WO2000043027A1 - Utilisation d'une proteine inhibitrice du complement viral dans le traitement et le diagnostic de la maladie d'alzheimer - Google Patents

Utilisation d'une proteine inhibitrice du complement viral dans le traitement et le diagnostic de la maladie d'alzheimer Download PDF

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WO2000043027A1
WO2000043027A1 PCT/US2000/001115 US0001115W WO0043027A1 WO 2000043027 A1 WO2000043027 A1 WO 2000043027A1 US 0001115 W US0001115 W US 0001115W WO 0043027 A1 WO0043027 A1 WO 0043027A1
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protein
vcp
complement
cells
binding
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PCT/US2000/001115
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Girish J. Kotwal
James Daly, Iv
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University Of Louisville Research Foundation, Inc.
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Priority to US09/889,624 priority Critical patent/US7084106B1/en
Priority to AU27294/00A priority patent/AU2729400A/en
Publication of WO2000043027A1 publication Critical patent/WO2000043027A1/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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention provides a novel treatment for senile dementia (Alzheimer's Type), comprising administering an anti-complement protein to a patient in need of such treatment in an amount sufficient to inhibit the complement cascade and thereby inhibit the production of amyloid plaques in the brain of the patient.
  • AD Alzheimer's Disease
  • Alzheimer reported a case of what he termed "presenile dementia” in a 51 year old patient at a psychiatric meeting in Southwest Germany. He recognized certain characteristics that he felt differentiated it from the usual diagnosis of dementia. First was the early onset of the disease in an otherwise healthy young woman. More importantly, however, were the histological changes he found in sections of brain tissue from the patient. Alzheimer described seeing amyloid (starch-like) plaques and coarse-fibered proliferations of neurofibhls under the microscope. Several other researchers in years following reported similar findings of presenile dementia, and in 1910 a textbook of psychiatric disorders defined this form of dementia as "Alzheimer's disease.” The eponym was adopted in the literature and became the standard. It is perhaps fitting, since Alzheimer's original observations are still the main criteria of diagnosis for the disease.
  • plaques and neurofibhls described by Alzheimer which are today called senile plaques and neurofibrillary tangles (NFT), are used as a definitive diagnosis of AD ( Figure 1 ) (Palmert, M.R. et al. (1996) Science 24:11080-11084).
  • the plaques and tangles are seen primarily in the hippocampus, amygdala, and the cerebral cortex (Van Broeckhoven, C.L. (1995) Eur. Neurol. 35:8-19).
  • Evidence for either a molecular or immunological disease origin may be found in the plaques and tangles, depending upon a researcher's point of reference.
  • Presenilins 1 and 2 are integral membrane proteins coded for on different chromosomes, that when mutated are responsible for up to 90% of the cases of autosomal dominant early-onset familial Alzheimer's disease (FAD) (Thinakaran, G. et al. (1996) Neuron 17(1 ):181-19O). Although FAD accounts for only 10%, of all cases of AD, there is evidence of an interaction between the presenilins and APP (Weidemann, A. et al. (1997) ⁇ /af. Med. 3(3):328-332).
  • apoE apolipoprotein E
  • apoE4 apolipoprotein E
  • AD histopathological investigations into AD also point to the immune response having an important role in disease progression.
  • APP is capable of binding specifically to C1q, which can trigger activation of the classical pathway of the complement cascade in an antibody-independent manner (Webster, S. et al. (1995) Biochem. Biophys. Res. Comm. 217:869-875).
  • Deposition of complexes and formation of immunomodulators by the cascade have been credited with activating the microglia - macrophages of the brain - which in turn cause a progression and maintenance of the inflammation.
  • Local tissue destruction follows, along with a further persistence and increase in inflammation.
  • plaques are the most distinctive feature found in the brain tissue of patients with AD. These characteristic plaques are composed of an aggregation of the A ⁇ peptide, which is a by-product of APP metabolism. Morphologically distinct varieties of A ⁇ deposits have been described from conventional silver staining of histological sections:
  • Diffuse plaques are found in nondemented persons over the age of 65 years and are immunoreactive with a variety of different anti-AP antibodies. However, they generally are not stained with dyes like Congo red and thioflavin S.
  • amyloid plaques also referred to as neuritic plaques because they contain cells and subcellular components such as astrocytic processes, dystrophic neurites, microglia, neurons with or without neurofibrillary tangles and proteins such as complement components, apolipoprotein E and alpha-1-chymotrypsin) develop at later stages of AD. As seen in the autopsied brain tissue in Figure 1 , they appear dark in the center when stained with silver, and also stain with both Congo red and thioflavin S.
  • AY plaques have been reported. This third type of plaque is similar to the amyloid plaques but have no central amyloid core (Schmidt, M.L. et al. (1995) Am. J. Pathol. 147:503- 515. Besides plaques, the intracellular neurofibrillary tangles are often also characteristic of AD brain tissue. The plaques and neurofibrillary tangles are the primary diagnostic features of AD. However, immunohistochemical staining is becoming more useful as additional antibodies specific to plaque components are developed. An immunodominant region of APP has been localized to the C-terminal tail.
  • This region of the processed APP had been postulated to remain intracellular and was recently shown to accumulate specifically in the neuronal cells of the hippocampus and amygdala of AD patients, but not in similar tissue of the normal age-matched patients (Kotwal, G.J. et al. (1997) Soc. Neurosci. Abstr. 22:502).
  • a high titer antibody to the immunodominant region can give rise to specific intracellular immunohistochemical staining in the amygdala, which may someday find routine usage to confirm diagnosis in conjunction with clinical history and silver staining.
  • the presence of the C-terminus demonstrates that the C-terminal tail of APP accumulates intracellularly in neural tissue of those suffering from AD.
  • a ⁇ accumulation is a necessary step in the pathogenesis of AD, but does not account for all the pathogenesis of AD.
  • the gene that is implicated as a risk factor in late-onset sporadic AD and associated with increased risk for AD is the lipid carrier, apolipoprotein E (apoE) epsilon 4 allele (Stritmatter, W.J. & Roses, A.D. (1996) >4w?. Rev. Neurosci. 19:53). It is suggested that apoE4 is less efficient at cell repair than the alleles E2 and E3.
  • apoE4 apolipoprotein E
  • the apoE4 is thought not only to contribute to the formation of ⁇ -amyloid plaques, but also to account for the neurofibrillary tangles. It has been suggested that apoE may bind to A ⁇ and after exiting astrocytes may get trapped into low-density lipoprotein (LDL)-related proteins present on the surface of neurites of apoptotic neurons. ApoE is further suggested to provide protective binding to the tau protein, thus preventing homodimerization. The homodimerization creates paired helical filament formation, which in turn forms the neurofibrillary tangles.
  • LDL low-density lipoprotein
  • PS1 and PS2 The most recent group of proteins discovered to have an involvement in AD are the two presenilins (PS1 and PS2). Point mutations in these proteins are sufficient to cause early-onset FAD. The roughly 30 mutations in PSI and 2 in PS2 account for at least 50% of the early-onset FAD cases. These proteins are highly conserved with each other.
  • APP is an integral membrane glycoprotein with a large extracellular amino terminus, a single passage through the cell membrane, and a short carboxy-terminal tail, as illustrated in Figure 2. It is expressed ubiquitously throughout the body and is found in several tissue-specific isoforms due to variant splicing.
  • the largest isoform (APP 770 ) contains a putative kunitz protease inhibitor (KPI); however, the form found predominantly in neuronal tissue, APP 695 does not contain the KPI region.
