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WO1999041279A2 - Procedes et compositions comprenant l'utilisation d'un peptide b-amyloide bloque - Google Patents

Procedes et compositions comprenant l'utilisation d'un peptide b-amyloide bloque Download PDF

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WO1999041279A2
WO1999041279A2 PCT/US1999/003231 US9903231W WO9941279A2 WO 1999041279 A2 WO1999041279 A2 WO 1999041279A2 US 9903231 W US9903231 W US 9903231W WO 9941279 A2 WO9941279 A2 WO 9941279A2
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peptide
amyloid
blocked
amyloid peptide
terminus
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PCT/US1999/003231
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WO1999041279A3 (fr
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David G. Lynn
Stephen C. Meredith
Timothy S. Burkoth
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Arch Development Corporation
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Publication of WO1999041279A3 publication Critical patent/WO1999041279A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to fibrillogenesis. More particularly, it concerns the use of blocked A ⁇ compositions for the inhibition of fibril formation. Particular aspects of the present invention describe the methods and compositions of inhibition of plaque formation in, for example, Alzheimer's Disease.
  • Amyloidoses are characterized by the tissue deposition of proteins, which are believed to have a ⁇ -sheet conformation in common despite the lack of amino acid sequence homology (Carrel and Lomas, 1997; Horwich and Weissman et al, 1997). Amyloidosis is defined by the deposition of amyloid into tissues of diseases typified by Alzheimer's Disease (AD) and Down's Syndrome. Systemic amyloidoses are characterized by amyloid deposition throughout the viscera. The amyloid of animals is a complex material composed partly of protein fibrils. This protein varies from disease to disease. Among these proteins is A ⁇ , involved in the pathological progression of Alzheimer's Disease (Glenner and Wong, 1984).
  • AD is now the fourth-largest killer of adults 65 and older. This disease impacts one of every three families in the United States (Gonzalez-Lima, 1987), and affects over 13 million people world-wide. As the population trends lead to an increase in the number of older people, this figure will only increase. Thus, it is an important goal of medical science to identify methods of preventing, alleviating or abrogating this disease.
  • the histopathology of AD is characterized by the presence of extracellular plaques and largely intracellular tangles within the cerebral cortex, hippocampus and the diffuse subcortical projection system.
  • Plaques are made up of a rim of dystrophic neurites surrounding a core of amyloid ⁇ -protein formed from abnormally processed amyloid precursor protein (APP).
  • APP is a membrane spanning found in all nerve cells. Tangles occur from abnormally phosphorylated tau protein. Duplication of the APP gene are found in Trisomy 21 (Down's Syndrome; DS) and leads to an Alzheimer's type pathology in the cerebral cortex of individuals with DS (Rosser, 1993).
  • a ⁇ is a 40-43 amino acid proteolytic fragment of the transmembrane APP (Kang, 1987; Goldgaber, 1987; Tanzi, 1987). This protein rapidly and irreversibly associates into insoluble fibrils (Kirschner et al, 1987; Hilbich, 1991; Hilbich et al, 1991 ; Burdick et al, 1992; Castano et al, 1986).
  • the mechanisms of this aggregation, and the structure of the final fibrillar products are not known in detail, however, in this form, the peptides are believed to be neurotoxic. Although these peptides are found in normal brains, they are at higher concentrations in brains from patients with Alzheimer's disease, and these insoluble fibrils are believed to be pathogenic as they form insoluble plaques and tangles in nerves tissues.
  • the present invention provides, in one aspect, a method of inhibiting amyloid fibrillogenesis comprising contacting tissue with a composition comprising an amyloidogenic peptide that has been blocked.
  • the peptide is blocked at one or more sites selected from the group consisting of the C-terminus, the N-terminus and a side chain of the peptide.
  • the peptide is blocked at the C-terminus of the peptide.
  • peptide is blocked at the N-terminus of the peptide.
  • the peptide is blocked at a side chain of the peptide.
  • the peptide is blocked using a polymer.
  • the polymer is polyethylene glycol.
  • the amyloidogenic peptide may be selected from the group consisting of cystatin C, AApoA-I, AApoA-II, AScr or PrP- 27, A ⁇ peptide, amyloid A, amyloid kappa L-chain, amyloid lambda L-chain, A ⁇ 2M, ATTR, AIAPP, amylin atrial naturetic factor, procalcitonin and gelsolin.
  • the tissue may be selected from the group consisting of pancreas, brain, muscle and heart.
  • the tissue is from a subject having a pathological state selected from the group consisting of Alzheimer's Disease, Down's Syndrome, Dutch-Type Hereditary Cerebral Hemorrhage Amyloidosis, Reactive (Secondary) Amyloidosis, Familial Mediterranean Fever, Familial Amyloid Nephropathy With Urticaria And Deafness (Muckle- Wells Syndrome), Idiopathic (Primary) Myeloma, Macroglobulinemia-Associated Myeloma, Familial Amyloid Polyneuropathy (Portuguese, Japanese, Swedish), Familial Amyloid Cardiomyopathy (Danish), Isolated Cardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes, Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma Of The Thyroid, Familial Amyloidosis (Finnish), Hereditary Cerebral Hemorrhage With Amyloidosis (Icelandic),
  • the subject may be selected from the group consisting of human, sheep and cow. In preferred embodiments, the subject is a human.
  • Also contemplated by the present invention is a method of inhibiting amyloid fibrillogenesis comprising contacting tissue with a composition comprising a ⁇ - amyloid peptide that has been blocked at an end terminal or a side chain.
  • the peptide is blocked using a polymer.
  • the polymer may be polyethylene glycol.
  • the A ⁇ peptide may be blocked at one or more sites selected from the group consisting of the C-terminus, the N-terminus and a side chain of the peptide.
  • the A ⁇ peptide is a full length A ⁇ peptide.
  • the A ⁇ peptide comprises a partial sequence of a full length A ⁇ peptide.
  • the A ⁇ peptide may comprise a peptide having amino acid residues 10 through to 35 A ⁇ peptide.
  • the A ⁇ peptide has the sequence of SEQ ID NO: 1.
  • the A ⁇ peptide has the sequence of SEQ ID NO:2.
  • the polyethylene glycol has a molecular weight of between about 1000 and 6000 Da. In particularly preferred embodiments, the polyethylene glycol has a molecular weight of about 3000 Da.
  • a ⁇ peptide that is blocked by conjugation.
  • the A ⁇ peptide is blocked at one or more sites selected from the group consisting of the C-terminus, the N-terminus and a side chain of the peptide.
  • the A ⁇ peptide is blocked independently at the C-terminus, the N-terminus or a side chain of the peptide.
  • the A ⁇ peptide is a full length A ⁇ peptide.
  • the A ⁇ peptide comprises a peptide having amino acid residues 10 through to 35 A ⁇ peptide.
  • the peptide is blocked by conjugation to a polymer.
  • the polymer is polyethylene glycol.
  • the polyethylene glycol has a molecular weight of about 3000.
  • the present invention also contemplates a method of inhibiting amyloid plaque formation in a subject comprising administering a pharmaceutical composition comprising a A ⁇ peptide blocked by conjugation to a second compound and a pharmaceutically acceptable buffer, solvent or diluent.
  • the second compound is a polymer.
  • the polymer is polyethylene glycol.
  • the A ⁇ peptide is blocked at one or more sites selected from the group consisting of the C-terminus, the N-terminus and a side chain of the peptide.
  • the administering is effected by regional delivery of the pharmaceutical composition.
  • the administering comprises delivering the pharmaceutical composition endoscopically, intratracheally, percutaneously, or subcutaneously.
  • the subject is a mammal. In still further embodiments, the subject is a human. In certain embodiments, the subject suffers from amyloidosis. In particularly preferred embodiments, the subject has Alzheimer's disease. In other preferred embodiments, the subject has Down's Syndrome.
  • a pharmaceutical composition comprising a A ⁇ peptide blocked by conjugation to a second compound and a pharmaceutically acceptable buffer, solvent or diluent.
  • the second compound may be a polymer and the polymer may be polyethylene glycol.
  • the pharmaceutical composition is defined by having a A ⁇ peptide that is blocked at one or more sites selected from the group consisting of the C-terminus, the N-terminus and a side chain of the peptide.
  • FIG. 1 Unlabeled peptides were cross-linked by tTg, then analyzed on
  • Tris-Tricine SDS-PAGE Tris-Tricine SDS-PAGE.
  • Cross-linked peptide species ranged from dimeric to hexameric, depending upon the amount of tTg included. In some studies, an extremely faint band consistent with a heptameric structure was seen; however, higher order peptide oligomers could not be observed.
  • FIG. 2A and FIG. 2B FIG. 2A: an antiparallel pair of ⁇ -strands.
  • FIG. 2A an antiparallel pair of ⁇ -strands.
  • FIG. 3A, FIG. 3B and FIG. 3C Measurable double quantum relaxation exists at both position 15 (Q) and position 16 (K) in the fibrils.
  • FIG. 3 A and FIG. 3B are spectra taken of 50 mg 1- C-K-A ⁇ fibrils mixed with 10 mg unlabeled HMB.
  • FIG. 3 A Unfiltered CP/MAS spectrum.
  • FIG. 3B Double quantum filtered spectrum, showing a strong signal for the labeled peptide, but no signal for the unlabeled control, HMB.
  • FIG. 3A, FIG. 3B and FIG. 3C Measurable double quantum relaxation exists at both position 15 (Q) and position 16 (K) in the fibrils.
  • FIG. 3 A and FIG. 3B are spectra taken of 50 mg 1- C-K-A ⁇ fibrils mixed with 10 mg unlabeled HMB.
  • FIG. 3 A Unfiltered CP/MAS spectrum.
  • FIG. 3B Double quantum filtered spectrum, showing a strong signal for the labeled peptide,
  • 3C Double quantum efficiency was quantified for l- 13 C-Q(15)-A ⁇ , l- 13 C-K(16)-A ⁇ , the HMB controls, and for 13 C-labeled standards, malonic and succinic acid. Comparison of the labeled A ⁇ results to the standards predicts an interstrand contact with a distance of 5 A ( ⁇ 1 A) for the l- I3 C-Q(15)-A ⁇ and l- 13 C-K(16)-A ⁇ .
  • FIG. 4 Inter-strand DRAWS data fits 5 A, 3-spin model. The DRAWS study was run for a series of mixing times (0 to 22 ms).
  • FIG. 5 Proposed model of A ⁇ fibrils. A six-stranded parallel ⁇ -barrel,
  • FIG. 6A Preparation of C-PEG-A ⁇ (10-35). Schematic representation of the C-terminal PEGylation of A ⁇ (10-35).
  • FIG. 7A Circular Dichroism Spectroscopy.
  • FIG. 7B Circular Dichroism Spectroscopy: Circular dichroic (CD) spectra show [ ⁇ ] 2 ⁇ as functions of pH concentration.
  • FIG. 7C Circular Dichroism Spectroscopy: Circular dichroic (CD) spectra show [ ⁇ ] 217 as functions of peptide concentration.
  • FIG. 8A, FIG. 8B and FIG. 8C The amino acid sequence of A ⁇ can be separated into distinct domains: a hydrophilic N-terminus (aa 1-16), a central hydrophobic region (aa 17-21) and a long hydrophobic C-terminus (aa 29-43). These domains are shown to be conserved in A ⁇ (10 . 35) and A ⁇ -PEG.
  • FIG. 8B Summary of the inter peptide 13 C carbonyl contracts observed for A ⁇ (10 . 35) by solid state NMR. Inter peptide distances were measured using DRAWS at positions V 12 , Q ⁇ 5 -V, 8 , V 24 , G 26 , G 29 , G 33 , and L 34 .
  • FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E and FIG. 9F Electron Micrographs of A ⁇ (10 . 35) titrated with A ⁇ -PEG at pH 5.7.
  • FIG. 9E. 1 :4 A ⁇ -PEG, and FIG. 9F 100% A ⁇ -PEG.
