.0) to 50 μl aliquots, followed by reading on the fluorometer as previously described (Castillo et al, J. Neurochem. 69:2452-2465, 1997). The effect of various concentrations of P400 (5- lOOnM) on Aβ fibrillogenesis (25 μM) is also tested in a similar manner as described above. The effect of various concentrations of P400 (5-100nM) on another amyloid protein called amylin (25 μM; Bachem Inc.)(which accumulates in the pancreas of 90% of patients with type II diabetes) is also determined to give an idea of amyloid specificity Previous studies (Castillo et al, J Neurochem 69.2452-2465. 1997) indicate that increasing concentration of fibπllar Aβ in this assay gives a proportional increase in fluorescence in the presence of 100 μM Thioflavin T, ruling out the presence of any disproportionate inner filter effects in these studies. The increase in Thioflavin T fluorescence with time is proportional to the increase in the amount of amyloid fibπls with time. Initial data indicates that P400 does not contπbute to Thioflavin T fluorescence when measured alone
In another fluorometry expeπment, several different concentrations of P400 are used to determine the dose-dependent effects of P400 on dissolution of pre-formed Aβ amyloid fibπls For this fibπl dissolution assay, 250 μM of Aβ (1-40) are allowed to fibπlhze for 1 week following incubation at 37°C In addition, 250 μM of Aβ 1-42 (which does not require pre-fibπllation) is used Aliquots are then made up to 25 μM with vaπous concentrations of P400 and incubated for 4 days at 37°C, and analyzed using Thioflavin T fluorometry assays as described above. 5-10 μl aliquots from vaπous incubation mixtures are also be spotted on gelatin-coated glass slides and air-dπed overnight, before staining with Congo red the next day (Puchtler et al, J Histochem Cytochem. 10:355-364, 1962). Whether a postulated decrease in fibπl foπnation coπelates with a decrease in Congo red staining is determined. P400 potential inhibition of Aβ amyloid fibril formation as determined by Thioflavin T fluorometry and Congo red staining assays are confirmed by negative stain electron microscopy For these studies, negatively stained fibπls are prepared by floating pioloform. carbon-coated grids on peptide solutions (1 to 2 mg/ml) in the presence or absence of increasing concentrations of P400 (to be determined empirically) To control for pH changes, peptides are dissolved in buffered solutions of 20 mM glycine (for pH 2 to 3 and pH 9 to 10) or 20 mM Tπs -HCL (for pH 6 to 8) After the gπds are blotted and air-dπed, the samples are stained with either 2% (w/v) uranyl acetate or 1 % (w/v) phosphotungstic acid and visualized, and photographed with a JEM 1200 EX II (JEOL Ltd, Tokyo, Japan), using 80kV accelerating voltage
Example 7 P400 as a Specific Diagnostic Marker For Alzheimer's Disease Rationale: The data suggests that P400 is reduced in the biological fluids in Alzheimer's disease and may therefore potentially serve as a specific biochemical marker. Studies utilizing Aβ-hgand blotting techniques followed by scanning densitometry can quantitate changes relative intensity P400 among Alzheimer's disease versus normal aged-matched control patients. In addition, sandwich or competitive ELISAs are used to determine whether P400 is useful as a specific marker protein for Alzheimer's disease and/or its progression Methodology: Aβ-hgand blotting techniques are used to assess levels of P400 in biological fluids of Alzheimer's disease versus normal aged-matched controls Biotinylation of Aβ 1-40, and assessment of serum and CSF samples by Aβ-hgand blotting is as described in example 5. Following transfer of serum and CSF proteins to PVDF membranes as descπbed in example 5, membrane blots are probed for 2 hrs with 50 nM biotinylated AB (1-40) in TTBS which is heated to 90°C for 5 minutes pnor to use. The membranes are then nnsed three times (5 seconds each) with TTBS, probed for 30 minutes with streptavidin alkaline phosphatase conjugate (Vectastain), nnsed again (as descπbed above), followed by the addition of an alkaline phosphatase substrate solution (Vectastain). Following color development, flushing the membranes with distilled deiomzed water stops the reaction. The relative intensity of P400 bands is determined by scanning the blots and quatifying using a Biorad scanning densitometer, as previously descnbed (Maresh et al, Soc Neurosci. Abst. 23.2221, 1997)
If specific polyclonal antibodies against P400 are obtained as descnbed in example 5, then a sandwich ELISA to quantitate levels of P400 in serum and CSF from Alzheimer's disease versus normal aged patients is employed. For these studies, Aβ is immobilized on the plate and serves as the capture molecule. CSF or serum is then applied, and any P400 bound to Aβ is detected using the antι-P400 antibody More specifically, Nunc plates (Maxisorb) containing immobilized Aβ 1-40 or 1-42 (Bachem Inc., Toπance CA) is prepared by incubating each well overnight with 2 μg of AB in 40 μl Tns-buffered saline (TBS. 100 mM Tπs-HCl. 50 mM NaCl, and 3 mM NaN3, pH 7.4) and blocking the next day with 300 μl TBS containing 0.05% Tween-20 (TTBS) plus 2% bovine serum albumin (BSA) Wells without immobilized Aβ are also blocked with TTBS containing 2 % albumin as descnbed above (i.e. blank wells). Serum samples diluted 1 100 are applied in tnphcate wells in the presence or absence of immobilized Aβ (1-40), and then incubated overnight. Known amounts of punfied P400 are also applied to serve as an internal quantitative standard. The next day, the wells are nnsed 3 times with TTBS and probed for 30 min with 100 μl of a secondary probe consisting of peroxidase conjugated anti-rabbit antibody diluted 1:500 (out of 2 μg/ml )(Jackson immunoresearch, Westgrove, PA) in TTBS containing 0.1 % BSA. The wells are then nnsed 3 times with TTBS, and 100 μl of substrate solution (OPD-Sigma Fast from Sigma Chemical Co., St. Louis. MO.) is added to each well and allowed to develop for 10 minutes or until there is a significant color difference. The reaction is stopped with 50 μl of 4 N Sulfuπc acid and read at 490 nm on an ELISA plate reader. The optical density of vanous concentrations of standard P400 is plotted, and the concentrations of P400 in CSF and serum samples is determined based on the standard curve. If polyclonal antibodies against P400 are not available, levels of