  • KPI putative kunitz protease inhibitor
  • the normal functions of APP have been proposed to include action as a cell receptor, involvement in cell-cell interactions, and inhibition of proteases (KPI), among others (Selkoe, D.J. (1997) Sc/ ' ence 275:630-631).
  • APP contains a signal peptide on its N-terminus and is membrane- anchored, so it is processed through the secretory pathway after translation (see Figure 3). This pathway begins with cotranslational translocation across the membrane of the endoplasmic membrane (ER) into its lumen, with anchorage in the membrane occurring at the transmembrane domain. Processing of the protein to maturity, including glycosylation and sulfation, continues through the ER and into the Golgi network (Adams, C. (1997) Gerontology 43:8-19).
  • Proteolytic cleavage of APP can also occur as part of its processing through the secretary pathway. Somewhere between the trans-Golgi network and its localization on the cell surface, APP is cleaved at a region on the extracellular side of APP near the membrane in the region that could otherwise form A ⁇ (Sisodia, S.S. et al. (1990) Science 248:492-495), thus prohibiting its formation.
  • the membrane-bound endoprotease responsible for the cleavage has been dubbed ⁇ -secretase and appears to require for its specificity no more than an ⁇ -helical conformation and a specified distance of 12-13 residues from the membrane (Sisodia, S.S. (1992) Proc. Nat. Acad. Sci.
  • a ⁇ is likely to be formed during this process, probably in the early endosome, due to proteolytic cleavage by enzyme(s) called ⁇ -secretase and Y-secretase (see Figure 2). Recycling to the surface would result in release of A ⁇ into the extracellular medium. It has been demonstrated that A ⁇ production requires APP to be membrane bound and localized to a slightly acidic vesicle, such as the early endosome or the late trans-Golgi (Selkoe, D.J. (1996) Ann. N.Y. Acad. Sci. 777:57-64). The entire processing pathway is tightly controlled.
  • PLC protein kinase C
  • muscarinic receptors For instance, upregulation of the protein kinase C (PKC) pathway by muscarinic receptors results in an increase in the ⁇ -secretase cleavage and a decrease in A ⁇ production. Serum levels of ligands for these receptors, such as acetylcholine and interleukin-1 (IL-1 ), have been reported to be abnormal in AD patients (Buxbaum, J.D. & Greengard, P. (1996) Ann. N.Y. Acad. Sci. 777:327-331 ). An intracellular increase in calcium will also upregulate ⁇ -secretase activity (Adams, C. (1997) Gerontology 43:8-19).
  • PPC protein kinase C
  • mutating APP such as occurs in some cases of early- onset FAD, is sufficient to cause AD.
  • a ⁇ in diffuse plaques (precluding symptom onset), its ability to form insoluble filaments in vitro, trisomy 21 in Down's syndrome (patients develop early-onset AD), the exhibited direct neurotoxicity, and the interaction with numerous molecules believed to be involved with AD including immune molecules, all point to APP's, and specifically A ⁇ 's, pivotal role in AD pathogenesis (Spillantini, M.G. et al. (1996) /.cfa Neuropath. (Berl.) 92:42-48).
  • a final proposed role of APP in AD involves its C-terminal region. It has been demonstrated that a specific region of the APP terminus (amino acids 657-676) is able to specifically bind and activate heterotrimeric G proteins (see Figure 2) (Selkoe, D.J. (1996) J. Biol. Chem. 271 :18295-
  • NFTs neurofibrillary tangles
  • microtubule destabilization has been proposed to disrupt axonal transport which causes dying back of axons, impairing synaptic transmission (Peskind, E.R. (1996) J. Clin. Psychiatry 57 (Suppl. 14): 5-8).
  • Such a mechanism of AD pathogenesis would not require an interaction with A ⁇ and therefore would not necessitate plaque formation.
  • comparison of a recently discovered presenile dementia lacking plaques but containing tangles shows tau forms and NFTs to be identical to those in AD according to several different analytical methods (Spillantini, M.G. et al. (1996) Ada Neuropath. (Berl.) 92:42-48).
  • Hyperphosphorylated tau binds itself, forming long filaments (PHFs), which accumulate intracellularly to form the recognized NFTs. It his been shown that carbamoylation or glycation of cationic tau residues will result in NFTs like those seen in AD. These cationic sites, particularly lysine residues, appear to be important for microtubule binding. Therefore, the phosphorylation of tau probably blocks the normal binding sites of tau to microtubules, preventing interaction (Farias, G. et al. (1997) Mol. Cell. Biochem. 168:59-66). NFTs may themselves be pathogenic if they accumulate to such an extent within the cell that they impair normal cellular processes.
  • isoforms of apoE may interact with tau as well and inhibit or enhance NFT formation (discussed above).
  • ApoE plays a role in at least sporadic AD, and perhaps FAD as well.
  • the ApoE4 isoform increases the likelihood and decreases the age of onset of AD (Corder, E.H. et al. (1993) Science 261 :921-923).
  • ApoE is a 34-kDa protein found circulating throughout the body as well as the central nervous system.
  • ApoE scavenges lipid from degenerating neurons and redistributes it to branching neurites via uptake by the low-density Hpoprotein (LDL) or LDL- related protein (LRP) receptors (Mahley, R.W. et al. (1996) Ann. N.Y. Acad. Sci. 777:139-145). ApoE also binds A ⁇ , and so along with its receptor may be a means of mopping up extracellular A ⁇ (Rebeck, G.W. (1995) Ann. Neurol. 37:211-217).
  • LDL low-density Hpoprotein
  • LRP LDL-related protein
  • Presenilins 1 and 2 are also involved in AD.
  • PS1 has been shown to be membrane-anchored and contain eight membrane-spanning regions. The amino and carboxyl termini are both located on the cytoplasmic side of the membrane, along with a large hydrophilic loop between transmembrane domains six and seven. This loop is proteolytically cleaved during maturation of the protein to produce an approximately 25-28 kDa N-terminal and an approximately 16-19 kDa C-terminal protein. The shape is hypothesized to be a barrel within the membrane, with the loop acting as a gate (Rohan de Silva, H.A., & Patel, A.J. (1997) Neuroreport 8(8):i-xii).
  • the mature form has been shown to localize primarily to the perinuclear membrane regions (ER and Golgi), with a small percentage in the surface membrane (Tanzi, R.E. et al. (1996) Alzheimer's Dis. Rev. 1 :190-198).
  • the presenilins show close homology to two proteins of Caenorhabditis elegans, SPE4 and SEL12.
  • SPE4 mediates the docking of a Golgi-derived organelle which stores and transports polypeptides (Takeshima, A. et al. (1996) Biochem. Biophys. Res. Comm. 227:423-426). This suggests a role in protein trafficking through the secretary pathway for the presenilins.
  • SEL12 facilitates signaling by the Notch family of receptors (Beyreuther, K. & Masters, C.L. (1997) Nature Med. 3:723-725). These receptors are involved in determination of cell fate during development. This may indicate a role in cell signaling for the presenilins, or perhaps SEL 12 controls transport of second messengers between the nuclear and surface membrane. The latter possibility would again suggest a role for the presenilins in protein trafficking.
  • a role in cell signaling or protein trafficking relates to AD pathogenesis in the following manner. It is known that mutations in either of the presenilins result in an increase in the expression of A ⁇ 42 without an increase in APP expression (Citron, M. et al. (1997) Nature Med. 3:67-72). Also, immunoprecipitation of coexpressed PS2 and APP in cell culture results in complexes of PS2/immature APP (Weidemann, A. et al. (1997) ⁇ 7af. Med. 3(3):328-332). Together, these two pieces of evidence, along with homology and localization studies, suggest that presenilins regulate trafficking and processing of APP through the secretary pathway. When mutations occur in presenilins, it is likely that conformational changes induce a change of function or a new function with regard to APP processing, which results in an increase in the amount of A ⁇ produced.