  • FIG. 10A, FIG. 10B and FIG IOC SANS data fit to a modified Guinier plot for a rod-like form of SANS data of A ⁇ (10 . 35) and A ⁇ -PEG in 99% D 2 O buffer. The average radii were calculated to be 46 ( ⁇ 5)A and 65( ⁇ 1)A, respectively.
  • FIG. 10B SANS data fit to a modified Guinier analysis for a rod-like form of A ⁇ (]0 _ 35) and A ⁇ -PEG in 12% D 2 O buffer that eliminates the coherent scattering from PEG. The radii were calculated to be 46 ( ⁇ 5)A and 47( ⁇ 5)A, respectively.
  • FIG. IOC Schematic representation of the assembly of peptide and PEG in the fibril.
  • FIG. 11 A, FIG. 11B, FIG. 11C and FIG. 11D Three-dimensional structure of A ⁇ (10 . 35) .
  • FIG. 11 A Electron Micrograph of a typical paired helical fiber observed for A ⁇ (10 . 35) at pH 7.4.
  • FIG. 1 IB Enhanced EM showing the paired assembly.
  • FIG. l lC Structure of the paired fibrils incorporating the 1100 A repeat composed of 220 ⁇ -strands, each with a 1.6° offset to define the twist of the ⁇ -helices. Orienting the peptides in parallel and in register would place the hydrophobic C-terminal amino acids entirely along one edge of the ⁇ -sheet driving their association.
  • FIG. 11D Three-dimensional structure of A ⁇ (10 . 35) .
  • FIG. 11 A Electron Micrograph of a typical paired helical fiber observed for A ⁇ (10 . 35) at pH 7.4.
  • FIG. 1 IB Enhanced EM showing the paired assembly.
  • FIG. l lC
  • AD Alzheimer's disease
  • AD the third leading cause of death in the elderly in the US (Hardy and Allsop, 1991)
  • AD is manifested neuropathologically by dense, extracellular, proteinaceous plaques, composed mainly of the A ⁇ peptide (Glenner and Wong, 1984).
  • the number of plaques correlates directly with the severity of the disease (Selkoe et al, 1994).
  • the inventors have used DRAWS C solid state nuclear magnetic resonance (NMR) studies (Gregory et al. , 1997) to measure precise distances between individual A ⁇ peptides in the plaques.
  • NMR DRAWS C solid state nuclear magnetic resonance
  • the inventors developed a series of peptides modified by derivatization or blocking at several loci on the beta amyloid peptides or fragments of these peptides, including the termini and side chain derivatives.
  • the inventors employed polyethylene glycol to block or modify the side chains and the C- and N-termini of amyloid peptides of varying length.
  • these PEG-peptides showed enhanced solubility.
  • PEG-beta amyloid peptides showed inhibited fibrillogenesis, suggesting that PEGylated peptides or analogues thereof might be used as inhibitors of fibrillogenesis in vivo.
  • the peptides have most of the salient structural features of the nonmodified peptides and therefore can be used as structural probes.
  • C- terminal modified PEG- A ⁇ (10-35) shares all known structural features of the nonmodified peptide, yet the former is sufficiently soluble for detail two-dimensional NMR studies that can elucidate the entire structure of the peptide.
  • inventors report the synthesis and characterization of plaque-competent A ⁇ peptides, derivatized at the C-terminus, N- terminus or containing sides chain modification with polyethyleneglycol (PEG).
  • PEG polyethyleneglycol
  • These modifications while preserving the salient structural features of the non- modified peptide, markedly enhances solubility of A ⁇ , which can allow for obtaining detailed structural information.
  • the formation of fibrils by A ⁇ is fully reversible through changes in pH and protein concentration, again in contrast with the nonmodified peptide.
  • the present invention provides a strategy for dissecting the steps in the process of amyloid fibrillogenesis, and may be exploited in the development of site-specific inhibitors of this process.
  • Amyloidosis refers to a pathological condition characterized by the presence of amyloid.
  • Amyloid is a generic term referring to a group of diverse but specific extracellular protein deposits which are seen in a number of different diseases. Though diverse in their occurrence, all amyloid deposits have common morphologic properties, stain with specific dyes (e.g., Congo red), and have a characteristic red- green birefringent appearance in polarized light after staining. They also share common ultrastructural features and common x-ray diffraction and infrared spectra.
  • specific dyes e.g., Congo red
  • Amyloidosis can be classified clinically as primary, secondary, familial and/or isolated. Primary amyloidosis appears de novo without any preceding disorder. Secondary amyloidosis is that form which appears as a complication of a previously existing disorder. Familial amyloidosis is a genetically inherited form found in particular geographic populations. Isolated forms of amyloidosis are those that tend to involve a single organ system. Different amyloids are also characterized by the type of protein present in the deposit.
  • neurodegenerative diseases such as scrapie, bovine spongiform encephalitis, Creutzfeldt- Jakob disease and the like are characterized by the appearance and accumulation of a protease-resistant form of a prion protein (Hope and Baybutt, 1991 ; Prusiner, 1991 ; Chesbro, 1991) in the central nervous system.
  • Alzheimer's disease is characterized by congophilic angiopathy, neuritic plaques and neurofibrillary tangles, all of which have the characteristics of amyloids.
  • the plaque and blood vessel amyloid is formed by the beta protein.
  • Other systemic diseases such as adult-onset diabetes, complications of long- term hemodialysis and sequelae of long-standing inflammation or plasma cell dyscrasias are characterized by the accumulation of amyloids systemically. In each of these cases, a different amyloidogenic protein is involved in amyloid deposition.
  • the present invention pertains to methods and compositions useful for treating amyloidosis.
  • the methods of the invention involve administering to a subject a therapeutic compound which inhibits amyloid deposition. More particularly, the present invention involves the use of blocked amyloid peptides so that the aggregation of these peptides into plaques is inhibited.
  • “Inhibition of amyloid deposition” is intended to encompass prevention of amyloid formation, inhibition of further amyloid deposition in a subject with ongoing amyloidosis and reduction of amyloid deposits in a subject with ongoing amyloidosis.
  • Inhibition of amyloid deposition may be determined relative to an untreated subject or relative to the treated subject prior to treatment.
  • the present invention provides for the inhibition of fibril formation by ⁇ -amyloid peptide.
  • Amyloid deposition or fibrillogenesis is inhibited by inhibiting an interaction between monomers of the amyloidogenic protein. In more defined aspects this is achieved by blocking the end termini or side chains of the amyloidogenic protein so that the monomers can not interact. Such "blocking" may be achieved using, for example, PEG.
  • the methods of the present invention in a broad aspect are useful for treating amyloidosis associated with any disease in which amyloid deposition occurs. Clinically, amyloidosis can be primary, secondary, familial or isolated.
  • Amyloids have been categorized by the type of amyloidogenic protein contained within the amyloid. Exemplary amyloidogenic proteins that may be modified by the methods of the present invention include those listed in Table 1. The pathological states associated with aberrant expression or mutation in these peptides are also listed.
  • peptides include fibrinogen-associated amyloid and lysozyme-associated amyloid.
  • the present invention is directed to the modification of ⁇ -amyloid peptides such that the formation of insoluble fibrils is inhibited.
  • the compositions and methods described herein will thus be useful in the inhibition, abrogation or alleviation or the deleterious effects of plaque formation in Alzheimer's disease, Down's syndrome, and Dutch hereditary cerebral hemorrhage amyloidosis.
  • amyloidosis including but not limited to atrial and brain natriuretic peptide (Pucci et al, 1991; Kaye et al, 1986; Benvenga and Facchiano 1995); gelsolin (Steiner et al, 1995; Kivela et al, 1994; Maury et al, 1994) Cystatin (Abrahamson, 1996; Jacobson and Buxbaum, 1991; Bobek et al, 1991 Abrahamson et al, 1990) Apolipoprotein A-I (Johnson et al, 1992 Soutar et al, 1992; Rader et al, 1992) and Apolipoprotein A-II (Higuchi et al, 1991).
  • amyloid kappa L-chain (Genbank:P04430; Idiopathic (Primary) or P01617; L33854) or Macroglobulinemia-Associated amyloid lambda L-chain Myeloma
  • Systemic Senile Amyloidosis AIAPP (Genbank X68830; PI 0997) or Adult Onset Diabetes amylin (Genbank: M27503) Insulinoma atrial naturetic factor Isolated Atrial Amyloid procalcitonin (Genbank P06881) Medullary Carcinoma Of The Thyroid gelsolin (Genbank: U28044; M36927; Familial Amyloidosis (Finnish) X75629; X75630) cystatin C (Genbank: X52255; M58167, Hereditary Cerebral Hemorrhage With M58169) Amyloidosis (Icelandic)
  • AApoA-I (Genbank P02647; J00098) Familial Amyloidotic Polyneuropathy
  • amyloid angiopathy amyloid angiopathy characteristic of AD and certain the other disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of about 39-43 amino acids designated the ⁇ -amyloid peptide (A ⁇ , or sometimes ⁇ -AP, A ⁇ P or ⁇ /A4. ⁇ AP).
  • a ⁇ was first purified and a partial amino acid sequence reported in Glenner and Wong (1984). The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829.
  • Patent 4,912,206 describes a cDNA clone for the Alzheimer's Amyloid peptide and the open reading frame of the sequenced clone as well as mapping the gene, which is highly conserved, to human chromosome 21. Both of these patents have been incorporated herein by reference.
  • a ⁇ is a small fragment of a much larger precursor protein, referred to as the ⁇ -amyloid precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans.
  • APP ⁇ -amyloid precursor protein
  • Knowledge of the structure of the gene encoding APP has demonstrated that A ⁇ arises as a peptide fragment that is cleaved from APP by as- yet-unknown enzymes (proteases).
  • protes enzymes
  • the precise biochemical mechanism by which the A ⁇ fragment is cleaved from APP and subsequently deposited as amyloid plaques in the cerebral tissue and in the walls of cerebral and meningeal blood vessels is currently unknown.
  • Several lines of evidence indicate that progressive cerebral deposition of A ⁇ plays a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades (for review, see Selkoe, 1991).
  • a ⁇ is an approximately 4.2 kD protein which, in the brains of AD, Down's Syndrome, HCHWA-D and some normal aged subjects, forms the subunit of the amyloid filaments comprising the senile (amyloid) plaques and the amyloid deposits in small cerebral and meningeal blood vessels (amyloid angiopathy).
  • a ⁇ can occur in a filamentous polymeric form (in this form, it exhibits the Congo-red and thioflavin-S dye-binding characteristics of amyloid described in connection therewith).
  • a ⁇ can also occur in a non-filamentous form ("preamyloid” or “amorphous” or “diffuse” deposits) in tissue, in which form no detectable birefringent staining by Congo red occurs.
  • U.S. Pat. No. 4,666,829 describes the insoluble form of A ⁇ .
  • a ⁇ is an approximately 39-43 amino acid fragment of a large membrane-spanning glycoprotein, referred to as the ⁇ -amyloid precursor protein (APP), encoded by a gene on the long arm of human chromosome 21.
  • APP ⁇ -amyloid precursor protein
  • a ⁇ is further characterized by its relative mobility in SDS-polyacrylamide gel electrophoresis or in high performance liquid chromatography (HPLC). Its 43 -amino acid sequence is given in SEQ ID NO: 1 or a sequence that is substantially homologous thereto.
  • the A ⁇ peptide is itself toxic to cultured neurons (Yankner et al, 1989; Yankner et al, 1990; Yankner, et al., 1990; Roher et al., 1991).
  • Several animal models have also been produced in which A ⁇ is over-produced, though the results of these models have been ambiguous (Yankner, 1996).
  • a ⁇ fibrils appears to proceed through one or more soluble, aggregated oligomeric intermediates (Jarrett et al, 1994; Lomakin et al., 1996; Jarrett and Lansbury, 1993).