P400 in biological fluids are quantitated by a competitive ELISA format. The competitive ELISA is performed based on the competition for binding of biotinylated P400 versus nonbiotinylated P400 (in serum or CSF) to immobilized Aβ. In this scenario, the amounts of biotinylated P400 bound to immobilized Aβ is inversely proportional to the amounts of P400 in the biological sample. More specifically, Nunc plates (Maxisorb) containing immobilized Aβ (1-40 or 1-42; Bachem Inc., Toπance, CA) is prepared by incubating each well overnight with 2 μg of Aβ in 40 μl Tris-buffered saline (TBS; 100 mM Tris-HCl, 50 mM NaCl, and 3 mM NaN3, pH 7.4) and blocking the next day with 300 μl TBS containing 0.05% Tween-20 (TTBS) plus 2% bovine serum albumin (BSA). Wells without immobilized Aβ are also blocked with TTBS containing 2 % albumin as described above (i.e. blank wells). Various known standard concentrations of nonbiotinylated P400 and serum or CSF samples with unknown concentrations of P400 are mixed with fixed concentration of biotinylated-P400. The fixed concentration of biotinylated-P400 is chosen so that it is close to saturating all of the immobilized Aβ sites in each well. These solutions are applied in triplicate wells in the presence or absence of immobilized Aβ (1-40) and incubated overnight. The next day the wells are rinsed 3 times with TTBS and probed for 30 min with 100 μl of secondary probe made up with streptavidin-peroxidase (1 :500 of 2 μg/ml) in TTBS containing 0.1 % BSA. The wells are then rinsed 3 times with TTBS and 100 μl of the substrate solution ( OPD-Sigma Fast from Sigma Chemical Co., St. Louis, MO.) is added to each well and allowed to develop for 10 minutes or until there is a significant color differences. The reaction is stopped with 50 μl of 4 N Sulfuπc acid and read at 490 nm. A standard curve is then plotted from the optical density results of the standard concentrations of P400, which is an inverse relationship. The concentrations of P400 in CSF and serum is determined based on the standard curve.
Further Aspects and Utilizations of the Invention
P400-Derived Protein Fragments and Polypeptides
One therapeutic application of the present invention is to use P400, P400 protein fragments which bind Aβ or other amyloid proteins, and/or P400 polypeptides derived from amino acid sequencing of the P400 fragments which bind Aβ or other amyloid proteins, as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses. The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease and Down's syndrome (wherein the specific amyloid is refeπed to as beta-amyloid protein or Aβ), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediteπanean Fever (wherein the specific amyloid is refeπed to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is refeπed to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is refeπed to as amylin or islet amyloid), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is refeπed to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is refeπed to as beta2-microglobulin amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is refeπed to as transthyretin or prealbumin), and the amyloid associated with endocrine tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is refeπed to as variants of procalcitonin).
The polypeptides refeπed to above may be a natural polypeptide, a synthetic polypeptide or a recombinant polypeptide. The fragments, derivatives or analogs of the polypeptides to any P400 fragment refeπed to herein may be a) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be encoded by the genetic code, or b) one in which one or more of the amino acid residues includes a substituent group, or c) one in which the mature polypeptide is fused with another compound, such as a compound used to increase the half-life of the polypeptide (for example, polylysine), or d) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of the invention.
The tertiary structure of proteins refers to the overall 3-dimensional architecture of a polypeptide chain. The complexity of 3-dimensional structure arises from the intrinsic ability of single covalent bonds to be rotated. Rotation about several such bonds in a linear molecule will produce different nonsuperimpossable 3-dimensional aπangements of the atoms that are generally described as confoπnations.
Protein conformation is an essential component of protein-protein, protein-substrate, protein- agonist, protein-antagonist interactions. Changes in the component amino acids of protein sequences can result in changes that have little or no effect on the resultant protein conformation. Conversely, changes in the peptide sequences can have effects on the protein conformation resulting in reduced or increased protein-protein, etc. interactions. Such changes and their effects are generally disclosed in Proteins: Structures and Molecular Properties by Thomas Creightonm W.H Freeman and Company, New York, 1984 which is hereby incoφorated by reference
"Conformation" and "conformation similaπty" when used in this specification and claims refers to a polypeptide's ability (or any other organic or inorganic molecule) to assume a given shape, through folding and the like, so that the shape, or conformation, of the molecule becomes an essential part of it's functionality, sometimes to the exclusion of its chemical makeup It is generally known that m biological processes two conformational similar molecules may be interchangeable in the process, even the chemically different "Conformational similaπty" refers to the latter mterchangeability or substitutabi ty For example, P400 and P400-deπved protein fragments are among the subjects of the invention because they have been shown to bind the Aβ protein and render it inactive in fibπl foπnation; it is contemplated that other molecules that are conformationally similar to P400, or any claimed P400 fragment or polypeptide, may be substituted in the claimed method to similarly render the Aβ inactive in fibnllogenesis and other amyloid processes In general it is contemplated that levels of conformational similanty at or above 70% are sufficient to assume homologous functionality in the claimed processes, though reduced levels of conformational similarity may be made to serve as well. Conformational similar levels at or above 90% should provide some level of additional homologue functionality.