  • the neuritic/amyloid plaques which are the hallmark of AD consist of aggregated A ⁇ , along with a number of complement components and complement control proteins such as C1 inhibitor and other noncomplement proteins.
  • the neuritic plaques are associated with damaged (dystrophic) neuronal processes, activated microglia, and reactive astrocytes.
  • active regions of several of the microbial immunomodulatory proteins which target the complement cascade at various sites, can be employed for therapeutic intervention in inflammatory responses due to activation of the complement cascade (Kotwal, G.J. (1996) Immunologist 4:157-164).
  • the inflammation modulatory protein (IMP) a small complement-binding protein that has been shown to be functionally similar to CR1 , has been shown in in vivo experiments to cause a diminished specific swelling response (Miller, CG. et al. (1997) Virology 22:126-133).
  • CM I cell- mediated immunity
  • B and T cells which give rise to anti-neuronal antibody and cytotoxic T- cell (CTL) activity, respectively. This somehow signals across the blood- brain barrier and leads to a chronic phase in the brain.
  • the CD8 + CTLs either directly through CMI or indirectly through glia activation (microglial and astroglial cells), induce target cell cytotoxicity.
  • the activation of the glial cells then causes nonspecific tissue damage.
  • the neuron-specific degeneration characteristic of AD is due to the CMI directed by specific autoantibodies.
  • a large number of autoantibodies and immune abnormalities, some of which are inherited, have been cataloged by Singh (id.)
  • a major approach to the treatment of AD has involved attempts to augment the cholinergic function of the brain.
  • An early approach was the use of precursors of acetylcholine synthesis, such as choline chloride and phosphatidyl choline (lecithin). Although these supplements are generally well tolerated, randomized trials have failed to demonstrate any clinically significant efficacy.
  • Direct intracerebroventricular injection of cholinergic agonists such as bethanacol appears to have some beneficial effects, although this requires surgical implantation of a reservoir connecting to the subarachnoid space and is too cumbersome and intrusive for practical use.
  • a somewhat more successful strategy has been the use of inhibitors of acetylcholinesterase (AChE), the catabolic enzyme for acetylcholine.
  • AChE acetylcholinesterase
  • Physostigmine a rapidly acting, reversible AChE inhibitor, produces improved responses in animal models of learning, and in patients with AD some studies have demonstrated mild transitory improvement in memory following physostigmine treatment.
  • the use of physostigmine has been limited because of its short half-life and tendency to produce symptoms of systemic cholinergic excess at therapeutic doses.
  • tacrine acridine derivative tacrine
  • COGNEX® 1 ,2,3,4- tetrahydro-9-aminoacridine
  • Tacrine was first synthesized nearly fifty years ago, and the pharmacology of this agent has been the subject of numerous studies. It is a potent centrally acting inhibitor of AChE.
  • the side effects of tacrine may be significant and dose- limiting: abdominal cramping, nausea, vomiting, and diarrhea are observed in up to one-third of patients receiving therapeutic doses. Tacrine may also cause hepatotoxicity, as evidenced by the elevation of serum transaminases observed in up to 20% of patients treated. Because of the relatively small improvements that result from tacrine treatment and the significant side- effect profile, its clinical usefulness is limited.
  • the present invention features a Vaccinia virus encoding a complement control protein which can block the complement pathway, binding to complement components. This property can be used in the treatment and diagnosis of Alzheimer's disease.
  • the viral protein termed VCP, can block the complement activation in AD and can bind to plaques in pathological samples.
  • the present invention relates to a method for treating Alzheimer's disease comprising administering to a patient in need of such treatment a therapeutic amount of the protein of Formula (I):
  • the invention further provides a pharmaceutical formulation for the treatment of Alzheimer's Disease, which comprises a protein of the Formula (I) together with one or more pharmaceutical acceptable diluents, carriers or excipients therefore.
  • AD Brain Tissue The left photograph shows a conventional silver stained section of brain tissue from postmortem AD patient showing the characteristic hallmark of AD, the amyloid/neuritic plaques (indicated by arrows). The central portion of a plaque is a dense, dark brown to black region surrounded by a clear ring and then a ring of particulate matter comprising cell debris, activated microglial cells, and several different proteins.
  • the right photograph shows immunohistochemical staining of a microsection of the amygdala (a brain structure that plays a major role in emotions such as fear and anxiety and in the startle response) from a postmortem AD patient.
  • a high-titer antibody raised in a rabbit against a synthetic peptide whose sequence was derived from a computer-generated immunodominant region within the putative intracellularly localized C- terminal tail of APP was used as a primary antibody. This was followed by a peroxidase-conjugated anti-rabbit secondary antibody. Brown color developed on addition of DAB reagent. The nuclei were finally stained with hematoxylin. the numerous brown threads seen specifically in the AD tissue are indicative of the presence of the cytoplasmic C-terminal tail of APP in the neuropils (dead/dying neurons).
  • FIG. 1 A detailed diagram of the major isoform of amyloid precursor protein (APP 695 ) present in neurons.
  • the 17-amino acid signal peptide is cotranslationally cleaved in the endoplasmic reticulum.
  • the APP has a cysteine-rich region (8 cysteines within a span of 150 amino acids), as well as an acidic region with a high percentage of the acidic amino acids glutamic acid and aspartic acid.
  • a threonine-rich region is marked Thr.
  • Altematively spliced mRNA may have an insertion of up to 225 nucleotides that encodes a 56-amino acid Kunitz protease inhibitor region (KPI) to form the isoform APP 751 , and another additional 19-amino acid region (isoform APP 770 ).
  • KPI 56-amino acid Kunitz protease inhibitor region
  • the APP anchors to the cell surface via a membrane-spanning region.
  • the cell surface APP can either be digested by ⁇ - secretase and gamma-secretase to give rise to ⁇ -APPs (584 amino acids) and a small P3 protein, which are soluble and are released into the extracellular space, with the C-terminus staying behind (as shown in the middle diagram).
  • ⁇ -APPs 584 amino acids
  • a small P3 protein which are soluble and are released into the extracellular space, with the C-terminus staying behind (as shown in the middle diagram).
  • it may be digested by ⁇ -secretase and gamma-secretase to give rise to A ⁇ containing a region (40-42 amino acids) that is secreted along with ⁇ -APPs (578 amino acids), with the C-terminal tail remaining cell associated.
  • the A ⁇ aggregates to form the nucleus of the amyloid plaque and has a region that can bind C1q and activate the classical complement pathway.
  • the A ⁇ can also couple to C3 via an ester linkage and activate the alternative pathway.
  • the cytoplasmic C-terminal tail has been speculated to trigger a cascade of events contributing to the apoptosis of the APP-producing neurons. Such a cascade is thought to be initiated by the binding of the C-terminal region to a particular family of GTP binding proteins (G 0 ).
  • FIG. 3 The aberrant biosynthesis of APP accounts for some of the amyloid plaque formation. This is complicated by other proteins regulating the amount of APP synthesized and process, such as the presenillins 1 and 2.
  • the neurofibrillary tangles may be formed with the tau protein homodimerizes in the presence of the epsilon 4 allele of apoE, statistically found more often in persons susceptible to neurodegenerative disorders.
  • the second and third alleles may be able to block the sites on tau that contribute to the formation of homodimers, thereby contributing to the normal cytoskeletal structure.