  • Water soluble A ⁇ oligomers have been isolated from the media of cultured neuronal cell lines expressing endogenous or transfected ⁇ APP (Seubert et al., 1992; Shoji et al, 1992). Similar or identical soluble oligomers have been isolated from CSF of Alzheimer's Disease patients and normal subjects (Haass et al., 1992; Busciglio et al, 1993; Soto and Castano, 1996).
  • the CD curve of the oligomer is not the classic ⁇ sheet curve observed by Greenfield and Fasman (Greenfield and Fasman, 1969), i.e. with a minimum of « -18,000 deg-cm /dmol at 217-218 nm.
  • the secondary structure (as defined by CD) can be altered by changing solvent conditions e.g., the peptide is a helical when dissolved in 50% (v/v) trifluoroethanol (Soto et al, 1995).
  • a ⁇ was neurotoxic only under solvent conditions in which the ⁇ sheet structure is present (Buchet et al, 1996; Sato et al, 1995; Li et al., Simmons et al, 1994).
  • a characteristic finding of A ⁇ in common with all other amyloid-forming proteins and is in fact, the amyloid-defining finding, is its ability to bind Congo Red dye which displays green birefringence (Puchtler et al, 1962; Ashburn et al, 1996).
  • a ⁇ also binds thioflavin S, in common with other amyloids.
  • a mainstay of aggregation studies is the use of turbidimetry to measure solubility changes, and quasielectric light scattering to monitor aggregation in real time.
  • CD spectroscopy has been used to help define the ⁇ sheet-like character of the aggregates.
  • X-ray diffraction studies have also demonstrated a ⁇ -stranded structure, but have thus far failed to yield detailed structural information (Kirschner et al,
  • FTIR has the advantage of being able to study insoluble aggregates, and has also shown ⁇ sheet-like structure (Hilbich et al, 1991 ; Fraser et al, 1991).
  • a partial structural elucidation of the A ⁇ (amino acids 1-28) in DMSO has also been reported.
  • This peptide was found to have ⁇ helical character; this is in contrast to the inventors' data on A ⁇ (amino acids 10-35) in DMSO which is highly structured and, importantly, has NOE signatures typical of ⁇ strand.
  • this peptide was chosen for further studies presented herein. Also when dissolved in water at pH 2.1, some of the peptide precipitated, but the remainder stayed in solution indefinitely, i.e. for the duration of the NMR studies or longer. NMR data was obtained using a 750 mHz instrument; it showed essentially a ⁇ strand-like structure and some NOEs suggestive of a central turn. Because of the limited solubility of this peptide, a full structure determination was not possible. Lansbury et al. took a solid state NMR approach towards a C-terminal nonapeptide of A ⁇ and concluded that it contained a ⁇ turn (Lansbury et al., 1995). The inventors have for the first time, definitively characterized the molecular arrangement of A ⁇ peptide. These results are discussed in relation to plaque formation in AD.
  • Alzheimer's amyloid plaques consist of a highly ordered, fibrillar structure which can be replicated in vitro using synthetic peptides.
  • the basic secondary structure of these fibrils is considered to be a ⁇ -strand; however, the precise arrangement of the individual peptides in these fibrils has eluded researchers, primarily because the insoluble nature of these fibrils renders them unsuitable for solution NMR or x-ray crystallographic techniques.
  • the inventors have developed a generalizable approach, using solid state NMR and specifically labeled synthetic peptides, by which determination of the specific structure of the peptide in its plaque-conformation is possible. Jarrett et al, (1994) have proposed the following schema to describe the process of amyloid fibrillogenesis
  • a first step in addressing some of these questions is to define some of these forms structurally.
  • the determination of a form does not necessarily mean the determination of the unique form: there may be numerous pathways and structures that lead to fibril formation.
  • the present invention used the truncated form of A ⁇ described by Lee et al, i.e. A ⁇ (10-35), since this peptide was shown to add to bona fide amyloid plaques from Alzheimer's Disease brains.
  • the inventors' goal was to circumvent the blocks and to determine the structures of some of the forms of A ⁇ (10-35) in the above pathway.
  • the inventors have identified the molecular arrangement of A ⁇ in fibrils using crosslinking by tissue transglutaminases and by NMR studies. The results of these investigations are described herein below and in the Examples.
  • Tissue transglutaminases are able to crosslink full length A ⁇ at a specific site:
  • transglutaminase links A ⁇ amino acid side chains that are proximate in space. This was demonstrated by the fact that of the crosslink is exclusively between Glnl5 and Lysl6, never between Gin 15 and Lys28. Further, the secondary structure most compatible with this crosslink is a ⁇ -stranded structure. This is demonstrated by the fact that Glnl5 and Lysl6 are not crosslinked within a single chain, even though the spacing for such a crosslink would be possible in an ⁇ helical, ⁇ -turn or random coil conformation.
  • the fibril may be composed of units of 6 A ⁇ chains, or some multiple of 6 A ⁇ chains.
  • an interdigitating fibril such as collagen or hemoglobin S, forms a continuous array of higher oligomers upon crosslinking (Adachi et al, 1983; Mirchev and Ferrone, 1997; Rossler et al, 1995; Reiser et al, 1992; Eyre et al, 1984; Bezrukov, 1979). It will be of interest to know the structure of the hexamer, and to test which of the crosslinked aggregates are competent to form fibrils, as judged by EM or binding of dyes like Congo Red or Thioflavin S.
  • amyloid fibril is composed of units of 6n chains of A ⁇ , then the monomer, dimer, trimer, tetramer and hexamer could form fibrils, but the pentamer (not an even divisor of 6) should not.
  • the inventors examined A ⁇ ( 10-35) in the fibrillar state by solid state NMR spectroscopy as described herein below.
  • the DRAWS (Dipolar Recoupling with Windowless Sequence) pulse sequence was devised and developed, respectively, by David Gregory (Gregory et al., 1996; Gregory et al, 1997) and Robert Botto. It serves to reintroduce dipolar coupling between two nuclei, and to suppress chemical shift anisotropy (CSA).
  • CSA chemical shift anisotropy
  • a 26-amino acid peptide, comprising residues 10-35 of the A ⁇ peptide was synthesized (SEQ ID NO:2). This peptide was chosen because it has been demonstrated that while it is more soluble, thus much more amenable to laboratory studies, than the native peptide, it still retains the ability to add to authentic plaques isolated from AD patients (Lee et al, 1995). Furthermore, like the native peptide (Dudek and Johnson, 1994), the inventors found that this peptide could be specifically cross linked by the enzyme tissue tranglutaminase (tTg) at the positions of Lys- 15 and Gin- 16 of adjacent peptides.
  • tissue tranglutaminase tissue tranglutaminase
  • the inventors have found a specific, repeating alignment of the ⁇ -strands in the AD amyloid fibril.
  • the single-label approach employed here greatly simplifies quantum mechanical analysis of the data and allows for accurate solid state NMR studies on repeating, polymeric structures such as the A ⁇ fibril, and moves the use of solid state NMR of proteins away from theoretical uses in highly specialized laboratories into an area where it can be used as a general technique for anyone interested in structural biology.
  • the inventors have determined the arrangement of a domain of the fibril in the context of the entire fibril. In this manner, it is now possible to continue through the sequence and elucidate the arrangement of the entire structure, thus breaking through one of the major developments of Alzheimer's disease.
  • fibrillogenesis is the process by which a soluble biopolymer (most commonly a protein) forms an insoluble aggregate.
  • the aggregate is usually composed of linear, interwound strands, with crosslinks between the strands.
  • Collagen fibrillogenesis Collagen is secreted from fibroblasts, osteoblasts and other cells as a precursor protein called procollagen. Although collagen, like all proteins, is synthesized in a cell, it is made for the production of extracellular matrix; hence, it is secreted from cells and undergoes fibrillogenesis in the extracellular compartment. Most types of procollagen contain three polypeptide chains called pro- ⁇ chains. Procollagen undergoes many post-translational modifications in the cell in which it is synthesized, and these include oxidative deamination of select lysine side chains to yield aldehyde functions ("allysines").
  • the procollagen undergoes a series of limited proteolytic steps by which the globular N- and C-terminal domains are removed. This leaves a triple helical tropocollagen molecule which is now able to associate into microfibrils through non-covalent interactions. Within the microfibril, covalent bonds form between select lysine side chains and the allysyl aldehyde functions. The collagen microfibrils then undergo further levels of aggregation into fibrils, composed of multiple, linearly and laterally associated microfibrils; and then into large fibers which make up the strands of tendons, the bone matrix, etc. The degree of lateral self-association and covalent sidechain-sidechain crosslinking renders the collagen fibrils and fibers insoluble and relatively resistant to proteolytic enzymes.
  • Fibrin deposition Fibrinogen is soluble plasma protein involved in blood coagulation. During the formation of a thrombus or blood clot, proteolytic enzymes are activated in the plasma which lead to the activation of thrombin (from prothrombin). Thrombin makes selective proteolytic cleavages in fibrinogen.
  • Fibrinogen is a large rod-shaped protein composed of 6 polypeptide chains ( ⁇ 2 ⁇ 2 ⁇ 2). The rod-shape of fibrin can be further defined as having three smaller globular domains connected in series by linear connecting domains. Thus, this shape, a "double dumbbell", has a directionality, i.e. a "head” and a "tail”.
  • Thrombin cleaves the ⁇ chains and the ⁇ chains in one position each, to liberate two small peptides called fibrinopeptides A and B, respectively.
  • fibrin self-associates into large, insoluble aggregates, called fibrin polymers or fibrin fibrils.
  • the self-association occurs in two dimensions: linear and lateral.
  • the linear self-association makes head-to-tail contacts between adjacent fibrin molecules. These linear aggregates can also associate laterally to make broad fibrils.
  • linear aggregation yields a long, highly asymmetrical, and hence highly viscous solution, but lateral self-association renders the aggregate insoluble.
  • fibrinogen is synthesized in the liver, circulates as a plasma protein (i.e. it is extracellular), and then its conversion to fibrin and subsequent steps of fibrillogenesis all occur in the extracellular compartment.
  • Fibers of Hemoglobin S Sickle cell anemia is a mutation of one amino acid hemoglobin: the 6th amino acid in the two ⁇ chains is mutated from Glu to Val. Under low oxygen conditions, Hemoglobin S undergoes a conformational change to a fiber-forming state. The fibers form through self-association of the ⁇ chains containing the solvent-exposed hydrophobic Val6 residue. As in the previous two examples, aggregation can occur linearly or laterally. As in the former examples, too, the lateral association yields large insoluble fibrillar aggregates, sometimes called "tactoids". These tactoids are not only insoluble, but rigid and therefore distort and disrupt red blood cells.
  • Amyloid fibrillogenesis The steps of amyloid fibrillogenesis are still being elucidated.
  • a ⁇ is a series of peptide, 39-43 amino acids in length, derived from a normal transmembrane protein, the A ⁇ precursor protein ( ⁇ -APP).
  • a series of proteases the identity of which is not known, can cleave ⁇ -APP to yield A ⁇ .
  • a ⁇ is found in the cerebrospinal fluid, which is extracellular. In this compartment, A ⁇ can self-associate to form amyloid fibrils. There are other pathways by which amyloid fibrils might form as well.
  • a ⁇ can bind to an extracellular protein found in the cerebrospinal fluid (and also in the blood) called apolipoprotein E.
  • apolipoprotein E an extracellular protein found in the cerebrospinal fluid (and also in the blood) called apolipoprotein E.
  • the receptor proteins for apo E include the LDL receptor, the LDL receptor-related protein (LRP), and GP330, among others.
  • LRP LDL receptor-related protein
  • GP330 GP330
  • the main site of amyloid fibrillogenesis is extracellular, and the fibrils per se, whether complexed to apolipoprotein E or not, are internalized by cells and then become neurotoxic.
  • the latter hypothesis puts the site of fibrillogenesis as the extracellular compartment.