Thus, one skilled in the art would envisage that changes can be made to the P400 sequence, or fragments or polypeptides thereof, that would increase, decrease or have no effect on the binding of P400 or fragments thereof, to Aβ amyloid In addition, one skilled in the art would envisage vaπous post- translational modifications such as phosphorylation, glvcosylation and the like would alter the binding of P400, P400 fragments or P400 polypeptides to Aβ amyloid.
The polypeptides of the present invention include the polypeptides or fragments of P400 and fragments thereof, as well as polypeptides which have at least 70% similanty (preferably 70 % identity) and more preferably a 90% similanty (more preferably a 90% identity) to the polypeptides of P400.
Fragments or portions of the polypeptides or fragments of P400 of the present invention may be employed for producing the coπesponding full-length polypeptides by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full length polypeptides.
The polypeptides of the present invention may be a naturally punfied product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bactenal, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant procedure, the polypeptides of the present invention may be glycosylated or may be non glycosylated Polypeptides of the invention may also include an initial methiomne amino acid residue
Chemical polypeptide synthesis is a rapidly evolving area in the art, and methods of solid phase polypeptide synthesis are well-descπbed in the following references, hereby entirely incoφorated by reference (Mernfield, J Amer Chem Soc 85 2149-2154, 1963, Mernfield, Science 232 341-347, 1986, Fields, Int J Polypeptide Prot Res 35, 161, 1990)
Recombinant production of P400 polypeptides can be accomplished according to known method steps Standard reference works seting forth the general pnnciples of recombinant DNA technology include Watson, Molecular Biology of the Gene, Volumes I and II, The Benjamin/Cummings Publishing Company Ine , publisher, Menlo Park, Calif 1987, Ausubel et al, eds , Cuπent Protocols in Molecular Biology, Wiley Interscience, publisher, New York, N Y 1987, 1992, and Sambrook et al, Molecular Cloning A Laboratory Manual, Second Edition, Cold Spπng Harbor Laboratory, publisher, Cold Spπng Harbor, N.Y 1989, the entire contents of which references are herein incoφorated by reference
The polypeptides of the present invention may also be utilized as research reagents and mateπals for discovery of treatments and diagnostics for human diseases
Antibodies
Antibodies generated against the polypeptides coπesponding to specific sequences recognizing the P400 fragments of the present invention hich bind Aβ or other amyloid proteins can be obtained by direct injection of the polypeptides into an animal or by administeπng the polypeptides to an animal, preferably a nonhuman The antibody so obtained will then bind the polypeptides itself In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides Such antibodies can then be used to isolate the polypeptides from tissue expressing that polypeptide
The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies, chimeπc antibodies, anti-idiotypic antibodies to antibodies specific for P400 deπved protein fragments or polypeptides of the present invention
Polyclonal antibodies are heterogeneous populations of antibody molecules denved from the sera of animals immunized with an antigen
A monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybndo a technique (Kohler and Milstem, Nature 256:495-497, 1975), the tπoma technique, the human B-cell hybndoma technique (Kozbor et al. Immunology Today 4 72, 1983), and the EBV-h bπdoma technique to produce human monoclonal antibodies (Cole et al, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp 77-96, 1985) Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE. IgA, GILD and any subclass thereof
Chimenc antibodies are molecules different portions of which are deπved from different animal species, such as those having vanable region denved from a munne monoclonal antibody and a human immunoglobulin constant region, which are pπmaπly used to reduce immunogenicity in application and to increase yields in production Chimeπc antibodies and methods for their production are known in the art (ex. Cabilly et al. Proc Natl Acad Sci U S A 81 :3273-32"77, 1984, Harlow and Lane Antibodies' A Laboratory Manual. Cold Spπng Harbor Laboratory 1988)
An anti-idiotypic antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An anti-iodiotypic antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the monoclonal antibody with the monoclonal antibody to which an anti-iodiotypic antibody is being prepared. The immunized animal will recognize and respond to the ldiotypic determinants of the immunizing antibody by producing an antibody to these ldiotypic determinants (the anti-idiotypic antibody). See, for example. U S Patent No 4,699.880. which is herein incoφorated by reference
The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as. for example. Fab and F(ab')2, which are capable of binding antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less nonspecific tissue binding than an intact antibody (Wahl et al, J Nucl Med. 24-316-325. 1983)
The antibodies or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect P400 or P400-deπved fragments in a sample or to detect presence of cells which express a P400 polypeptide of the present invention.This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometπc or fluorometπc detection
One of the ways in which a P400 antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme lmmunoassay (EIA). This enzyme, in turn, when later exposed to an appropπate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometnc, fluorometπc, or by visual means. Enzymes which can be used detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroιd isomerase, yeast alcohol dehydrogenase, alpha glycerophosphate dehydrogenase, tπose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparagmase, glucose oxidase. beta-galactosidase. πbonuclease, urease, catalase, glucose-6-phosphate dehydrogenase. glucoamylase and acetylcholinesterase The detection can be accomplished by colometπc methods which employ a chromogenic substrate for the enzyme Detection can be accomplished by colometnc methods which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual companson of the extent of enzymatic reaction of a substrate with similarly prepared standards (see Harlow and Lane, Antibodies. A Laboratory Manual. Cold Spnng Harbor Laboratory 1988; Ausubel et al, eds., Cuπent Protocols in Molecular Biology. Wiley Interscience, N.Y. 1987, 1992)
Detection may be accomplished using any of a vanety of other immunoassays. For example, by radiolabeling of the antibodies or antibody fragments, it is possible to detect R PTPase through the use of a radioimmunoassay (RIA) A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work et al, North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, incoφorated entirely by reference herein The radioactive isotope can be detected by such means as the use of a gamma-counter, a scintillation counter or by autoradiography.