  • ApoE besides having sites for binding to receptor, has a lipid binding site to which A ⁇ can bind. A ⁇ can thus be transported from astrocytes to the neuronal surface and recycled via endosomes and lysosomes to remain cytoplasmic and become incorporated in the neurofibrillary tangles.
  • FIG. 4 Antibody-independent activation of the classical complement pathway (CCP and initiation of inflammatory events in AD.
  • a ⁇ , its aggregate, or the amyloid plaque can trigger the CCP by binding to C1q, shown in blue.
  • the binding to C1q occurs via the first few amino acids of A ⁇ (4-11 ), with the aspartate in position 7 being the most critical for binding.
  • a ⁇ or its aggregate can form ester links to the C3 component and can activate the alternate complement pathway (ACP), shown in yellow.
  • Complement activation can result in the release of chemotactic factors such as C3a, C4a, and C5a, shown here in green. These factors can cause an influx of microglial cells and astrocytes which contribute to the inflammatory response. These cells are also present in the neuritic plaques.
  • FIG. 5 Generation of recombinant plasmid pAPPc PCR was performed on a modified pUC 18 vector, pFB68L, containing the Ap Pe 95 C-terminus (nucleotidesl786-3207) incorporated at the EcoRI restriction site.
  • the primers were designed so that the 1. I kbp PCR product would contain the two new restriction sites A/col and Sa/I. Digestion of the PCR product and pTM3 with ⁇ /col and Sail and subsequent ligation produced plasmid pAPPc.
  • MCS multiple cloning site
  • T7 bacteriophage promoter pT7
  • T7t termination sequence
  • EMC encephalomyocarditis virus "translational enhancer” leader sequence
  • GTT guanosyl phosphoribosyl transferase
  • p7.5K 7.5 kbp vaccinia promoter
  • TK thymidine kinase
  • origin of replication ori
  • ampicillin resistance gene ampicillin resistance gene
  • FIG. 6 Sequence of pAPPc within insert and flanking regions
  • the DNA and amino acid sequences of pAPPc and the flanking pTM3 sequence is shown with primer sets, restriction sites and important regions noted.
  • The. arrows indicate the location and direction of the five sets of primers synthesized for DNA sequencing.
  • mice Thirty-two animals were injected in a series of three experiments with ei-ther Trisbuffered saline or Tris buffer, control product (pTM3) or C-100 (APP), or AB peptide. The mice were sacrificed at either five (5h) or 48 (48h) hours after injection. C5+/+ and C5-/- mice were used. Numbers in parenthesis indicate the number of individual mice in each group.
  • FIG. 8 Connective tissue Air pouch cell counts
  • Cell count bars are means of all gfids counted from animals injected with 100 microliters of sample listed on x-axis, plus or minus standard deviation shown as an error bar.
  • 4A is a summary of counts from mice sacrificed five h post injection.
  • 4B is a summary of counts from mice sacrificed 48 h post injection.
  • Air pouch connective tissue section composite Representative photos of single-layer connective tissue spreads from one experiment.
  • Left column panels (5A, C, E and G) are from C-5-/- mice injected, whereas right column panels (58, D, F and H) are from C5+/+ mice injected.
  • Panels 5A, B, C and D are from mice sacrificed five hours after injection.
  • Panels 5E, F, G and H are from mice sacrificed 48 h after infection.
  • Mouse sections in panels 5A, B, E and F were from animals injected with in vitro transcription/translation product encoded by control pTM3 DNA, and those in 5C, D, G and H were injected with product from in vitro transcription/translation reaction encoded by pAPPc DNA.
  • Migrating cells found most frequently in epithelial air pouch sections are indicated: resident fibroblast, ( «-) neutrophil, ( ⁇ ) eosinophil, ( ⁇ ) monocytes. The magnification used in 400X.
  • FIG. 10 Immunoassay for complement activation by A ⁇ 6A.
  • Internal controls for quantitation of results in ng/mi included standards A, B and C; and high (HPC) and low (LPC) positive controls.
  • external controls of heat activated IgG (HAG) and zymosan were included for confirmation of intact complement pathway in normal (NHS) and agammaglobulinemic (AHS) human sera.
  • 6B Measurement of NHS complement activation in the presence or absence of 50 ⁇ M A ⁇ peptide (A-beta(50)). Inhibition of complement activation by AB in NHS was determined by the addition of purified VCP. Background activation is demonstrated by the incubation of NHS with TBS. Standard deviation is indicated by error bars. 6C. Measurement of AHS complement activation in the presence or absence of 50 ⁇ M A ⁇ peptide, with or without added VCP. Background activation is demonstrated by the incubation of NHS with TBS.'The addition of MEM to all reactions was necessary because VCP is solubilized in it and also MEM seems to enhance complement activation by A ⁇ .
  • FIG. 11 Intervention by VCP into proposed inflammatory cascade due to A ⁇ complement activation. Aggregated A ⁇ activates the complement cascade that results in the deposit of complement components in the nascent plaque as well as releasing chemotactic factors. These factors such as C5a can stimulate an immune response that results in the activation and influx of microglial cells. The accumulated microglia cause more local tissue damage and amplify and perpetuate the inflammation, which results in plaque growth. VCP inhibits both pathways of complement activation by binding to complement components C3 and C4, which results in the diminished preferential release of the chemotactic factor C5a, as demonstrated by the X superimposed over the arrow. Therapeutically, this could alleviate some of the tissue damage caused by a self-perpetuating inflammatory response.
  • Figure 12 Representative histograms showing that VCP is able to reduce mouse antihuman HLA class I antibody binding to HUVECs cells.
  • the results of flow microflourimetric analysis are as follows: A) Nonspecific mouse monoclonal antibody (isotype-matched mouse lgG2a mAb) binding to HUVECs cells (negative control) B) Mouse monoclonal antihuman HLA- ABC antibody binding to HUVECs cells (positive control) C) Mouse monoclonal antihuman HLA-ABC antibody binding to HUVECs cells in the presence of 5 ⁇ l (2 ⁇ g) of VCP D) Mouse antihuman HLA-ABC antibody to HUVECs cells in the presence of 20 ⁇ l (5 ⁇ g) of VCP.
  • FIG 13. PAGE analysis of the heparin binding activity of BSA, HBP, lysozyme, and MIP-1 alpha.
  • various proteins BSA, HBP, lysozyme, and MIP-1 ⁇
  • S.M. starting material
  • Unb. unbound fraction
  • W wash.
  • Figure 14 PAGE analysis of the heparin binding activity of VCP and rVCPs.
  • VCP and various rVCPs were passed through separate HiTRAP heparin columns and eluted with sodium chloride concentrations ranging from 250 mM to 2.5 M. The fractions were collected, run on SDS PAGE, silver stained, and the band densities measured. The results are shown as follows: A) recombinant rVCP SCR (1 ,2) B) recombinant rVCP SCR (2,3) C) recombinant rVCP SCR (3,4) D) recombinant rVCP E) VCP from the natural infection process.
  • M molecular weight marker
  • S.M. starting material
  • Unb. unbound fraction
  • W wash
  • VCP VCP from natural infection.
  • Figure 15 Sequence alignment including termini of rVCP constructs and putative heparin binding sites. Multiple alignment of the four short consensus repeats (SCR) from orthopoxviruses VAC-COP (vaccinia virus, Copenhagen strain) (Goebel et al., 1990 Virology 179:247-263), VAC-WR (vaccinia virus, western reserve strain) (Kotwal, G.J. et al. 1989 Virology 171 :579-587), CPV-GRI (cowpox virus, Russian isolate from human patient) (Schelkunov, S.N., et al.