  • a ⁇ itself is found in the cerebrospinal fluid (i.e. extracellularly) as a free peptide or extremely limited solubility; and that this peptide rapidly self-associates into insoluble fibrils.
  • the aggregation occurs in two dimensions: linearly and laterally.
  • the inventors studies work on PEG-A ⁇ (10-35) presented herein demonstrate that the linear association generates long, viscous aggregates that are soluble, but the lateral association renders these aggregates insoluble.
  • the present invention provides compositions of soluble peptide derivatives of amyloidogenic peptides.
  • the present invention shows that modification of the amyloidogenic peptide to render the side chains and end termini of the peptide unavailable for polymerization results in the generation of modified peptides that act as inhibitors of amyloidogenesis.
  • N-terminal of the A ⁇ peptide were involved in fibril formation.
  • a test of this model, and a potentially expedient way to obtain detailed structural data would be to derivatize the termini of A ⁇ in such a way that linear aggregation is blocked by steric hindrance. This was accomplished through use of polyethylene glycol derivatization of N- or C-termini of A ⁇ (10-35).
  • the inventors have developed a series of peptides modified by PEG derivatization at several loci on the beta amyloid peptides or fragments of these peptides, including the termini and side chain derivatives. Compared to nonmodified peptides, PEG-peptides show enhanced solubility.
  • PEG-A ⁇ peptides show inhibited fibrillogenesis, suggesting that these peptides or analogues thereof will be useful as inhibitors of fibrillogenesis in vivo. Furthermore, the peptides have most of the salient structural features of the nonmodified peptides and therefore can be used as structural probes, for site-directed delivery of medicaments.
  • the A ⁇ peptide contains a hydrophilic N-terminus, a central hydrophobic region (aa 17-21) and a long and very hydrophobic C-terminus (aa 29-43).
  • Residues 10-35 represents an apparent minimal structure competent to add to bona fide plaques from brains of patients with Alzheimer's Disease (Lee et al, 1995).
  • the inventors have used solid state NMR to demonstrate that the central hydrophobic region forms a parallel ⁇ -sheet. Hypothesizing that the C-terminal hydrophobic region mediates an irreversible step in fibril formation, the inventors synthesized the C-terminal PEG- derivatized peptide 1 :
  • the data presented herein provide for the first time a congener of the pathogenic A ⁇ peptide which forms native fibrils in a reversible manner and allow structural events of fibrillogenesis to be characterized in detail. Such modifications can be used to probe regional peptide-peptide interactions, and furthermore may lead to the development of inhibitors of different phases of the fibrillogenesis process. Structural proposals such as those made above can now be defined in this and other amyloidogenic peptides in the early events in nucleation and propagation of fibrils so critical in many disease states.
  • PEG-proteins such as PEG-adenine deaminase
  • PEG-adenine deaminase are used because they are functional, and yet have an increased half-life in the plasma (Hershfield et al, 1992; Weinberg et al, 1993; Bax et al, 1996; Hershfield , 1995; Hershfield , 1993).
  • the inventors hypothesized that PEGylation of A ⁇ might increase the solubility N-terminal PEG- A ⁇ (10-35), and/or retard fibrillogenesis, while retaining many or all of the structural features of the native peptide.
  • the inventors synthesized A ⁇ (10-35) derivatized with PEG on the N- or C- terminus. Further, derivatives containing PEG on both termini of A ⁇ (10-35) are also contemplated.
  • the PEG- A ⁇ may comprise the full length sequence of A ⁇ or comprise a portion of the A ⁇ as exemplified by the A ⁇ (10-35) peptide as used in certain examples herein. It should be understood that mutants of the full length or truncated A ⁇ can also be used the only limiting factor for the purposes of the present invention is that that A ⁇ is recognizable as a plaque-competent A ⁇ . That is to say in the absence of the solubilization by conjugation to PEG the A ⁇ used will form fibrils and/or plaques.
  • N-terminal PEG- A ⁇ is more than an order of magnitude greater than the underivatized peptide.
  • preliminary NMR data demonstrated that these peptides have NOEs characteristic of an extended structure, and quite similar to those the inventors have measured of A ⁇ (10-35) in DMSO and to those reported by Lee et al, (1995) for A ⁇ (10-35) in acidic media. Increases in turbidity over time are also dramatically retarded, with barely any increase observable after a week.
  • Solution phase 2-D NMR confirmed a transition from a soluble extended conformation to an oligomeric species, and demonstrated the structural similarity between the modified and unmodified peptides. Electron microscopy demonstrated that C-PEG modification specifically inhibits the lateral aggregation of fibrils into bundles, but not the longitudinal growth of fibrils, indicating participation of the C- terminus in the transition from soluble to insoluble ⁇ sheet.
  • the present invention suggests that derivatization of A ⁇ (10-35) at either terminus would retard or prevent longitudinal aggregation, but not lateral aggregation.
  • Side chain PEGylated A ⁇ (10-35) for example, derivatized on the e-amino group of Lysl6, might then interfere with lateral aggregation.
  • the inventors observed, further, that some aggregation of N-PEG- A ⁇ (10-35) occurred at neutral pH, but not at pH 3.2. It may be that lateral aggregation does still occur in this peptide, though it can be controlled by the pH.
  • the one exception proves the rule, i.e. A ⁇ (1-42) > A ⁇ (1-43), but residue 43 is the hydrophilic residue, Thr.
  • the formation of oligomers and/or higher aggregates may proceed through interactions between C-terminal domains. Accordingly, steric hindrance of these interactions may be best at retarding polymerization of soluble oligomers into fibrils.
  • N-terminal and C-terminal PEG- A ⁇ (10-35) employs techniques well known to those of skill in the art N-terminal PEG- A ⁇ (10-35) was synthesized by the manual addition of carboxy-PEG to deprotected A ⁇ (10-35) still attached to the resin. Coupling was carried out by the repeated addition of carboxy- PEG + 4 equivalents of BOP reagent, until manual qualitative ninhydrin test revealed no remaining free amines.
  • the amino acid sequence is designated as SEQ ID NO:2.
  • Compound 1 shows the C-PEG- A ⁇ (10-35) synthesized herein.
  • Compound 1 was synthesized using standard FMOC protocols on PAP Tenta-Gel purchased from Rapp Polymer. PAP resin is cleaved by TFA yielding a linear polyethylene glycol 3000 average molecular weight covalently bound to the carboxyl terminus of the peptide (Bayer and Rapp, 1992; Rapp et al, 1990). The last 10 residues were double coupled to an overall crude coupling yield of 96%. After cleavage from the resin and side-chain deprotection, the peptide had an modal molecular weight of 6212 (2900 peptide + 3000 ave. mol. wt. PEG) by MALDI-TOF mass spectroscopy, was a single peak by RP-HPLC, and one band by SDS-PAGE.
  • amyloidogenic peptides include but are not limited to A ⁇ peptide, amyloid A, amyloid kappa L-chain, amyloid lambda L-chain, A ⁇ 2M, ATTR, AIAPP, amylin, atrial naturetic factor, procalcitonin, gelsolin, cystatin C, AApoA-I, AApoA-II, and AScr or PrP-27.
  • a ⁇ peptide amyloid A
  • amyloid kappa L-chain amyloid lambda L-chain
  • a ⁇ 2M ATTR
  • AIAPP amylin
  • atrial naturetic factor procalcitonin
  • gelsolin cystatin C
  • cystatin C AApoA-I
  • AApoA-II AApoA-II
  • AScr or PrP-27 AScr or PrP-27.
  • the gene encoding such an amyloidogenic protein can be inserted into an appropriate expression system.
  • the gene can be expressed in any number of different recombinant DNA expression systems to generate large amounts of the polypeptide product, which can then be purified and used as described herein.
  • amino acid sequence variants of a polypeptide can be prepared. These may, for instance, be minor sequence variants of a polypeptide that arise due to natural variation within the population or they may be homologues found in other species. They also may be sequences that do not occur naturally but that are sufficiently similar that they function similarly and/or elicit an immune response that cross-reacts with natural forms of the polypeptide. Sequence variants can be prepared by standard methods of site-directed mutagenesis such as those described below in the following section.
  • Amino acid sequence variants of a polypeptide can be substitutional, insertional or deletion variants.
  • Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity, and are exemplified by the variants lacking a transmembrane sequence described above.
  • Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage. Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • Insertional variants include fusion proteins such as those used to allow rapid purification of the polypeptide and also can include hybrid proteins containing sequences from other proteins and polypeptides which are homologues of the polypeptide.
  • an insertional variant could include portions of the amino acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species.
  • Other insertional variants can include those in which additional amino acids are introduced within the coding sequence of the polypeptide. These typically are smaller insertions than the fusion proteins described above and are introduced, for example, into a protease cleavage site.
  • major amyloidogenic determinants of the polypeptide are identified by an empirical approach in which portions of the gene encoding a given polypeptide are expressed in a recombinant host, and the resulting proteins tested for their ability to elicit fibrillogenesis, amyloidogenesis or plaque formation.
  • PCRTM can be used to prepare a range of cDNAs encoding peptides lacking successively longer fragments of the C-terminus of the protein (PCRTM technology is disclosed in U.S. Patent 4,683,202 and U.S. Patent 4,682,195, each incorporated herein by reference).
  • the amyloidogenic activity of each of these peptides then identifies those fragments or domains of the polypeptide that are essential for this activity. Further studies in which only a small number of amino acids are removed at each iteration then allows the location of the amyloidogenic determinants of the polypeptide.
  • Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al, (1993).
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions.
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • Successful applications of the peptide mimetic concept have thus far focused on mimetics of ⁇ -turns within proteins.
  • Likely ⁇ -turn structure within a polypeptide can be predicted by computer-based algorithms. Once the component amino acids of the turn are determined, peptide mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.
  • Modification and changes may be made in the structure of a gene and still obtain a functional molecule that encodes a protein or polypeptide with desirable characteristics.
  • the following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • the amino acid changes may be achieved by change the codons of the DNA sequence, according to the following data.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.
  • hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). TABLE 2
  • the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (- 0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (- 4.5).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine *-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis is well known in the art.
  • the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
  • site-directed mutagenesis is performed by first obtaining a single- stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein.
  • An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared.
  • This primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • E coli polymerase I Klenow fragment DNA polymerizing enzymes
  • a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained.
  • recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • DNA expression plasmids may be designed to optimize production of the heterologous amyloidogenic proteins and mutants thereof described in the previous section. These expression plasmids include a number of enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in cells. Elements designed to optimize messenger RNA stability and translatability in cells are defined.
  • expression construct is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • the transcript may be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.
  • the nucleic acid encoding a gene product is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for
  • RNA synthesis The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co- operatively or independently to activate transcription.
  • the particular promoter that is employed to control the expression of a nucleic acid encoding a particular gene is not believed to be important, so long as it is capable of expressing the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the gene of interest.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a gene of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • Tables 3 and 4 list several elements/promoters which may be employed, in the context of the present invention, to regulate the expression of the gene of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression but, merely, to be exemplary thereof. Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
  • enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • Eukaryotic promoters can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • NC AM Neural Cell Adhesion Molecule
  • SAA Human Serum Amyloid A
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • viruses to enter cells via receptor-mediated endocytosis and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).
  • the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).
  • a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed.
  • the inventors have employed the human Growth Hormone and SV40 polyadenylation signals in that they were convenient and known to function well in the target cells employed.
  • Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • the delivery of a nucleic acid in a cell may be identified in vitro or in vivo by including a marker in the expression construct.
  • the marker would result in an identifiable change to the transfected cell permitting easy identification of expression.
  • a drug selection marker aids in cloning and in the selection of transformants, for example, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol.
  • enzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic) or chloramphenicol acetyltransferase (CAT) (prokaryotic) may be employed.
  • Immunologic markers also can be employed.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
  • the expression construct In order to effect expression of gene constructs, the expression construct must be delivered into a cell. For protein production, this delivery may be accomplished in vitro, in laboratory procedures for transforming cells lines. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • viruses The ability of certain viruses to enter cells via receptor- mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).