It is also possible to label a P400 polypeptide antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythnn, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. commercially available, e.g , from Molecular Probes, Ine (Eugene, Oregon, U S A )
The antibody can also be detectably labeled using fluorescence emitting metals such as 152EU, or other of the lanthanide senes. These metals can be attached to the antibody using such metal groups as diethylenetnamine pentaacetic acid (EDTA)
The antibody can also be detectably labeled by coupling it to a chemiluminescent compound The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that anses duπng the course of a chemical reaction, Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acndinium ester, lmidazole, acπdinium salt, and oxalate ester
Likewise, a bioluminescent compound may be used to label the antibody of the present invention Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for puφoses of labeling are lucifenn, luciferase and aequonn. The antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of a P400 fragment of the present invention In situ detection may be accomplished by removing a histological specimen from a patient, and providing the labeled antibody of the present invention to such a specimen The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of a P400 fragment polypeptide but also its distπbution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide vanety of histological methods (such as staining procedures) can be modified in order to achieve such m situ detection
In accordance with yet a further aspect of the present invention there are provided antibodies against P400, P400 fragments and/or P400-deπved polypeptides which interact with Aβ or other amyloid proteins, or deπvatives thereof These antibodies can be used for a number of important diagnostic and/or therapeutic applications as descπbed herein. In one aspect of the invention, polyclonal and/or monoclonal antibodies made against P400, P400 fragments and/or P400-deπved polypeptides which bind Aβ or other amyloid proteins, may be utilized for Western blot analysis (using standard Western blotting techniques knowledgeable to those skilled in the art) to detect the presence of amyloid protein-binding P400 fragments or amyloid protein-binding P400 polypeptides in human tissues and in tissues of other species. Western blot analysis can also be used to determine the apparent size of each amyloid protein-binding P400 fragment In addition, Western blotting following by scanning densitometrv (known to those skilled in the art) can be used to quantitate and compare levels of each of the P400 fragments in tissue samples. biological fluids or biopsies obtained from individuals with specific diseases (such as the amyloid diseases) in comparison to tissue samples, biological fluids or biopsies obtained from normal individuals or controls. Biological fluids, include, but are not limited to, blood, plasma, serum, cerebrospinal fluid, sputum, saliva, uπne and stool
In yet another aspect of the invention, polyclonal and/or monoclonal antibodies made against P400, P400 fragments and or P400-deπved peptides which bind Aβ or other amyloid proteins, can be utilized for immunoprecipitation studies (using standard immunoprecipitation techniques known to one skilled in the art) to detect P400, P400 fragments and/or P400-denved peptides which bind Aβ or other amyloid proteins, in tissues, cells and/or biological fluids. Use of the P400, P400 fragments and/or P400- denved peptide antibodies for immunoprecipitation studies can also be quantitated to determine relative levels of P400, P400 fragments and/or P400-deπved peptides which interact with Aβ or other amyloid proteins, in tissues, cells and/or biological fluids. Quantitative immunoprecipitation can be used to compare levels of P400, P400 fragments and/or P400 amyloid protem-bmding peptides in tissue samples, biological fluids or biopsies obtained from individuals with specific diseases (such as the amyloid diseases) in comparison to tissue samples, biological fluids or biopsies obtained from normal individuals or controls.
Therapeutic Applications
Yet another aspect of the present invention is to make use of P400, P400 fragments and/or P400- derived polypeptides as amyloid inhibitory therapeutic agents. The P400-derived peptide sequences or fragments can be synthesized utilizing standard techniques (ie. using an automated synthesizer). P400, P400 fragments and/or P400-derived peptides which bind Aβ or other amyloid proteins, can be used as potential blocking therapeutics for the interaction of P400 in a number of biological processes and diseases (such as in the amyloid diseases described above). In a prefeπed embodiment, specific peptides made against P400 or fragments thereof may be used to aid in the inhibition of amyloid formation, deposition, accumulation, and /or persistence in a given patient. Likewise, in another prefeπed embodiment anti-idiotypic antibodies made against P400, P400 fragments and/or P400-derived peptides (as described above) may be given to a human patient as potential blocking antibodies to disrupt continued amyloid formation, deposition, accumulation and/or persistence in the given patient.
Preparations of P400-derived polypeptides for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain axillary agents or excipients which are known in the art. Pharmaceutical compositions such as tablets, pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositories, sterile injectable solutions, sterile packaged powders, can be prepared according to routine methods and are known in the art.