  • SCR short consensus repeats
  • K+R positively charged amino acids
  • %K+R percentage of positively charged amino acids
  • FIG. 1 VCP model showing the heparin binding sites.
  • Front (A) and back (B) views of the modeled structure of VCP SCR (1-4) showing the heparin binding sites (differentially colored). In order to differentiate the extents of the individual modules, they are shaded appropriately.
  • the present invention provides a method for treating Alzheimer's disease comprising administering to a patient in need of such treatment a therapeutic amount of the protein of Formula (I):
  • amino acid abbreviations are set forth below:
  • Base pair (bp) - refers to DNA or RNA.
  • the abbreviations A,C,G, and T correspond to the 5'-monophosphate forms of the nucleotides (deoxy)adenine, (deoxy)cytidine, (deoxy)guanine, and (deoxy)thymine, respectively, when they occur in DNA molecules.
  • the abbreviations U,C,G, and T correspond to the 5'-monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules.
  • base pair may refer to a partnership of A with T or C with G.
  • base pair may refer to a partnership of T with U or C with G.
  • Chelating Peptide - An amino acid sequence capable of complexing with a multivalent metal ion.
  • EDTA an abbreviation for ethylenediamine tetraacetic acid.
  • ED50 an abbreviation for half-maximal value.
  • FAB-MS an abbreviation for fast atom bombardment mass spectrometry.
  • Immunoreactive Protein(s) - a term used to collectively describe antibodies, fragments of antibodies capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived, and single chain polypeptide binding molecules as described in PCT Application No. PCT/US 87/02208, International Publication No. WO 88/01649. mRNA - messenger RNA.
  • MWCO - an abbreviation for molecular weight cut-off.
  • Patient - a patient is any animal, usually a mammal, preferably a human.
  • PMSF an abbreviation for phenylmethylsulfonyl fluoride.
  • Reading frame the nucleotide sequence from which translation occurs "read” in triplets by the translational apparatus of tRNA, ribosomes and associated factors, each triplet corresponding to a particular amino acid. Because each triplet is distinct and of the same length, the coding sequence must be a multiple of three. A base pair insertion or deletion (termed a frameshift mutation) may result in two different proteins being coded for by the same DNA segment. To insure against this, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e. the correct "reading frame” must be maintained. In the creation of fusion proteins containing a chelating peptide, the reading frame of the DNA sequence encoding the structural protein must be maintained in the DNA sequence encoding the chelating peptide.
  • Recombinant DNA Cloning Vector any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
  • Recombinant DNA Expression Vector any recombinant DNA cloning vector in which a promoter has been incorporated.
  • Replicon - A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.
  • RNA - ribonucleic acid A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.
  • RP-HPLC an abbreviation for reversed-phase high performance liquid chromatography.
  • Transcription the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.
  • Treating - describes the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. Treating obesity therefor includes the inhibition of food intake, the inhibition of weight gain, and inducing weight loss in patients in need thereof.
  • Vector - a replicon used for the transformation of cells in gene manipulation bearing polynucleotide sequences corresponding to appropriate protein molecules which, when combined with appropriate control sequences, confer specific properties on the host cell to be transformed. Plasmids, viruses, and bacteriophage are suitable vectors, since they are replicons in their own right. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. vectors include Recombinant DNA cloning vectors and Recombinant DNA expression vectors.
  • X-gal - an abbreviation for 5-bromo-4-chloro-3-idolyl beta-D- galactoside.
  • SEQ ID NO: 1 refers to the sequence set forth in the sequence listing and means a complement-inhibiting protein of the formula: M K E V S V T F L T L L G I G C V L S C C T I P S R P I N M K F N S V E T D A N A N Y N I G D T I E Y L c L P G Y R K Q K M G P I Y A K C T G T G
  • the present invention provides a method for treating Alzheimer's disease comprising administering to an organism an effective amount of a compound of Formula (I) in a dose between about 1 and 1000 ⁇ g/kg.
  • a preferred dose is from about 10 to 100 ⁇ g/kg of active compound.
  • a typical daily dose for an adult human is from about 0.5 to 100 mg.
  • compounds of the Formula (I) can be administered in a single daily dose or in multiple doses per day.
  • the treatment regime may require administration over extended periods of time.
  • the amount per administered dose or the total amount administered will be determined by the physician and depend on such factors as the nature and severity of the disease, the age and general health of the patient and the tolerance of the patient to the compound.
  • the instant invention further provides pharmaceutical formulations comprising compounds of the Formula (I).
  • the proteins preferably in the form of a pharmaceutically acceptable salt, can be formulated for parenteral administration for the therapeutic or prophylactic treatment of obesity.
  • compounds of the Formula (I) can be admixed with conventional pharmaceutical carriers and excipients.
  • the compositions comprising claimed proteins contain from about 0.1 to 90% by weight of the active protein, preferably in a soluble form, and more generally from about 10 to 30%.
  • the present proteins may be administered alone or in combination with other anti-Alzheimer's agents or agents useful in treating dementia (i.e., improving cognitive function).
  • Preferred agents for use in combination with the protein of the present invention for treating Alzheimer's disease include acetylcholine precursors (e.g., choline chloride and phosphatidyl choline (lecithin)); cholinergic agonists (e.g., acetylcholine, acetyl-L-camitine, anatoxine a, arecoline, bethanecol, carbachol, decamethonium, 1 ,1-dimethyl-4-phenyl-piperazinium, c/s-Dioxolane, epibatidine, epiboxidine, methacholine, methylcarbamylcholine, methylfurtrethonium, metoclopramide, muscarine, nicotine, oxotremorine, pilocarpine, and pharmaceutically acceptable salts thereof); cholinesterase inhibitors (e.g., ambenonium, adrophonium, methylphysostigmine, n
  • the protein is administered in commonly used intravenous fluid(s) and administered by infusion.
  • intravenous fluids as, for example, physiological saline, Ringer's solution or 5% dextrose solution can be used.
  • a sterile formulation preferably a suitable soluble salt form of a protein of the Formula (I) , for example the hydrochloride salt
  • a pharmaceutical diluent such as pyrogen-free water (distilled), physiological saline or 5% glucose solution.
  • a suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.
  • the proteins for use in the presently claimed invention may be prepared by construction of the DNA encoding the claimed protein and thereafter expressing the DNA in recombinant cell culture.
  • the mutations that might be made in the DNA encoding the protein of the present invention must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See DeBoer et al., EP 75.444A (1983).
  • the protein of the present invention may be produced either by recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods.
  • the synthesis of the protein of the present invention may proceed by solid phase peptide synthesis or by recombinant methods.
  • the principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area such as Dugas, H. and Penney, C, Bioorganic Chemistry, Springer-Verlag, New York, pgs. 54-92 (1981).
  • peptides may be synthesized by solid-phase methodology utilizing an PE-Applied Biosystems 430A peptide synthesizer (commercially available from Applied Biosystems, Foster City California) and synthesis cycles supplied by Applied Biosystems. Boc amino acids and other reagents are commercially available from PE-Applied Biosystems and other chemical supply houses.
  • Tyr, 4-bro ⁇ mo carbobenzoxy Boc deprotecfion may be accomplished with trifluoroacetic acid (TFA) in methylene chloride.
  • TFA trifluoroacetic acid
  • Formyl removal from Trp is accomplished by treatment of the peptidyl resin with 20% piperidine in dimethylformamide for 60 minutes at VC.
  • Met(O) can be reduced by treatment of the peptidyl resin with TFA/dimethylsulfide/conHC1 (95/5/1 ) at 25°C for 60 minutes.