  • the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.
  • the expression vector comprises a genetically engineered form of adenovirus.
  • retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the El region (El A and EIB) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
  • MLP major late promoter
  • TPL 5'-tripartite leader
  • adenovirus generation and propagation of replication deficient adenovirus vectors, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al, 1987), providing capacity for about 2 extra kb of DNA.
  • the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El-deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).
  • MOI multiplicities of infection
  • Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.
  • the preferred helper cell line is 293.
  • Racher et al (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
  • natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.
  • Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • the typical vector according to the present invention is replication defective and will not have an adenovirus El region.
  • the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
  • the polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al, (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.
  • Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 9 -10 ⁇ plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991 ; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991 ; Stratford-Perricaudet et al, 1990; Rich et al, 1993).
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990).
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
  • a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983).
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
  • retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al, 1981).
  • Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.
  • new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al, 1988; Hersdorffer et al, 1990).
  • viral vectors may be employed as expression constructs in the present invention.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
  • Non-viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
  • the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well (Dubensky et al, 1984).
  • a naked DNA expression construct into cells may involve particle bombardment.
  • This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987).
  • Several devices for accelerating small particles have been developed.
  • One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990).
  • the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al, 1990; Zelenin et al, 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i. e. ex vivo treatment.
  • DNA encoding a particular gene may be delivered via this method and still be incorporated by the present invention.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
  • Wong et al, (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.
  • Nicolau et al, (1987) accomplished successful liposome- mediated gene transfer in rats after intravenous injection.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989).
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991).
  • HMG-1 nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
  • a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a particular gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990).
  • ASOR asialoorosomucoid
  • transferrin Wang and Wu, 1990
  • Primary mammalian cell cultures used for the production of amyloidogenic proteins may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented and are disclosed herein by reference (Freshner, 1992).
  • One embodiment of the foregoing involves the use of gene transfer to immortalize cells for the production of proteins.
  • the gene for the protein of interest may be transferred as described above into appropriate host cells followed by culture of cells under the appropriate conditions.
  • the gene for virtually any polypeptide may be employed in this manner.
  • the generation of recombinant expression vectors, and the elements included therein, are discussed above.
  • the protein to be produced may be an endogenous protein normally synthesized by the cell in question.
  • Examples of useful mammalian host cell lines are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN and MDCK cells.
  • a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and process the gene product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the foreign protein expressed.
  • Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e. a monolayer type of cell growth).
  • Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products.
  • suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent T-cells.
  • the airlift reactor also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture.
  • the gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor.
  • the main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1.
  • the airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.
  • Protein purification techniques to isolate amyloidogenic proteins fro use in the present invention.
  • Such proteins may be recombinant proteins produced as described herein above, or they may be isolated from natural sources.
  • Protein purification techniques are well known to those of skill in the art. These techniques tend to involve the fractionation of the cellular milieu to separated the amylin form other components of the mixture. Having separated amyloidogenic proteins from the other plasma components the amyloidogenic peptide sample may be purified using chromatographic and electrophoretic techniques to achieve complete purification. Analytical methods particularly suited to the preparation of a pure peptides are ion-exchange chromatography, HPLC, FPLC and the like.
  • Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein. It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al, Biochem.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain and adequate flow rate. Separation can be accomplished in a matter of minutes, or a most an h. Moreover, only a very small volume of the sample is needed because the particles are so small and close- packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography is a special type of partition chromatography that is based on molecular size.
  • the theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size.
  • the sole factor determining rate of flow is the size.
  • molecules are eluted from the column in decreasing size, so long as the shape is relatively constant.
  • Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
  • Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
  • Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The methods for the generation of antibodies that would be suitable for use in accord with the present invention are well known to those of skill in the art.
  • a ⁇ is an exemplary amyloidogenic peptide and is responsible for amyloid deposits in small cerebral and meningeal blood vessels (amyloid angiopathy).
  • a ⁇ is an exemplary amyloidogenic peptide and is responsible for amyloid deposits in small cerebral and meningeal blood vessels (amyloid angiopathy).
  • compositions of modified A ⁇ that inhibit fibrillogenesis and in a broader aspect amyloidogenesis.
  • Amyloidogenesis may be monitored using an assay that employs dyes such as Congo-red and thioflavin-S dye because amyloidogenic peptides in their aggregated form become stained whereas those in their soluble form do not become stained.
  • methods are provided for screening for inhibitors of amyloidogenesis.
  • Such methods may use cells that comprise an amyloidogenic phenotype either as adherent cells on a culture dish, as part of an alginate biomatrix, in suspension culture or in any other form that permits the polypeptide form amyloid plaques and for such formation to be monitored for example using staining with the dyes discussed above. These cells are then used as reagents to monitor the effects of the fibrillogenesis inhibiting compositions produced by the present invention.
  • the present invention provides methods of screening for inhibitors of amyloidogenesis, by monitoring amyloidogenic function in the absence of the candidate substance and comparing such results to the assay performed in the presence of candidate amyloidogenesis inhibitors.
  • the present invention concerns a method for identifying such inhibitors. It is contemplated that this screening technique will prove useful in the general identification of a compound that will serve the purpose of decreasing, inhibiting or otherwise abrogating the formation of amyloidogenic plaques and fibrils secretion of, for example, any amyloidogenic polypeptide that has been modified so that the C-terminal, the N-terminal and the side chains thereof are not free to form aggregates as described according to present invention.
  • amyloidogenic peptide such as for example, Alzheimer's Disease, Downs Syndrome, and other amyloidoses for example those listed in Table 1.
  • the present invention is directed to a method for determining the ability of a candidate PEGylated amyloidogenic peptide to inhibit the amyloidogenesis around cell that is either naturally amyloidogenic or has been engineered to possess amyloidogenic properties for screening purposes as described herein.
  • a sample having amyloidogenic plaques may be an adherent cell that has the ability to form amyloid plaques or fibril aggregates.
  • amyloidogenic peptides are insoluble in aqueous solution.
  • a simpler assay format that may be employed to test whether a composition is capable of inhibiting fibril formation may comprise a solution of amyloidogenic peptide.
  • a sample having amyloidogenic plaques comprises a solution of amyloidogenic peptide in which amyloidogenesis is exhibited as turbidity of the sample.
  • the method including generally the steps of:
  • step (c) measuring the amyloid plaques of said sample; and (d) comparing the amyloidogenic plaques of the sample in step (c) with the amyloidogenic plaques of the sample of step (a).
  • a candidate substance as being capable of inhibiting amyloid plaque formation in the assay above, one would measure or determine the amyloid plaque formation in the absence of the added candidate substance by determining for example the turbidity of the solution, or the presence of the number of plaques and such. One would then add the candidate substance to the cell and determine the amyloidogenesis in the presence of the candidate substance.
  • a candidate substance which decreases the amyloidogenesis or fibril forming capacity relative to the function in its absence, is indicative of a candidate substance with inhibitory capability.
  • Fibrillogenesis is the process by which fibrils appear in aggregates the extracellular matrix of tissue.
  • fibrillogenic aggregates increase the turbidity of a solution.
  • the presence of these aggregates may be monitored and quantified using any of a number of techniques.
  • the turbidity of a solution can be measured spectrophotometrically, the presence of aggregates may be measured and monitored using microscopy and amyloidogenesis can be monitored using ELISA based techniques that employ dyes such as Congo red for the detection of plaque forming peptides.
  • US Patent 5,538,845 is incorporated herein by reference, in that it provides methods for identifying compounds capable of inhibiting the production of A ⁇ peptide in cells. Such methods may be used in conjunction with the A ⁇ inhibitors generated herein to assess the ability of such compound to inhibit wild-type A ⁇ function in plaque formation.
  • the method involves first binding a dye to a purified amyloid protein.
  • the dye is Congo red.
  • the composition comprising a dye bound to a purified amyloid protein is mixed with serum or tissue homogenate to form a reaction mixture.
  • the activity of the fibrillogenesis inhibiting compositions of the present invention may also be monitored using the methods set forth in U.S.
  • Patent 5,474,893 wherein degradation of the amyloid plaque by the inhibitors produced herein will disrupt the dye/amyloid bond causing dye to be released into a supernatant solution where its concentration and amount are spectrophotometrically determined. The amount of the amyloid degraded is calculated based upon the quantity of the dye found in the supernatant.
  • U.S. Patent 5,348,963 also provides methods that may be useful in combination with the present invention when screening for inhibitors of amyloid formation.
  • Fibril formation may also be monitored using HPLC.
  • HPLC HPLC
  • the disappearance of the monomer peak is indicative of aggregation. This may also be confirmed by electron microscopy.
  • This HPLC method to measure fibrillogenesis uses light scattering. Examples of the use of quasielastic light scattering to measure fibrillogenesis of A ⁇ has been extensively studies and is well known to those of skill in the art (Walsh et al, 1997; Wood et al, 1996; Lomakin et al, 1997; Wood et al, 1996; Lomakin et al, 1996; Shen et al, 1993).
  • High performance size exclusion chromatography with multiangle light scattering detector (HPSEC-MALLS) is performed using the Showa Denko Shodex Protein KW-804 size exclusion column, on a Wyatt Technologies DAWN Laser photomer/OPTILAB refractometer system
  • the molecular weight is calculated using the procedure described in the Wyatt Technologies manual using identical conditions of pH, ionic strength and peptide concentration as in the CD analysis.
  • scattering of other types of radiation can be used for similar purposes, e.g. , small angle X-ray diffraction or small angle neutron scattering.
  • the term “candidate substance” refers to any molecule that is capable of inhibiting amyloidogenic function.
  • the candidate substance may be a protein or fragment thereof, that has been modified to prevent the formation of aggregates. It may prove to be the case that the most useful pharmacological compounds for identification through application of the screening assay will be compounds that are structurally related to other known amyloidogenic peptides.
  • the active compounds may include fragments or parts of naturally- occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive. However, prior to testing of such compounds in humans or animal models, it will be necessary to test a variety of candidates to determine which have potential.
  • the candidate screening assays are simple to set up and perform. Thus, in assaying for a candidate substance, after obtaining an amyloidogenic sample, one will admix a candidate substance with the sample, under conditions which would allow measurable amyloidogenesis to occur. In this fashion, one can measure the ability of the candidate substance to inhibit the amyloidogenic parameter in the absence of the candidate substance. In this fashion the ability of the candidate inhibitory substance to reduce, abolish, or otherwise diminish amyloidogenesis may be detected.
  • Effective amounts in certain circumstances are those amounts effective to reproducibly inhibit amyloidogenesis or fibril formation in comparison to their normal levels. Compounds that achieve significant appropriate changes in activity will be used.
  • the present invention further comprises pharmaceutical compositions incorporating a PEGylated amyloidogenic compound described herein above and including a pharmaceutically acceptable carrier.
  • Such pharmaceutical compositions should contain a therapeutic or prophylactic amount of at least one compound identified by the method of the present invention.
  • pharmaceutically acceptable refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • compositions just described are suitable for systemic administration to the host, including both parenteral, topical, and oral administration.
  • the pharmaceutical compositions may be administered parenterally, i.e. subcutaneously, intramuscularly, or intravenously.
  • the present invention provides compositions for administration to a host, where the compositions comprise a pharmaceutically acceptable solution of the identified compound in an acceptable carrier, as described above.
  • injectibles are prepared either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectible use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectible solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Sterile injectible solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the preparation of more, or highly, concentrated solutions for local injection also is contemplated.
  • the use of DMSO as solvent is preferred as this will result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the pharmaceutically acceptable carrier can be any compatible, non-toxic substance suitable to deliver the compounds to an intended host.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectible compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • compositions may also be incorporated into the pharmaceutical compositions.