In yet another aspect of the invention, P400, P400 fragments and/or P400-derived peptides may be used as an effective therapy to block amyloid formation, deposition, accumulation and/or persistence as observed in the amyloid diseases. For example, the invention includes a pharmaceutical composition for use in the treatment of amyloidoses comprising a pharmaceutically effective amount of a P400, P400 fragments and/or P400-derived peptide anti-idiotypic antibody and a pharmaceutically acceptable carrier. The compositions may contain P400, P400 fragments and/or P400-derived peptide anti-idiotypic antibody, either unmodified, conjugated to a potentially therapeutic compound, conjugated to a second protein or protein portion or in a recombinant form (ie. chimeric or bispecific P400, P400 fragment and/or P400 polypeptide antibody). The compositions may additionally include other antibodies or conjugates. The antibody compositions of the invention can be administered using conventional modes of administration including, but not limited to, topical, intravenous, intra-arterial, intraperitoneal, oral, intralymphatic, intramuscular or intralumbar. Intravenous administration is prefeπed. The compositions of the invention can be a variety of dosage forms, with the prefeπed form depending upon the mode of administration and the therapeutic application. Optimal dosage and modes of administration for an individual patient can readily be determined by conventional protocols.
P400, P400 fragments and/or P400-derived peptides, or antibodies of the present invention may be administered by any means that achieve their intended puφose, for example, to treat P400 involved pathologies, such as Alzheimer's disease and other amyloid diseases, or other related pathologies, using a P400-derived polypeptide described herein, in the form of a pharmaceutical composition.
For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal or buccal routes. Alternatively, or concuπently, administration may be by the oral route. Parenteral administration can be by bolus injection or by gradual perfusion over time.
A prefeπed mode of using a P400-derived polypeptide, or antibody pharmaceutical composition of the present invention is by oral administration or intravenous application.
A typical regimen for preventing, suppressing or treating P400-involved pathologies, such as Alzheimer's disease amyloidosis, comprises administration of an effective amount of P400-derived polypeptides, administered over a period of one or several days, up to and including between one week and about 24 months.
It is understood that the dosage of the P400-derived polypeptides of the present invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concuπent treatment, if any, frequency of treatment, and the nature of the effect desired. The most prefeπed dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.
The total dose required for each treatment may be administered by multiple doses or in a single dose. A P400-derived polypeptide may be administered alone or in conjunction with other therapeutics directed to P400-involved pathologies, such as Alzheimer's disease or amyloid diseases, as described herein.
Effective amounts of a P400-derived polypeptide or composition, which may also include a P400- fragment derived antibody, are about O.Olμg to about lOOmg/kg body weight, and preferably from about 10 μg to about 50 mg/kg body weight, such as 0.05, 0.07, 0.09, 0.1, 0.5, 0.7, 0.9. , 1, 2, 5, 10, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/kg. Preparations for parenteral administration include steπle aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain axillary agents or excipients which are known in the art Pharmaceutical compositions compπsing at least one P400 denved polypeptide, such as 1-10 or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 P400-denved polypeptides, of the present invention may include all compositions wherein the P400- deπved polypeptide is contained in an amount effective to achieve its intended puφose In addition to at least one P400-deπved polypeptide, a pharmaceutical composition may contain suitable pha naceutically acceptable earners, such as excipients, earners and/or axillanes which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
Pharmaceutical compositions compπsing at least one P400-deπved polypeptide or antibody may also include suitable solutions for administration intravenously, subcutaneously, dermally, orallv , mucosally, rectally or may by injection or orally, and contain from about 0.01 to 99 percent, preferably about 20 to 75 percent of active component (i.e. polypeptide or antibody) together with the excipient. Pharmaceutical compositions for oral administration include pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, and syrups.
The P400, P400 fragments and or P400-denved peptides for Alzheimer's disease and other central nervous system amyloidoses may be optimized to cross the blood-brain banner. Methods of introductions include but are not limited to systemic administration, parenteral administration i.e., via an intrapeπtoneal, intravenous, penoral. subcutaneous, intramuscular, mtraartenal, intradermal, intramuscular, mtranasal, epidural and oral routes. In a prefeπed embodiment, P400, P400 fragments and/or P400-denved peptides may be directly administered to the cerebrospinal fluid by intraventπcular injection. In a specific embodiment, it may be desirable to administer P400, P400 fragments and/or P400-denved peptides locally to the area or tissue in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion duπng surgery, topical application, by injection, by infusion using a cannulae with osmotic pump, by means of a catheter, by means of a suppository, or by means of an implant.
In yet another embodiment P400, P400 fragments and/or P400-denved peptides may be delivered in a controlled release system, such as an osmotic pump. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, ie. the brain, thus requmng only a fraction of the systemic dose.