  • the peptides may be further deprotected and cleaved from the resin with anhydrous hydrogen fluoride containing a mixture of 10% m-cresol or m-cresol/10% p-thiocresol or m-cresol/p- thiocresol/dimethylsulfide.
  • Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at zero degrees Centigrade or below, preferably -20°C for thirty minutes followed by thirty minutes at 0°C.
  • the peptide/resin is washed with ether.
  • the peptide is extracted with glacial acetic acid and lyophilized.
  • the protein of the present invention may also be produced by recombinant methods. Recombinant methods are preferred if a high yield is desired.
  • the basic steps in the recombinant production of protein include: a) construction of a synthetic or semi-synthetic (or isolation from natural sources) DNA encoding the protein of the present invention, b) integrating the coding sequence into an expression vector in a manner suitable for the expression of the protein either alone or as a fusion protein, c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector, and d) recovering and purifying the recombinantly produced protein.
  • the gene encoding the claimed protein may also be created by using polymerase chain reaction (PCR).
  • the template can be a cDNA library (commercially available from CLONETECH or STRATAGENE) or mRNA isolated from human adipose tissue.
  • the claimed protein may be made either by direct expression or as fusion protein comprising the claimed protein followed by enzymatic or chemical cleavage.
  • a variety of peptidases e.g. trypsin
  • which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g. diaminopeptidase) of the peptide chain are known.
  • particular chemicals e.g. cyanogen bromide
  • the skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi- synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e.g., Carter P., Site Specific Proteolysis of Fusion Proteins, Ch. 13 in Protein Purification: From Molecular Mechanisms to Large Scale Processes, American Chemical Soc, Washington, D.C. (1990).
  • the claimed protein is a relatively large protein.
  • a synthetic coding sequence is designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into these expression and amplification and expression plasmids.
  • the isolated cDNA coding sequence may be readily modified by the use of synthetic linkers to facilitate the incorporation of this sequence into the desired cloning vectors by techniques well known in the art.
  • the particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed.
  • the choice of restriction sites are chosen so as to properly orient the coding sequence with control sequences to achieve proper in- frame reading and expression of the claimed protein.
  • plasmid vectors containing promoters and control sequences which are derived from species compatible with the host cell are used with these hosts.
  • the vector ordinarily carries a replication site as well as marker sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (Bolivar, et al., Gene 2: 95 (1977)).
  • Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA technology.
  • the desired coding sequence is inserted into an expression vector in the proper orientation to be transcribed from a promoter and ribosome binding site, both of which should be functional in the host cell in which the protein is to be expressed.
  • An example of such an expression vector is a plasmid described in Belagaje et al., U.S. patent No. 5,304,493, the teachings of which are herein are incorporated by reference.
  • the gene encoding A-C-B proinsulin described in U.S. patent No. 5,304,493 can be removed from the plasmid pRB182 with restriction enzymes Nde ⁇ and SamHI.
  • the genes encoding the protein of the present invention can be inserted into the plasmid backbone on a Nde ⁇ IBam ⁇ restriction fragment cassette.
  • procaryotes are used for cloning of DNA sequences in constructing the vectors useful in the invention.
  • E. coli K12 strain 294 (ATCC No. 31446) is particularly useful, other microbial strains which may be used include E. coli B and E. co//X1776 (ATCC No. 31537). These examples are illustrative rather than limiting. Prokaryotes also are used for expression. The aforementioned strains, as well as E. co// W3110 (prototrophic, ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, and various Pseudomonas species may be used.
  • Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase (vector pGX2907 [ATCC 39344] contains the replicon and ⁇ - lactamase gene) and lactose promoter systems (Chang et al., Nature, 275:615 (1978); and Goeddel et al., Nature 281 :544 (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATH1 [ATCC 37695] is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter) and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282).
  • vector pGX2907 contains the replicon and ⁇ - lactamase gene
  • lactose promoter systems Chang et al., Nature, 275:615 (1978); and Go
  • the protein may be recombinantly produced in eukaryotic expression systems.
  • Preferred promoters controlling transcription in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. ⁇ -actin promoter.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. Fiers, et al., Nature, 273:113 (1978).
  • the entire SV40 genome may be obtained from plasmid pBRSV, ATCC 45019.
  • the immediate early promoter of the human cytomegalovirus may be obtained from plasmid pCMBP (ATCC 77177).
  • promoters from the host cell or related species also are useful herein.
  • Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent having been found 51 (Laimins, L. et al., PNAS 78:993 (1981 )) and 3' (Lusky, M. L, et. al., Mol. Cell Bio. 3:1108 (1983)) to the transcription unit, within an intron (Banerji, J. L. et al., Cell 33:729 (1983)) as well as within the coding sequence itself (Osborne, T.
  • enhancer sequences are now known from mammalian genes (globin, RSV, SV40, EMC, elastase, albumin, a-fetoprotein and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 late enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding protein. The 3' untranslated regions also include transcription termination sites.
  • Expression vectors may contain a selection gene, also termed a selectable marker.
  • selectable markers for mammalian cells are dihydrofolate reductase (DHFR, which may be derived from the Bal ⁇ /Hind ⁇ restriction fragment of pJOD-10 [ATCC 68815]), thymidine kinase (herpes simplex virus thymidine kinase is contained on the BamHl fragment of vP-5 clone [ATCC 2028]) or neomycin (G418) resistance genes (obtainable from pNN414 yeast artificial chromosome vector [ATCC 37682]).
  • DHFR dihydrofolate reductase
  • thymidine kinase herepes simplex virus thymidine kinase is contained on the BamHl fragment of vP-5 clone [ATCC 2028]
  • neomycin (G418) resistance genes obtainable from pNN414 yeast artificial chromos
  • the transfected mammalian host cell can survive if placed under selective pressure.
  • selectable markers are successfully transferred into a mammalian host cell
  • the first category is based on a cell.s metabolism and the use of a mutant cell line which lacks the ability to grow without a supplemented media.
  • Two examples are: CHO DHFR " cells (ATCC CRL-9096) and mouse LTK " cells (L-M(TK-) ATCC CCL-2.3). These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine.
  • the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, Southern P. and Berg, P., J. Molec. Appl. Genet. 1 : 327 (1982), mycophenolic acid, Mulligan, R. C. and Berg, P. Science 209:1422 (1980), or hygromycin, Sugden, B. et al., Mol. Cell. Biol. 5:410-413 (1985). The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.
  • a preferred vector for eucaryotic expression is pRc/CMV.
  • pRc/CMV is commercially available from Invitrogen Corporation, 3985 Sorrento Valley Blvd., San Diego, CA 92121.
  • the ligation mixtures are used to transform E. coli K12 strain DH5a (ATCC 31446) and successful transformants selected by antibiotic resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction and/or sequence by the method of messing, et al., Nucleic Acids Res. 9:309 (1981 ).
  • Host cells may be transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the techniques of transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), or Current Protocols in Molecular Biology (1989) and supplements.
  • Preferred suitable host cells for expressing the vectors encoding the claimed proteins in higher eukaryotes include: African green monkey kidney line cell line transformed by SV40 (COS-7, ATCC CRL-1651 ); transformed human primary embryonal kidney cell line 293, (Graham, F. L. et al., J. Gen Virol. 36:59-72 (1977), Virology 77:319-329, Virology 86:10-21 ); baby hamster kidney cells (BHK-21 (C-13), ATCC CCL-10, Virology 16:147 (1962)); Chinese hamster ovary cells CHO-DHFR- (ATCC CRL-9096), mouse Sertoli cells (TM4, ATCC CRL-1715, Biol. Reprod.