  • Preparation of pharmaceutical conditions incorporating active agents is well described in the medical and scientific literature. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 16th Ed., 1982, the disclosure of which is incorporated herein by reference. Dosages may vary according to the physician's diagnosis and the route of administration chosen. For example, one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to lOOOmL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated or diagnosed. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the cell preparations may further contain a preservative to prevent growth of microorganisms.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well-known parameters.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is diagnostically or therapeutically effective.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • these particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • direct injection into the plaque is contemplated.
  • the region of the amyloid plaque may be infused or perfused with the therapeutic compounds using any suitable delivery vehicle.
  • Local or regional administration, with respect to the plaque also is contemplated.
  • systemic administration may be performed. Continuous administration also may be applied where appropriate. Delivery via syringe or catherization is also contemplated.
  • Direct techniques usually involve placement of a drug delivery catheter into the host's ventricular system to bypass the blood-brain barrier.
  • Indirect techniques which are generally preferred, involve formulating the compositions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid-soluble drugs. Latentiation is generally achieved through blocking of the hydroxyl, carboxyl, and primary amine groups present on the drug to render the drug more lipid-soluble and amenable to transportation across the blood- brain barrier.
  • the delivery of hydrophilic drugs can be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood- brain barrier.
  • the concentration of the pharmacologically active compound in the pharmaceutical carrier may vary widely, i.e. from less than about 0.1 % by weight of the pharmaceutical composition to about 20% by weight, or greater.
  • Typical pharmaceutical composition for intramuscular injection would be made up to contain, for example, one to four ml of sterile buffered water and one ⁇ g to one mg of the compound identified by the method of the present invention.
  • the typical composition for intravenous infusion could be made up to contain 100 to 500 ml of sterile Ringer's solution and about 1 to 100 mg of the compound.
  • the pharmaceutical compositions of the present invention can be administered for prophylactic and/or therapeutic treatment of diseases related to the deposition of A ⁇ and other amyloidogenic peptides, such diseases including but not limited to Alzheimer's disease, Down's syndrome, and advanced aging of the brain.
  • diseases related to the deposition of A ⁇ and other amyloidogenic peptides such diseases including but not limited to Alzheimer's disease, Down's syndrome, and advanced aging of the brain.
  • the pharmaceutical compositions are administered to a host already suffering from the disease.
  • the pharmaceutical compositions will be administered in an amount sufficient to inhibit further deposition of A ⁇ plaque. In other embodiments, the size of the pre-existing amyloid plaque is diminished.
  • a therapeutically effective dose An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Such effective dose will depend on the extent of the disease, the size of the host, and the like, but will generally range from about 0.01 ⁇ g to 10 mg of the compound per kilogram of body weight of the host, with dosages of 0.1 ⁇ g to 1 mg/kg being more commonly employed.
  • the pharmaceutical compositions of the present invention are administered to a host susceptible to the A ⁇ -related disease, but not already suffering from such disease.
  • a host susceptible to the A ⁇ -related disease may be identified by genetic screening and clinical analysis, as described in the medical literature (Goate, 1991).
  • the pharmaceutical compositions will be able to inhibit or prevent deposition of the A ⁇ plaque at a symptomatically early stage, preferably preventing even the initial stages of the ⁇ -amyloid disease.
  • the amount of the compound required for such prophylactic treatment referred to as a prophylactically-effective dosage, is generally the same as described above for therapeutic treatment.
  • compositions of the present invention may be advantageously packaged into a kit comprising the active reagent(s) a suitable container means and even instructions for use of said kit.
  • the reagent(s) of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder.
  • the liquid solution is an aqueous solution.
  • the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide.
  • the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the reagents may be placed, and preferably suitably aliquoted. Where a second reagent is provided, the kit will also generally contain a second vial or other container into which this additional reagent may be placed.
  • the kits of the present invention will also typically include a means for containing the reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • EXAMPLE 1 A parallel ⁇ -strand domain of Alzheimer's A ⁇ fibrils determined using solid state NMR spectroscopy The present example describes the uses of DRAWS C solid state nuclear magnetic resonance (NMR) studies (Gregory et al, 1997) to measure precise distances between individual A ⁇ peptides in the plaques of the A ⁇ peptide that are
  • peptides were solubilized in water, the pH of which was approximately 3.0 due to residual trifiuoroacetic acid in the lyophilized powder; the pH was gradually adjusted to 7.4, and the samples were gently agitated at room temperature for 2-3 days under nitrogen. Under these conditions, Lee et al. found the 10-35 peptide to add to plaques isolated from Alzheimer's patients' brains (1995). Fibril formation was monitored by the disappearance of the monomer peak by HPLC and confirmed by electron microscopy. Once fibril formation was complete, the samples were either lyophilized and stored at -20°C or flash-frozen and immediately inserted into a NMR tube pre-chilled to -80°C.
  • the peptides generated include those given in Table 5.
  • the distance between two interstrand *Lysl6 residues was found to be 5 ⁇ 0.5 A. In contrast, the two Q15 were found to be «7 A apart.
  • the inventors synthesized two additional peptides 3 and 4 to test of the antiparallel arrangement.
  • Peptide 3 the interstrand distance between Glnl5 and Leu 17 should be «5. ⁇ A in the antiparallel ⁇ strand conformation, i.e. similar to the interstrand distance between Lysl6 residues.
  • Peptide 4 is a negative control, since the interstrand distances between Leul7 would be too far apart in an antiparallel ⁇ sheet to show coupling. Coupling would be seen in a parallel ⁇ conformation.
  • Peptide 8 serves as a secondary structure-sensitive distance control. In the ⁇ strand conformation, the intrastrand distance will be fixed by the secondary structure. In contrast, interstrand contacts will not result in coupling that can pass through a triple quantum filter.
  • Peptide 9 is a positive control for the triple quantum filter studies; the distances between labels is ⁇ 5 A, and the signal should be able to pass through a triple quantum filter.
  • Peptide purification and analysis For many peptides, first the scavengers and other low molecular weight contaminant are removed by a preliminary gel filtration chromatography step using Sephadex G-25. This is followed by preparative reverse phase HPLC. In the case of ⁇ A peptides, however, the inventors have modified this procedure because of unacceptable losses in the first step. In addition, the inventors observed that adequate removal of scavengers was achieved by repeated extractions using diethyl ether and/or ethyl acetate. Peptides are dissolved in 0.1% (v/v) TFA in water and injected onto C4 or C18 preparative reverse phase HPLC columns, typically from Rainin or Dynamax.
  • Alzheimer's amyloid plaques consist of a highly ordered, fibrillar structure which can be replicated in vitro using synthetic peptides.
  • the inventors have developed a generalizable approach, using solid state NMR and specifically labeled synthetic peptides, by which determination of the specific structure of the peptide in its plaque-conformation is possible.
  • solid state NMR has often been looked to as the "new solution” to solving structures of insoluble proteins, such as amyloids, or proteins difficult to crystallize, such as membrane proteins. Additionally, solid state NMR studies can determine distances to within 0.2 A (Gregory et al, 1997), more precise than the current resolution of any other technique. However, specific structures of insoluble proteins, such as amyloids, or proteins difficult to crystallize, such as membrane proteins. Additionally, solid state NMR studies can determine distances to within 0.2 A (Gregory et al, 1997), more precise than the current resolution of any other technique. However, specific structures of
  • DQDRAWS pulse sequence containing double quantum filter
  • the double quantum filter serves to remove any single quantum (non-coupled) signals from the study.
  • All of the inventors' experimental samples contained a small amount of unlabeled hexamethyl benzene (HMB) as an internal control. The only possible signals from the HMB are
  • FIG. 4B the inventors found the distance for both the 1- C-Gln-A ⁇ and l- 13 C-Lys-A ⁇ samples to be 5.0 A ( ⁇ 0.3); this was true both for repeated measurements of the same sample and for repeated sample preparations.
  • the fit to a 5.0 A distance was valid only for simulations in which each spin was coupled to 2 other spins at identical distances apart (FIG. 4A).
  • Models of 2 spins in isolation, at a distance of 4.5 to 5 A matched the range of DRAWS data collected, but not the shape of the curve (FIG. 4B).
  • models of 3 spins where one pair of contacts is close and the second pair is far such as might be seen in a linear arrangement of 3 spins, failed to fit the data.
  • N-terminal PEG- A ⁇ ( 10-35) was synthesized by the manual addition of carboxy-PEG to deprotected A ⁇ (10-35) still attached to the resin. Coupling was carried out by the repeated addition of carboxy-PEG + 4 equivalents of BOP reagent, until manual qualitative ninhydrin test revealed no remaining free amines.
  • the preparation of C-PEG- A ⁇ (10-35) was slightly less straightforward, and was carried out as shown in FIG. 6.
  • Circular dichroic (CD) spectra were measured at variable pH using peptide dissolved in a citrate/phosphate buffer system with the salt concentration held constant at 200mM. Data were collected on a Jasco J-600 spectropolarimeter at room temperature using either a 0.01 or 0.1 cm pathlength cuvette. The concentration of peptide by dry weight was 1.74 mM.
  • peptide was dissolved into a concentrated stock at 3.8 mM, and CD spectra measured of the stock and various dilutions made with phosphate/citrate buffer at pH 5.8 to achieve peptide concentrations from 0.022 to 3.8 mM.
  • Circular dichroic (CD) spectra show [ ⁇ ] 217 as functions of peptide concentration. For the concentration dependency, one can derive the equation:
  • K[ and K 2 are the constants for the two relevant dissociations.
  • lyophilized peptide was dissolved in 10% D 2 O/H 2 O and the pH was adjusted with NaOH/HCl, and the final volume adjusted to 600 ⁇ l . The samples were then centrifuged for 15 min at 14,000 X g. 1H
  • NMR data were collected on either a Varian 500 or 600 MHz NMR spectrometer with a 5mm probe using pulse field gradient solvent suppression. The solvent and polymer signal were saturated at a frequency which gave the best signal suppression of both peaks.
  • Typical 2D ⁇ NMR spectra were collected with 220 to 256 FIDs of 2 to 4K data points, 32 to 128 scans per HD, and spectral width of 5000 or 6000 Hz. Standard
  • Samples were prepared by dissolving dry peptide to concentrations in the mM range in citrate/phosphate buffer containing 0.1% (w/v) NAN 3 . Then the samples were centrifuged for 15 min at 14,000 X g and incubated for four days at room temperature. For EM, samples were applied to a glow discharge 400 mesh carbon- coated support film, followed by staining with 1 % uranyl acetate. Micrographs were recorded using Philips EM 300 at magnifications of 45,000. e. Separation of PEG-derivatized from non-derivativatized peptides.
  • the inventors employ a capping procedure. Free amino groups are biotinylated at pH 7 to target amino groups. Nonderivatized peptides are then be removed using immobilized streptavidin which binds the biotin groups. Although it is possible that a small amount of NH2 groups from lysine side chains will also be coupled to biotin, these will be removed by the same reaction with streptavidin.
  • An additional procedure for separating PEG peptides from underivatized peptides and unreacted PEG is equilibrium density gradient ultracentrifugation, as these species are of distinct and widely disparate densities.
  • NOESY and TOCSY spectra obtained give nearly identical results to those obtained by Lee et al (1995) for A ⁇ (10-35).
  • N-PEG- A ⁇ (10-35) stayed in solution indefinitely at pH 3.2, but slowly became insoluble at pH near neutrality (pH 6.8).
  • the major difference is the concentration of PEG- A ⁇ (10-35) that can be reached, resulting in a much higher quality spectra of the PEG- A ⁇ compared to nonderivatized A ⁇ .
  • Table 6 shown below is a chart comparing the conditions used by the inventors to those of Lee et al. (1995). Table 6 Comparison between Inventors data with that of Lee et al. 1995
  • N-PEG- A ⁇ (10-35) has a spectrum quite similar to that of non-derivatized A ⁇ (l-39), and undergoes a similar pH dependent shift in the spectrum: (FIG. 9B). This shows the feasibility of using N- PEG-A ⁇ (10-35) as a homologue of the non-derivatized peptide.