In yet another aspect of the present invention, peptidomimetic compounds modelled from P400, P400 fragments and/or P400-denved peptides identified as binding Aβ or other amyloid proteins, may serve as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses Peptidomimetic modelling is implemented by standard procedures known to those skilled in the art
In yet another aspect of the present invention, compounds that mimic the 3 dimensional Aβ binding site on P400 using computer modelling, may serve as potent inhibitors of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease and other amyloidoses. Design and production of such compounds using computer modeling technologies is implemented by standard procedures known to those skilled in the art
Recombinant DNA technology, including human gene therapy, has direct applicability to the P400 and their fragments, of this invention One skilled in the art can take the peptide sequences and create coπesponding nucleotide sequences that code for the coπesponding peptide sequences. These sequences can be cloned into vectors such as retroviral vectors, and the like. These vectors can, in turn, be transfected into human cells such as hepatocytes or fibroblasts, and the like. Such transfected cells can be introduced into humans to treat amyloid diseases. Alternatively, the genes can be introduced into the patients directly. The basic techniques of recombinant DNA technology are known to those of ordinary skill in the art and are disclosed in Recombinant DNA Second Edition, Watson, et al., W.H. Freeman and Company, New York, 1992, which is hereby incoφorated by reference
Diagnostic Applications
Another aspect of the invention is to provide polyclonal and or monoclonal antibodies against P400, P400 fragments and/or P400-denved peptides which bind Aβ or other amyloid proteins, which would be utilized to specifically detect P400. P400 fragments and/or P400-denved peptides in human tissues and/or biological fluids. In one prefeπed embodiment, polyclonal or monoclonal antibodies made against a peptide portion or fragment of P400. can be used to detect and quantify P400, P400 fragments and/or P400-denved peptides in human tissues and/or biological fluids. Polyclonal and/or monoclonal peptide antibodies can also be utilized to specifically detect P400 fragments and/or P400-denved polypeptides in human tissues and/or biological fluids. In a prefeπed embodiment, a polyclonal or monoclonal antibody made specifically against a peptide portion of P400 which binds Aβ (as descnbed herein), can be used to detect and quantify this P400 fragment in human tissues and/or biological fluids. In another prefeπed embodiment, a polyclonal or monoclonal antibody made specifically against a peptide portion or fragment of P400 which is present in human biological fluids and binds Aβ (as descnbed herein), can be used to detect and quantify this P400 fragment in human tissues and/or biological fluids
For detection of P400 fragments and or P400-denved polypeptides descnbed above in human tissues, cells, and/or in cell culture, the polyclonal and/or monoclonal antibodies can be utilized using standard lmmunohistochemical and lmmunocytochemical techniques, known to one skilled in the art.
For detection and quantitation of P400, P400 fragments and/or P400-denved peptides in biological fluids, including cerebrospinal fluid, blood, plasma, serum, unne, sputum, and/or stool, vanous types of ELISA assays can be utilized, known to one skilled in the art. An antibody molecule of the present invention may be adapted for utilization in an immunometnc assay, also known as a "two-site" or "sandwich" assay. In a typical immunometnc assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or earner, and a quantity of detectable labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid phase antibody, antigen, and labeled antibody
In a prefeπed embodiment, a "sandwich" type of ELISA can be used. Using this prefeπed method a pilot study is first implemented to determine the quantity of binding of each P400-fragment monoclonal antibody to microtiter wells. Once this is determined, aliquots (usually m 40 μl of TBS; pH 7.4) of the specific P400-fragment antibody are allowed to bind overnight to microtiter wells (Maxisorb C plate from Nunc) at 4oC. A series of blank wells not containing any P400-fragment specific monoclonal antibody are also utilized as controls. The next day, non-bound monoclonal antibody is shaken off the microtiter wells. All of the microtiter wells (including the blank wells) are then blocked by incubating for 2 hours with 300 μl of Tns-buffered saline containing 0.05% Tween-20 (TTBS) plus 2% bovine serum albumin, followed by 5 πnses with TTBS 200 μl of cerebrospinal fluid, blood, plasma, serum, unne, sputum, and/or stool and or any other type of biological sample is then diluted (to be determined empincally) in TTBS containing 2% bovine serum albumin and placed in wells (in tπphcate) containing bound P400 fragment antibody (or blank) and incubated for 2 hours at room temperature The wells are then washed 5 times with TTBS. A second biotinylated-monoclonal antibody against the same P400-denved fragment (but which is against a different epitope) is then added to each well (usually in 40 μl of TBS; pH 7.4) and allowed to bind for 2 hours at room temperature to any P400-fragment captured by the first antibody. Following incubation, the wells are washed 5 times with TTBS. Bound mateπals are then detected by incubating with 100 μl of peroxidase-avidm complex (1.250 dilution in TTBS with 0.1% BSA) for 1 hour on a rotary shaker. After 5 washes with TTBS, a substrate solution (100 μl, OPD-Sigma Fast from Sigma Chemical Co., St. Louis, MO, USA) is added and allowed to develop significant color (usually 8-10 minutes). The reaction is stopped with 50 μl of 4N sulfuπc acid and read on a standard spectrophotometer at 490 nm. This ELISA can be utilized to determine differences in specific P400 fragments (and/or AB- binding P400 fragments) in biological fluids which can serve as a diagnostic marker to follow the progression on a live patient dunng the progression of disease (i.e. monitonng of amyloid disease as an example). In addition, quantitative changes in P400 fragments can also serve as a prognostic indicator momtoπng how a live patient will respond to treatment which targets a given amyloid disease such as Alzheimer's disease. Such assays can be provided in a kit form
A competition assay may also be employed wherein antibodies specific to P400, P400 fragments and/or P400-denved peptides are attached to a solid support and labeled P400, P400 fragments and/or P400-deπved peptides and a sample deπved from a host are passed over the solid support and the amount of label detected attached to the solid support can be coπelated to the quantity of P400, P400 fragments and/or P400-denved peptides in the sample This standard technique is known to one skilled in the art