  • eukaryotic microbes such as yeast cultures may also be used.
  • Saccharomyces cerevisiae, or common baker's yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available.
  • the plasmid YRp7 for example, (ATCC-40053, Stinchcomb, et al., Nature 282:39 (1979); Kingsman et. al., Gene 7:141 (1979); Tschemper et al., Gene 10:157 (1980)) is commonly used.
  • This plasmid already contains the trp gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC no. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)).
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (found on plasmid pAP12BD ATCC 53231 and described in U.S. Patent No. 4,935,350, June 19, 1990) or other glycolytic enzymes such as enolase (found on plasmid pAC1 ATCC 39532), glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPCI ATCC 57090, 57091 ), zymomonas mobilis (United States Patent No.
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein (contained on plasmid vector pCL28XhoLHBPV ATCC 39475, United States Patent No. 4,840,896), glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose (GALI found on plasmid pRY121 ATCC 37658) utilization. Suitable vectors and promoters for use in yeast expression are further described in R.
  • yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae (found in conjunction with the CYC1 promoter on plasmid YEpsec-hllbeta ATCC 67024), also are advantageously used with yeast promoters.
  • a DNA molecule encoding the present invention is as shown below.
  • VCP can be used as a probe to detect amyloid plaques in situ or post mortem. In such an assay the VCP would bind to the complement components in the plaque. Subsequent detection of the label would be indicative of the presence of amyloid plaques.
  • VCP may be labeled with any suitable label known in the art. Particularly preferred labels include fluorescent labels (e.g., fluorescein) and enzymatic labels (e.g., peroxidase).
  • fluorescent labels e.g., fluorescein
  • enzymatic labels e.g., peroxidase
  • the amyloid plaque is the hallmark of Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • a ⁇ amyloid precursor protein
  • APP amyloid precursor protein
  • VCP vaccinia virus complement control protein
  • VCP vaccinia virus complement control protein
  • VCP Unlike the large and complex, human complement control proteins, VCP is much smaller and it retains the functionality of the human proteins. Functionally VCP resembles the first complement receptor (CR1 ) as it is able to bind both to the third component and the fourth component of the complement system (Kotwal, G.J. (1996) The Immunologist 4: 157-164; Kotwal, G.J. et al. (1990) Science 250:827830; McKenzie, R. et al. J. Infect. Dis. 166:1245-1250).
  • CR1 first complement receptor
  • VCP can effectively block the complement pathway at an early stage following complement activation and thereby potentially block the downstream events leading to the formation of C5a and membrane attack complex.
  • the homologue of VCP in cowpox virus has been termed as the inflammation modulatory protein (IMP) (Miller, CG. et al. (1997) Virology 229:126-133).
  • IMP inflammation modulatory protein
  • IMP has been shown in a mouse air-pouch model to significantly reduce the influx of inflammatory cells and drastically diminish the tissue damage elicited by cowpox virus infection (Kotwal, G.J., et al. (1998) Mol. Cell. Biochem. 185:39-46).
  • the A ⁇ is capable of causing a greater influx of immune cells in a mouse model, described earlier (id.), in the presence of the fifth complement component but not in its absence. This cause and effect relation suggests that the in vitro findings are biologically significant.
  • the A ⁇ activated complement activity can be blocked by VCP.
  • a ⁇ 2 100 ⁇ g of A ⁇ 2 (QCB, Hopkinton, MA) was slowly solubilized in 25 ⁇ l double distilled water. After solubilization was complete, 25 ⁇ l of 2 ⁇ TBS (Tris-buffered saline), pH 7.4, (100mM Tris, 300mM NaCl) was slowly titrated and mixed, giving a final A ⁇ 1 . 42 concentration of 2 ⁇ g/ ⁇ l (444 ⁇ M). The peptide was incubated at 24°C for approximately 2-3 days to allow for fibril formation.
  • TBS Tris-buffered saline
  • pH 7.4 100mM Tris, 300mM NaCl
  • a pUC18 plasmid containing the coding sequence for the C-terminal 100 amino acids of APP (C-100) inserted into an EcoRI restriction site (pFB68L) was used as a template for cloning into pTM3 ( Figure 5).
  • Forward (JD01 F) and reverse (JDOIR) primers were designed and synthesized (Bio- Synthesis, Lewisville, TX) for use in PCR with pFB68L as the template.
  • the primers were designed with two base pair changes so that the 1.1 kb PCR product would be a cDNA of the APP C-terminus with two new restriction sites, one at the 5'(Nco ⁇ ) and one at the 3' (Sa//I) end.
  • plasmid pTM3 After purification of the PCR product, it and plasmid pTM3 were digested with ⁇ /col and Sail and ligated with T4 DNA ligase (Gibco BRL, Gaithersburg, MD). This resulted in a recombinant plasmid, pAPPc, which contained within the original multiple cloning site (MCS) the APP C-terminus under the transcriptional control of a T7 bacteriophage promoter (pT7) and termination sequence (T7t).
  • MCS multiple cloning site
  • T7t7 T7 bacteriophage promoter
  • T7t termination sequence
  • An incorporated encephalomyocarditis virus translational enhancer leader sequence (EMC) served in lieu of a 5' cap for the mRNA.
  • EMC encephalomyocarditis virus translational enhancer leader sequence
  • plasmid include a guanosyl phosphoribosyl transferase (GPT) gene under the control of The 7.5 kbp vaccinia promoter (p7.5K) for positive selection of the plasmid in eukaryotic cells.
  • GPT guanosyl phosphoribosyl transferase
  • p7.5K The 7.5 kbp vaccinia promoter
  • the 5' and flanking sequences of the vaccinia virus thymidine kinase (TK) gene are required for homologous recombination to generate a recombinant TK-negative vaccinia virus.
  • a bacterial origin of replication (ori) is required for plasmid replication in transfected bacteria and an ampicillin-resistance gene (Amp r ) for selection of transformed bacteria.
  • the new recombinant plasmid, pAPPc was transformed into competent E. coli JM105 (Gibco BRL) and transformants were selected for their ability to form colonies on media containing ampicillin. Colonies were selected and further amplified before isolation of the plasmid DNA by alkaline lysis minipreparation (Maniatis, T. et al. (1984) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York, p. 363). A clone transformed with pAPPc, as determined by restriction analysis, was amplified and plated on selective media to analyze for purity.
  • a coupled wheat germ extract transcription/translation reaction system containing T7 RNA polymerase (Promega, Madison, Wl) was used for expression of pAPPc.
  • This system allows for transcription of a DNA under the control of a T7 promoter and translation of the produced mRNA all in one reaction tube.
  • the addition of a radiolabelled amino acid to the mixture allows for labeling of all newly synthesized polypeptides.
  • Manufacturer's suggested protocols were followed with reaction volumes of 50 ⁇ l, with or without the addition of 40 ⁇ Ci of [ 35 S]-methionine (Amersham, Arlington Heights, IL) and either 1 ⁇ g of pAPPc, pTM3, luciferase control DNA or no DNA added.
  • the reactions were incubated at 30°C for 90 min.
  • the amount of labeled polypeptide produced was measured in a liquid scintillation counter (Pharmacia, Uppsala, Sweden), and equal counts were run on a SDS-PAGE for confirmation of predicted
  • FIG. 7 is a flowchart demonstrating the distribution of animals for injection with various samples and the time points after injection of sacrifice. The time points were selected on the basis of expected times when an in vivo reaction would occur.