  • FIG. 7B shows the CD spectra showing [ ⁇ ] 217 as functions of pH concentration and FIG. 7C shows CD spectra show [ ⁇ ] 217 as functions of peptide concentration. From pH 3-7, 1 gave clear solutions while the native peptide under similar conditions of pH, ionic strength and concentrations formed gels or precipitates.
  • FIG. 7A shows a dramatic pH induced transition from random coil to a ⁇ sheet. There was a modal increase in ⁇ content (decreasing [ ⁇ ] 217 from pH 2.7 to 4.5, followed by the apparent loss of ⁇ content due to precipitation of peptide from pH 4.5 to 7.6 (FIG. 7B).
  • Electron micrographs prepared from 1 at pH 3.0 were virtually indistinguishable from those of nonmodified A ⁇ 10-35. Indeed, this is somewhat surprising in that the PEG moiety might be expected to increase the fibril diameter. Instead, the fibrils of PEG A ⁇ appeared as if negatively stained, and were no thicker than fibrils of nonmodified A ⁇ , consistent with the notion that the PEG coats exterior of the fibril. At pH 5.8 or 7.4, however, the nonmodified peptide fibrils aggregated laterally into bundles, whereas 1 remained as linear, non-aggregated fibrillar strands, and only slowly (3 days) formed occasional lateral aggregates at pH 7.4. Bundles of 1 were never observed at pH 5.8. EXAMPLE 3
  • the present example yields a high resolution description of fibers formed from
  • a ⁇ ( io- 35) peptides were studied both in solution and in fibrillar arrays.
  • the structure consists of four to six laminated ⁇ -helices propagating along the helical axis. Each peptide strand is oriented perpendicular to the helical axis in a parallel ⁇ structure, with each amino acid residue in register between the strands.
  • a ⁇ -PEG containing either 1- C-Leu or 1- C-Val was synthesized using standard fluorenyl-methoxy carbonyl protocols on PAP Tenta-Gel (Rapp Polymere) to introduce the label at position 17 or 24 (Burkoth et al, 1998). Cleavage by TFA and deprotection yielded a linear PEG 3000 covalently bound to the carboxyl terminus of the peptide. Peptide purity, determined by synthetic coupling yields and reverse phase HPLC of CNBr cleavage products, was >96%. Molecular masses of all peptides were verified by MALDI-TOF mass spectroscopy.
  • CP/MAS 13 C-NMR studies were performed on a Bruker Avance DSX spectrometer at 50.3 MHz using the DRAWS sequence (Benzinger et al, 1998; Gregory et al, 1997). Spectra were acquired on 10 mg samples with >500 scans for the fibril samples and >1800 scans for the non-fibrilized control sample of l- 13 C-Leu ⁇ -A ⁇ -PEG or 1- 13 C- Val 24 -A ⁇ -PEG. Hexamethylbenzene (8-12 mg) served as internal control.
  • the input parameters to the numerical calculation program included the chemical shift tensor (CSA) elements for the spin- 1/2 nuclei, the dipolar coupling strengths, Euler angles which rotate the CSA tensors from the molecular frame to their respective principle axis systems, an initial density matrix p(0), an observable, and any relevant relaxation parameters.
  • the Euler angles were set to zero, as it was determined that they had a negligible effect on the simulated curves.
  • the CSA parameters were taken from Ye et ⁇ /.(Ye et al, 1993).
  • the time increment was typically 2-3ms. Relaxation effects were modeled by multiplying the single quantum density matrix elements by an exponential factor at the end of each time increment. Data from the non-fibrilized sample was used to determine the appropriate single quantum relaxation constant.
  • the program also performs a powder average of 2000 randomly selected crystallite orientations.
  • Samples of A ⁇ ( io-35 ) /A ⁇ -PEG were prepared for EM by dissolving dry peptide to 2 mM in water containing 0.1% NaN 3 (pH ⁇ 3.0). The samples were mixed, centrifuged for 15 min at 14,000 X g and incubated for 1 h. The mixtures were then diluted to 1 mM in phosphate buffer at pH 5.7 and allowed to stand at room temp for 3 days. Samples were applied to a glow discharge 400 mesh carbon coated support film, followed by staining with 1% uranyl acetate. Micrographs were recorded using Philips EM 300 at magnifications of 100,000, 45,000, and 10,000.
  • the measurements on the samples in 12% D 2 O were made on SAD (Thiyagarajan et al, 1997) which is similar to SAND, but the Q range is limited to 0.005 - 0.25 A "1 .
  • the evolution of the scattering signal from the samples in 99% D 2 O was followed for several h at 22 °C, with each measurement lasting for 1 hr, to ensure that the samples had reached hermodynamic equilibrium at the length scales being measured.
  • Np is the number of particles in unit volume
  • Vp is the volume of the particles
  • F(Q) is the form factor describing the shape of the particles
  • p p and p r are the neutron scattering length density of the particle and the solvent, respectively.
  • the scattering length density p for any system can be calculated by using
  • b and M are the neutron scattering length and mass of individual atoms respectively, d is the density of the system and N A is Avogadro's number.
  • the difference in the scattering length densities of the particle and the matrix is known as the contrast.
  • I(Q) will be zero if the scattering length density of the particle is equal to that of the solvent. Since the scattering length density of H 2 O,
  • D 2 O, PEG, and the peptide are -0.56 x 1O 10 cm" 2 , 6.34 x 10 10 cm” 2 , 0.57 x 10 10 cm” 2 and 1.93 x lO 10 cm -2 , respectively, the coherent scattering from PEG can be eliminated if 16% D2O buffer is used (Thiyagarajan et al, 1995).
  • 12 % D 2 O buffer was used as a compromise to reduce the PEG scattering to an insignificant level, but still have sufficient contrast for the scattering from the peptide.
  • Absolute SANS data allow for the evaluation of mass-per-unit length for the rod-like particles from the y-intercept value of the modified Guinier plot (Ln[Q.I(Q). vs. Q plot] by using
  • I c (0) is the exponential of the y-intercept from the modified Guinier plot
  • c is the concentration of the sample in mg/ml determined by amino acid analysis
  • d is the density of the peptide (inverse of partial specific volume)
  • p p and p s are the neutron scattering length density values of peptide and solvent respectively
  • N A is the Avogadro's number.
  • the inventors define, using a combination of synthetic model peptides, solid- state NMR, and small angle neutron scattering (SANS), the three dimensional structure of the A ⁇ ( 10 - 35) fibril.
  • the amino acid sequence of A ⁇ can be separated into distinct domains: a hydrophilic N-terminus (aa 1-16), a central hydrophobic region (aa 17-21), and a long hydrophobic C-terminus (aa 29-43).
  • a ⁇ ( io- 3 5 ) a peptide comprising the core residues 10-35, preserves these characteristic domains (FIG. 8A) (Esler et al, 1996) and can be used to prepare homogeneous fibrillar arrays.
  • This parallel ⁇ -sheet orientation clusters the hydrophobic C-termini of each peptide and predicts the propagation of a hydrophobic face along the entire length of the fibril.
  • a priori such a solvent exposed hydrophobic surface would be expected to dominate the behavior of the fibrils.
  • a polyethylene glycol block was synthetically attached at the C-terminus of A ⁇ ( io- 35) , A ⁇ -PEG, as shown in FIG. 8A (Burkoth et al, 1998). This modification greatly improved solubility without removing the ability of the peptide to form fibrils.
  • FIG. 9 EM was used to examine the morphology of fibrils formed from mixtures of the two synthetic peptides.
  • the relative ratio of A ⁇ ( io-35 ) to A ⁇ -PEG was adjusted while total peptide concentration remained constant.
  • Fibrils composed entirely of A ⁇ ( io-3 5) contained primarily twisted, paired fibrils, -90 by -160 A across the narrowest and thickest dimensions, with a superhelical repeat distance of ⁇ 1100 A, and occasional monomeric fibrils -80 A diameter (FIG. 9A).
  • FIG. 9B, FIG. 9C, FIG. 9D and FIG. 9E As the A ⁇ -PEG component was increased, there was less and less lateral self-association of the fibrils.
  • a ⁇ - PEG fibrils (FIG. 9E) were entirely monomeric with poor edge contrast and diameters >80 A. These observations are consistent with A ⁇ -PEG and A ⁇ ( io-35 ) forming hetero-duplexes with PEG coating the surface of the A ⁇ fibril to prevent fibril-fibril association (Burkoth et al, 1998).
  • the hydrophobic C-terminus of A ⁇ ( ⁇ o-3 5) would also be localized to the edge of a monomeric fibril and thus exposed to solvent in the absence of pairing. For this reason, in the absence of PEG, non-paired single filaments would be expected to associate so as to sequester the hydrophobic edge away from solvent.
  • FIG. 10A shows that the average radius of A ⁇ ( io-3 5) in 99% D 2 O buffer at pH 5.6 was 46 ( ⁇ 5) A.
  • the slightly larger slope in the low Q region is consistent with the presence of rods of a larger radius, 70 ( ⁇ 10) A, similar to the fibril- fibril association observed by EM in FIG. 9A.
  • the SANS data for A ⁇ -PEG fibrils under the same conditions yielded a single species with a radius of 65 ( ⁇ 2) A.
  • the PEG moiety increases the radius of the fiber by about 20 A.
  • a fully extended A ⁇ ( ⁇ o-35) peptide would be -90 A in length.
  • the single fibril of A ⁇ -PEG in 12% D 2 O gave a mass-per- unit length of 2360 ( ⁇ 105) Da A.
  • the measured value for A ⁇ -PEG at 11.5 mg/ml in 16% D 2 O (the exact contrast match point for PEG) at pH 5.7 was 2,285 ( ⁇ 105) Da/A.
  • a 2,900 Da peptide arranged every 5 A in the sheet would predict a value of 2,320 Da/A with four laminated sheets.
  • the degree of lamination appears to be very dependent on the conditions used for fibril formation, particularly pH, and this value of four is assigned as a lower limit due of the unavoidable precipitation from highly concentrated peptide solutions and reduction in the concentration during the 20 hr of data collection.
  • the present data is therefore most consistent with a lamination of up to six sheets, positioned 10 A apart, for the ⁇ -helical A ⁇ ( io-35 ) fibril (FIG. 1 1).
  • This arrangement would predict a rectangular rod having dimensions of 90 x 70 A.
  • Such a structure accounts for the repeating 10 A and 5 A x-ray reflections commonly detected in many amyloids (Blake and Serpell, 1996), and most remarkably, confirms a structural model proposed of almost 25 years ago (Cooper, 1974).
  • Cooper based his model on polysaccharide dye binding agents, specifically I 2 and Congo Red, EM analyses (Glenner et al, 1968), and the initial x-ray diffraction results (Gueft, 1972).
  • 11B can be assigned as an antiparallel pair of ⁇ -helical bundles with dimensions of 180 x 70 A as shown in FIG. l lC.
  • An average duplex pitch of -1 100 A, shown in FIG. 11A and FIG. 1 IB, is made up of peptides within each ⁇ -helix spaced at 5 A intervals (FIG. 11 D), and the individual ⁇ -strands must be offset by 1.6°/peptide to define the twist of the ⁇ -helix.
  • Such a structural design of paired helices of opposite antiparallel orientation, spiraling to protect a hydrophobic interior is one of nature's most common and fundamental designs, most apparent in Watson-Crick DNA duplexes.
  • ⁇ -Helices are often composed of homologous repeating sequences, most notably Tyr, Phe, and bulky hydrophobic amino acids (Leu, He, Val) (Nesloney and
  • Leui 7 -Vali8, Phei9-Phe 20 , and He3i-Ile32 result in repeating aromatic and aliphatic stacks on both sides of every ⁇ -sheet.
  • the central core of hydrophobic residues, aa 17-21 forms a surface that would promote both vertical ⁇ -sheet elongation and sheet-sheet stacking.