Another object of the present invention is to use P400, P400 fragments and/or P400-denved peptides, in conjunction with P400, P400 fragment and/or P400 denved peptide antibodies, in an ELISA assay to detect potential P400, P400 fragment and/or P400-denved peptide autoantibodies in human biological fluids. Such a diagnostic assay may be produced in a kit form In a prefeπed embodiment, peptides containing the sequences of P400, P400 fragments and/or P400-denved peptides, as well as polypeptides which have at least 70% similanty (preferably 70 % identity) and more preferably a 90% similanty (more preferably a 90% identity) to the polypeptides descnbed above, will be used to initially bind to microtiter wells in an ELISA plate A pilot study is first implemented to determine the quantity of binding of each P400 fragment polypeptide to microtiter wells Once this is determined, aliquots (usually l-2μg in 40 μl of TBS, pH 7 4) of specific P400 fragment polypeptides (as descnbed herein) are allowed to bind overnight to microtiter wells (Maxisorb C plate from Nunc) at 4°C All the microtiter wells (including blank wells without the P400 fragment polypeptides) are blocked by incubating for 2 hours with 300 μl of Tns-buffered saline (pH 7 4) with 0.05% Tween-20 (TTBS), containing 2% albumin This is followed by 5 nnses with TTBS The patients' biological fluids (i.e., cerebrospinal fluid, blood, plasma, serum, sputum, unne, and/or stool) are then utilized and 200 μl are diluted (to be determined empincally) with TTBS containing 2% bovine serum albumin, and placed in microtiter wells (in tnphcate) containing a specific P400 fragment polypeptide or blank wells (which do not contain peptide), and are incubated at 1.5 hours at room temperature Any autoantibodies present in the biological fluids against the P400 fragment will bind to the substrate bound P400 fragment polypeptide (or fragments thereof). The wells are then nnsed by washing 5 times with TTBS 100 μl of biotinylated polyclonal goat anti-human IgGs (Sigma Chemical company, St. Louis, MO, USA), diluted 1:500 in TTBS with 0.1% bovine serum albumin, is then ahquoted into each well. Bound matenals are detected by incubating with 100 μl of peroxidase-avidin complex (1.250 dilution in TTBS with 0.1% bovine serum albumin) for 1 hour on a rotary shaker Following 5 washes with TTBS, substrate solution (100 μl, OPD Sigma Fast from Sigma Chemical Company, St. Louis, MO, USA) is added and allowed to develop significant color (usually 8-10 minutes). The reaction is stopped with 50 μl of 4N sulfuric acid added to each well and read on a standard spectrophotometer at 490 nm. This assay system can be utilized to not only detect the presence of autoantibodies against P400 fragments in biological fluids, but also to monitor the progression of disease by following elevation or diminution of P400 fragment autoantibody levels. It is believed that patients demonstrating excessive P400 fragment formation, deposition, accumulation and/or persistence as may be observed in the amyloid diseases will also caπy autoantibodies against the P400 fragments in their biological fluids. Various ELISA assay systems, knowledgeable to those skilled in the art, can be used to accurately monitor the degree of P400 fragments in biological fluids as a potential diagnostic indicator and prognostic marker for patients during the progression of disease (i.e. monitoring of an amyloid disease for example). Such assays can be provided in a kit form. In addition, quantitative changes in P400 fragment autoantibody levels can also serve as a prognostic indicator monitoring how a live patient will respond to treatment which targets a given amyloid disease.
Other diagnostic methods utilizing the invention include diagnostic assays for measuring altered levels of P400, P400 fragments and/or P400-derived peptides in various tissues compared to normal control tissue samples. Assays used to detect levels of P400, P400 fragments and/or P400-derived peptides in a sample derived from a host are well-known to those skilled in the art and included radioimmunoassays, competitive-binding assays, Western blot analysis and preferably ELISA assays (as described above).
Yet another aspect of the present invention is to use the antibodies recognizing P400, P400 fragments and/or P400-derived peptides for labellings, for example, with a radionucleotide, for radioimaging or radioguided surgery, for in vivo diagnosis, and/or for in vitro diagnosis. In one prefeπed embodiment, radiolabelled peptides or antibodies made (by one skilled in the art) against P400, P400 fragments and/or P400-derived peptides may be used as minimally invasive techniques to locate P400, P400 fragments and/or P400-derived peptides, and concuπent amyloid deposits in a living patient. These same imaging techniques could then be used at regular intervals (i.e. every 6 months) to monitor the progression of the amyloid disease by following the specific levels of P400, P400 fragments and/or P400- derived peptides.
Yet another aspect of the present invention is to provide a method which can evaluate a compound's ability to alter (diminish or eliminate) the affinity of a given amyloid protein (as described herein) or amyloid precursor protein, to P400, P400 fragments and/or P400-derived peptides. By providing a method of identifying compounds which affect the binding of amyloid proteins, or amyloid precursor proteins to such P400 derived fragments, the present invention is also useful in identifying compounds which can prevent or impair such binding interaction. Thus, compounds can be identified which specifically affect an event linked with the amyloid formation, amyloid deposition, and/or amyloid persistence condition associated with Alzheimer's disease and other amyloid diseases as described herein.
According to one aspect of the invention, to identify for compounds which allow the interaction of amyloid proteins or precursor proteins to P400-derived fragments or P400 polypeptides, either amyloid or P400 fragments are immobilized, and the other of the two is maintained as a free entity. The free entity is contacted with the immobilized entity in the presence of a test compound for a period of time sufficient to allow binding of the free entity to the immobilized entity, after which the unbound free entity is removed. Using antibodies which recognize the free entity, or other means to detect the presence of bound components, the amount of free entity bound to immobilized entity can be measured. By performing this assay in the presence of a series of known concentrations of test compound and, as a control, the complete absence of test compound, the effectiveness of the test compound to allow binding of free entity to immobilized entity can be determined and a quantitative determination of the effect of the test compound on the affinity of free entity to immobilized entity can be made. By comparing the binding affinity of the amyloid-P400 fragment complex in the presence of a test compound to the binding affinity of the amyloid-P400 fragment complex in the absence of a test compound, the ability of the test compound to modulate the binding can be determined.