  • the dramatic pH dependence for fibril formation clearly demonstrated with A ⁇ -PEG (Burkoth et al, 1998), is also a function of these side chain interactions. For example, Hisi3 and Hisi4, while on opposite faces of a single ⁇ -sheet, would directly interact with the respective His 14 and Hisi3 residues of the adjacent, laminated sheets.
  • amyloid fiber formed with A ⁇ (io- 35) has many of the features of the full length A ⁇ fibril, and structural features of this fibril maybe generalizable to other amyloid diseases.
  • this structure now represents the largest noncrystalline molecular assembly whose structure has been determined, and the methods outlined here provide the first general strategy for the characterization of such structural arrays. The methodology and the structural insight that it provides make it possible to develop more rational strategies to prevent amyloid formation.
  • EXAMPLE 4 Preclinical Models
  • the inhibitors of fibrillogenesis generated herein will be used in therapeutic applications.
  • prior to use as therapeutics in humans it may well be necessary to conduct efficacy testing in in vivo animal models.
  • the present Example provides methods for such testing.
  • U.S. Patent 5,387,742 describes a transgenic mice displaying the amyloid-forming pathology of Alzheimer's disease.
  • an amyloid enhancing factor is administered to an animal in order to enhance amyloidogenesis.
  • the preparation of amyloid enhancing factor has been described previously in Axelrad, M. A. et al. (1982).
  • animals are obtained in which one group is a control non-amyloid generating animal that remains untreated.
  • the remaining animals, which are amyloid- forming are divided into two groups 1) those which receive the therapeutic composition (i.e. for example the PEGylated ⁇ A) at 50 mg, 40 mg, 20 mg, or 10 mg by, for example, intraperitoneal injection every 12 h and 2) those which receive a buffered placebo by IP injection.
  • the animals are sacrificed at a time determined by the investigator.
  • the tissues suspected of having amyloid plaques are fixed for histochemical analysis. Following fixation, the tissues were embedded in paraffin, 8-10 micron sections were cut and stained with Congo Red (Puchtler et al, 1983). The histologic sections are then viewed under polarized light to assess by image analysis the percent of tissue occupied by amyloid. Of course other staining methods employing other dyes or antibody detection may also be used.
  • the viability of animals will also be indicative of the therapeutic potential of the PEGylated amyloidogenic peptide.
  • animals receiving only the placebo buffer should succumbed to illness or death prior to the termination of the study.
  • those animals receiving the therapeutic composition its is expected that more animals in this group survive than in the placebo group.
  • This example is concerned with the development of human treatment protocols using the inhibitors of amyloid fibrilliogenesis, such as for example the PEGylated A ⁇ peptides of the present invention, alone or in combination with other treatments for plaque related diseases.
  • These treatments will be of use in the clinical treatment of various diseases in which amyloidosis and plaque formation play a role, including Alzheimer's disease, Downs syndrome and other disease listed in Table 1 herein above.
  • patients may undergo placement of a catheter, or other suitable delivery device, in a cavity will provide an effective means of delivering a therapeutic compounds of the present invention and for sampling the individual for the presence of plaque-forming amyloiodgenic peptides.
  • the PEGylated peptide compound may be administered alone or in combination with other therapeutic drugs that are commonly used in the treatment of Alzheimer's Disease and other amyloidogenic diseases.
  • the administration may be regional, directly into the fibbrillogenic plaque, or in a systemic manner.
  • the starting dose may be 0.5mg/kg body weight.
  • Three patients may be treated at each dose level in the absence of grade > 3 toxicity.
  • Dose escalation may be done by 100% increments (0.5mg, lmg, 2mg, 4mg) until drug related grade 2 toxicity is detected. Thereafter dose escalation may proceed by 25% increments.
  • the administered dose may be fractionated equally into two infusions, separated by six h if the combined endotoxin levels determined for the lot of PEGylated peptide and the lot of second drug exceed 5EU/kg for any given patient.
  • the PEGylated peptide and anti-amyloidogenic drug combination may be administered over a short infusion time or at a steady rate of infusion over a 7 to 21 day period.
  • the PEGylated peptide compound infusion may be administered alone or in combination with the anti-amyloidogenic drug.
  • the infusion given at any dose level will be dependent upon the toxicity achieved after each administration. Hence, if Grade II toxicity was reached after any single infusion, or at a particular period of time for a steady rate infusion, further doses should be withheld or the steady rate infusion stopped unless toxicity improved.
  • Laboratory studies should include CBC, differential and platelet count, urinalysis, SMA-12-100 (liver and renal function tests), coagulation profile, and any other appropriate chemistry studies to determine the extent of disease, or determine the cause of existing symptoms. Also appropriate biological markers in serum should be monitored.
  • plaque size measurements are to be recorded every 4 wk.
  • Appropriate CAT scanning studies should be repeated every 8 wk to evaluate plaque response.
  • An urinalysis may be performed every 4 wk.
  • Clinical responses may be defined by acceptable measure. For example, a complete response may be defined by the disappearance of all measurable disease for at least a month. Whereas a partial response may be defined by a 50% or greater reduction of the sum of the products of perpendicular diameters of all evaluable fibrillogenic plaques or at least 1 month with no plaque sites showing enlargement. Similarly, a mixed response may be defined by a reduction of the product of perpendicular diameters of all measurable lesions by 50% or greater with progression in one or more sites.
  • the inhibition of fibrillogenesis is accomplished by blocking an amyloidogenic peptide with polyethylene glycol.
  • a ⁇ -PEG exploits the physical and biological characteristics of PEG.
  • PEG is a chemically inert, achiral, linear, homo-polymer of varying molecular weights, that has unique solubility characteristics; unstructured and soluble in both aqueous and organic solvents (Bailey and Koleske, 1967; Liu and Parsons, 1969; Maxfield and Shepherd, 1975). Because of these physical characteristics, PEG has been used in a variety of applications (Inada et al, 1995; Harris 1992).
  • PEG has been used as a solubilizing agent to expedite organic chemistry reactions (Han and Janda, 1997) and has been used to solubilize enzymes in apolar aprotic organic solvents (Kodera et al, 1994).
  • PEG can increase the lifetime of pharmacologically active proteins by blocking the surface of the bio-molecule from contact with the immune system and with proteases (Abuchowski et al, 1977).
  • PEG is believed to perturb protein protein interactions by acting as a stearic barrier.
  • the C-terminal A ⁇ -PEG block co-polymer is one solution to inhibiting AD fibril formation by perturbing protein-protein interactions and improving aggregate or fibril solubility. It is contemplated that the general features of this strategy can be extended to other specific inhibitor molecules in two general ways. The first is the use of a different chain length PEG moiety or a molecule that has similar characteristics to that of PEG. In this initial work a covalently attached heterogeneous PEG 3000 polymer was used, but a smaller or more homogeneous PEG molecule could be incorporated in a similar manner using solid phase synthetic methods. Moreover, the characteristics of PEG can be replaced with other solubilizing moieties.
  • Candidate solubilizing molecules considered are sugars, sulfate groups, phosphates or polar peptides/proteins. All of these modifications are available through the same solid phase synthesis methods developed for PEG attachment.
  • the second general strategy contemplated is the location of the covalently attached solubilizing and/or blocking agent.
  • the structure of the fibril described earlier shows that the attachment of a solubilizing molecule could also be incorporated at the N-terminus and on residue side-chain with little perturbation to the fibril structure.
  • solubilizing molecules such as sugars, sulfate groups, phosphates or polar peptides/proteins could be incorporated at the N-terminus and on residue side-chain of the fibril, potentially improving fibril solubility with little structural perturbations.
  • This generality will allow for versatility in tissue targeting and serum lifetime.
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Abstract

D'une manière générale cette invention concerne l'amyloïdogénèse, et d'une manière plus spécifique il s'agit de l'utilisation de protéines amyloïdogènes qui ont été bloquées au niveau des N- et/ou des C- termini et/ou des chaînes latérales pour empêcher leur accumulation sous forme de plaques amyloïdogènes. Des aspects particuliers de cette invention portent sur des procédés et des compositions qui empêchent la formation de plaques dans des maladies amyloïdes, telle que la maladie d'Alzheimer.
PCT/US1999/003231 1998-02-13 1999-02-12 Procedes et compositions comprenant l'utilisation d'un peptide b-amyloide bloque WO1999041279A2 (fr)

Priority Applications (1)

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AU26004/99A AU2600499A (en) 1998-02-13 1999-02-12 Methods and compositions comprising the use of blocked b-amyloid peptide

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US7465898P 1998-02-13 1998-02-13
US60/074,658 1998-02-13

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EP1700096A2 (fr) * 2003-12-04 2006-09-13 Areté Associates Detection de proteines et de prions alteres de maniere conformationnelle
EP1763364A2 (fr) * 2004-02-20 2007-03-21 AC Immune S.A. Procedes et compositions contenant des constructions supramoleculaires
JP2007527870A (ja) * 2004-02-20 2007-10-04 エーシー イミューン ソシエテ アノニム 超分子コンストラクトを含む方法および組成物
WO2008013859A2 (fr) * 2006-07-28 2008-01-31 Adlyfe, Inc. Sondes peptidiques pour des diagnostics et des produits thérapeutiques
WO2008070284A2 (fr) * 2006-10-16 2008-06-12 Johnnie B. Byrd, Sr. Alzheimer's Center And Research Institute Peptides bêta amyloïdes et procédés d'utilisations de ceux-ci
US7691639B2 (en) 2001-05-31 2010-04-06 Adlyfe, Inc. Misfolded protein sensor method
US8372593B2 (en) 2005-02-15 2013-02-12 Adlyfe, Inc. Method for detecting misfolded proteins and prions
US8409580B2 (en) 2003-07-25 2013-04-02 Ac Immune Sa Therapeutic vaccine targeted against P-glycoprotein 170 for inhibiting multidrug resistance in the treatment of cancers
US20150183858A1 (en) * 2011-10-31 2015-07-02 Daniel J. Capon Peptide-hinge-free flexible antibody-like molecule
US9289488B2 (en) 2010-08-12 2016-03-22 Ac Immune Sa Vaccine engineering
US9687447B2 (en) 2010-10-26 2017-06-27 Ac Immune S.A. Method for preparing liposome-based constructs
JPWO2021100744A1 (fr) * 2019-11-19 2021-05-27

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ESLER WP ET AL: "Point substitution in the central hydrophobic cluster of a human beta-amyloid congener disrupts peptide folding and abolishes plaque competence" BIOCHEMISTRY, XP002113078 cited in the application *
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WO2008013859A3 (fr) * 2006-07-28 2009-12-10 Adlyfe, Inc. Sondes peptidiques pour des diagnostics et des produits thérapeutiques
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US9289488B2 (en) 2010-08-12 2016-03-22 Ac Immune Sa Vaccine engineering
US9687447B2 (en) 2010-10-26 2017-06-27 Ac Immune S.A. Method for preparing liposome-based constructs
US20150183858A1 (en) * 2011-10-31 2015-07-02 Daniel J. Capon Peptide-hinge-free flexible antibody-like molecule
US9725503B2 (en) * 2011-10-31 2017-08-08 Daniel J. Capon Peptide-hinge-free flexible antibody-like molecule
US11345745B2 (en) 2011-10-31 2022-05-31 Biomolecular Holdings Llc Peptide-hinge-free flexible antibody-like molecule
JPWO2021100744A1 (fr) * 2019-11-19 2021-05-27
WO2021100744A1 (fr) * 2019-11-19 2021-05-27 Ｔａｏヘルスライフファーマ株式会社 PRODUIT RÉTICULÉ DE LA PROTÉINE AMYLOÏDE-β (Aβ) POUVANT ÊTRE SUBSTITUÉ PAR L'AMYLOSPHÉROÏDE (ASPD), ET ANALYSE DE L'ASPD

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WO1999041279A3 (fr) 1999-10-28

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