In the case in which the amyloid is immobilized, it is contacted with free P400 derived fragments or polypeptides, in the presence of a series of concentrations of test compound. As a control, immobilized amyloid is contacted with free P400-derived polypeptides, or fragments thereof in the absence of the test compound. Using a series of concentrations of P400-derived polypeptides, the dissociation constant (Kd) or other indicators of binding affinity of amyloid-P400 fragment binding can be determined. In the assay, after the P400-derived polypeptides or fragments thereof is placed in contact with the immobilized amyloid for a sufficient time to allow binding, the unbound P400 polypeptides are removed. Subsequently, the level of P400 fragment-amyloid binding can be observed. One method uses P400-derived fragment antibodies, as described in the invention, to detect the amount of specific P400 fragments bound to the amyloid or the amount of free P400 fragments remaining in solution. This information is used to determine first qualitatively whether or not the test compound can allow continued binding between P400- derived fragments and amyloid. Secondly, the data collected from assays performed using a series of test compounds at various concentrations, can be used to measure quantitatively the binding affinity of the P400 fragment-amyloid complex and thereby determine the effect of the test compound on the affinity between P400 fragments and amyloid. Using this information, compounds can be identified which do not modulate the binding of specific P400 fragments to amyloid and thereby allow the P400 fragments to reduce the amyloid formation, deposition, accumulation and/or persistence, and the subsequent development and persistence of amyloidosis.
Therefore a kit for practicing a method for identifying compounds useful which do not alter P400, P400 fragments and/or P400-derived peptides to an immobilized amyloid protein, said kit comprising a) a first container having amyloid protein immobilized upon the inner surface, b) a second container which contains P400, P400 fragments and/or P400-derived peptides dissolved in solution, c) a third container which contains antibodies specific for said P400, P400 fragments and/or P400-derived peptides, said antibodies dissolved in solution, and d) a fourth container which contains labeled antibodies specific for P400, P400 fragments and/or P400-derived peptides, said antibodies dissolved in solution.

Claims

We claim:
1. A pharmacological agent for binding to a beta-amyloid protein present in a biological fluid, wherein the pharmacological agent comprises a therapeutically effective amount of a component of the biological fluid
2. The pharmacological agent of claim 1 comprising a protein of molecular weight of approximately 350 to 450 kilodaltons (P400)
3 The pharmacological agent of claim 1 comprising P400, P400-deπved protein fragments, or P400-deπved polypeptides. 4 The pharmacological agent of claim 1 wherein the biological fluid is selected from a group of biological fluids consisting of blood, plasma, serum, cerebrospinal fluid, sputum, saliva, urine and stool. 5. The pharmacological agent of claim 1 wherein the biological fluid is of human origin.
6 A method of detection and/or quantification of P400, P400-denved protein fragments or P400-derιved polypeptides, m biological fluids wherein the method comprises the step of ligand blotting using biotmvlated-beta-amyloid protein
7 The method of claim 6 wherebv the method of detection and/or quantification further comprises the following steps a) use of SDS-PAGE to separate proteins present in biological fluids h ) transferring of said proteins to membranes used for ligand blotting, c) probing of transferred proteins using a beta-am vloid protein that ha^ been biotinylated d) detection of P400 P400-deπved protein fragments or P400- denved polypeptides using such ligandblottmg methods, ande) quantification of levels of P400, P400-deπveci protein fragments or P400-deπveα polypeptides. in a sample using scanning densitometrv or other detection methods 8 The method of claim 6 further comprising the use of antibodies generated against P400, P400-deπved protein fragments or P400-deπved polypeptides
9. The method of claim 8 further comprising ELISA based use of the antibodies for the detection of P400, P400-deπved protein fragments or P400-deπved polypeptides, in biological samples.
10. The ELISA method of claim 9 further comprising of the following steps: a) allowing a P400, P400-deπved protein fragment or P400-denved polypeptide monoclonal antibody to bind to microtiter wells for sufficient time to allow said binding, b) adding a quantity of biological fluid , c) adding to each well a second labeled monoclonal antibody against the same P400, P400-derιved protein fragments or P400-deπved polypeptides, but which is against a different epitope, to bind to any P400, P400- derived protein fragments or P400-deπved polypeptides captured by the first antibody, d) detection of bound materials by incubating with a substrate until a significant color reaction is allowed to develop
11 The method of claim 6 as applied to diagnose a disease or a susceptibility to a disease related to the levels of P400, P400-derιved protein fragments or P400-deπved polypeptides wherein elevated or diminished levels of P400, P400-deπved protein fragments or P400-deπved polypeptides will be indicative of the presence of a disease susceptibility to a disease, and progression of said disease
12. The method of claim 11 wherein said disease is selected from the group of disease consisting of Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage with amvloidosis of the Dutch type, and another disease that involves the deposition of a beta-amvloid protein
13 A method for the treatment of a patient having an identified clinical need to interfere with the pathological effects of amyloid, the method comprising administering to a patient a therapeutically effective amount of P400, P400-deπved protein fragments, or P400-derιved polypeptides.
PCT/US2000/006878 1999-03-15 2000-03-15 Therapeutic and diagnostic applications of p400: a newly discovered beta-amyloid binding protein present in human biological fluids WO2000055187A1 (en)

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