  • the air pouch consisting of a single cell layer of connective tissue, was surgically harvested, mounted and stained with May-Grunwald/Giemsa (Sigma, St. Louis, MO). Photographs of the slides were taken with a Zeiss Universal Research Microscope using a 40* oil immersion objective.
  • VCP The amount of VCP required to reduce complement-induced sSRBC hemolysis by half (CH 50 ) was determined using a microplate assay described earlier (Kotwal, G.J. et al. (1990) Science 250:827-830). Differing volumes (2, 1 , 0.5, 0.1 , or 0.01 ⁇ l) of VCP were added to 200 ⁇ l of sSRBC (Diamedix) and 1 ⁇ l of AHS or normal human sera (NHS) in a total volume of 250 ⁇ l and, incubated at 37°C for 1 h. Measurement of equal amounts of water-lysed sSRBCs was taken to be 100% hemolysis, whereas sSRBCs incubated in buffer without serum or VCP served as a negative hemolysis control.
  • Soluble membrane attack complex is a multiprotein complex composed of the activated terminal proteins of complement activation (C5-C8 and several C9).
  • This assay uses an immobilized monoclonal antibody specific for human C9 which captures SC5b-9 formed in solution on the surface of a microliter plate where it can bind a secondary antibody conjugated to horseradish peroxidase.
  • the addition of a chromogenic substrate produces a color change, which can be measured by a plate reader at an absorbance of 405 nm.
  • PCR amplification of pFB68L containing DNA coding for the C-terminal 100 amino acids of APP with primers JD01 F and JD01 R resulted in a cDNA with newly engineered ⁇ /col and Sail restriction endonuclease sites at its 5' and 3' ends, respectively.
  • a calculated size of 1.1 kbp was confirmed by agarose gel electrophoresis.
  • the cDNA and plasmid pTM3 were ligated together to form pAPPc.
  • This plasmid was transformed into competent E. coli, amplified, and purified using a maxiprep kit (Qiagen, Chatsworth, CA).
  • the pAPPc was sequenced and the precise open reading frame expressed along with the primers used for sequencing is shown in Fig. 6.
  • the pAPPc was then used in an in vitro wheat germ extract coupled transcription/ translation reaction.
  • a product of approximately 10 kDa termed C-100 was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and its identity was confirmed as corresponding to the APP C- terminus by immunoprecipitation with antibodies raised against the carboxyl terminus of APP.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • mice were employed to evaluate the effect of A ⁇ , with the experimenter blinded to the samples being investigated.
  • the number of mice injected in this study and the distribution of animals for injection with different samples are shown in Figure 7.
  • Thirty-two animals were injected over three different experimental runs with either TBS or Tris as background controls, products from in vitro coupled transcription/translation reactions containing either pTM3 or pAPPc DNA, or fibrillar A ⁇ peptide.
  • Samples were injected into air pouches in either C5+/+ or C5-/- mice. After injection, an incubation period of either five or 48 h was allowed before the animals were sacrificed and tissues were taken.
  • Figure 8 is a compilation of the results in graphical form, and represents the averages of the different experimental variations. At 5 h post-injection ( Figure 8A), the numbers of monocytes and eosinophils present in the different spreads were nearly the same. The neutrophil counts, however, showed a significant increase in connective tissue excised from C5+/+mice injected with C-100.
  • Serum from agammaglobulinemic patients is devoid of IgM and deficient in IgG, but allowed complement activation similar to the serum with normal levels of IgG and IgM. This confirmed earlier observations that activation of complement by A ⁇ is independent of either IgG or IgM anti-body. Discussion
  • amyloid deposition is capable of causing activation of the classical pathway of the complement cascade in an antibody-independent manner.
  • the complement system forms one of the first molecular lines of defense against infectious agents. Uncontrolled, it can trigger autoimmune destruction of healthy tissue.
  • Deposition of complexes and formation of immunomodulators by the cascade have been associated with activating microglia (Elkabes, S. et al. (1996): J. Neurosci. 16:2508-2521 ), the macrophages of the brain, which in turn cause a progression and maintenance of the inflammation, by secreting cytokines like IL- 1 which would attract other immune cells and astrocytes (Fig. 11 ).
  • Local tissue destruction follows, along with a further persistence of a chronic form of inflammation.
  • AD Alzheimer's disease
  • VCP vaccinia virus complement control protein
  • VCP vaccinia virus compliment control protein
  • HLA-ABC cell surface class I HLA-ABC molecules
  • triplicate wells (2 ml each) were trypsinized and washed with FTA hemagglutination buffer (Becton Dickinson) and stained for 30 min on ice with 0.25 ⁇ g of phycoerythrin-conjugated mouse antihuman HLA-ABC monoclonal antibody (Caltag, Buriingame, CA), or a mouse lgG2a mAb (an isotype-match negative control) in the presence or absence of 2 or 5 ⁇ g of VCP. After incubation, cells were washed three times in FTA buffer, and then fixed in Hank's balanced salt solution (HBSS) containing 2% paraformaldehyde.
  • HBSS Hank's balanced salt solution
  • VCP Fragments of VCP were expressed in Pichia pastoris using the secretory expression vector pPIC9.
  • Genomic DNA from vaccinia virus was used as template for the amplification of the DNA fragments encoding the above protein fragments by PCR.
  • rVCP SCR (3,4) In all cases except for rVCP SCR (3,4) the oligonucleotides used introduced a 5' EcoRI site and a 3' Notl site which were used for cloning the fragments into the expression vector.
  • cloning was carried out as described earlier (Wiles, P., et al. 1995. J. Biol. Chem. 270:25805-25811 ). Selection of clones and expression of rVCP SCR (3,4) has been described previously (Wiles, A.P., et al. 1997. J.Mol.Biol. 272:253-265).
  • rVCP SCR (1,2), rVCP SCR (2,3) and rVCP SCR (1-4) clones transformed with the expression vectors described above where selected on the basis of their ability to grow on histidine deficient medium.
  • Small-scale expression screening was performed by inoculating 5 ml of buffered minimal glycerol
  • rVCP SCR (1 ,2) and rVCP SCR (1-4)
  • 100 ml of BMG was inoculated with 5 ml of an overnight culture and grown at 30 °C with vigorous shaking overnight. This culture was then used to inoculate several litres of BMG. The cultures were grown for 48 hours until the OD reached approximately 20. The cells were spun down and resuspended in the same volume of BMM and grown for 4 to 5 days with vigorous shaking. Cells were fed methanol to a concentration of 1 % every 24 hours.
  • rVCP SCR (2,3) BMG media was inoculated with an overnight culture and grown at 30 °C with vigorous shaking until an OD of approximately 6 had been attained. The pellet was then harvested by centrifugation, resuspended in BMM media (40% of the total BMG volume) and grown for 5 days with daily addition of methanol to a concentration of 0.5%.
  • rVCP SCR Purification of rVCP SCR (3,4) has been described previously (Wiles, A.P., et al. 1997. J.Mol.Biol. 272:253-265).
  • media was concentrated down to a small volume using a combination of a Millipore Prep/Scale-TFF cartridge (3 kDa molecular weight cut-off) and Amicon stirred-cell (with 3 kDa molecular weight cut-off).
  • the sample was exchanged into 50 mM Tris-HCI (pH 9.0) using a Pharmacia PD-10 column and loaded onto a Pharmacia Mono-Q column, equilibrated in the same buffer.
  • the protein was then eluted with a NaCl gradient of 0 to 100% over twenty minutes.
  • Fractions containing rVCP SCR (1 ,2) were collected and concentrated using Amicon stirred-cell ultrafiltration and then loaded onto a Brownlee Aquapore C4 reverse-phase column and the protein eluted with an acetonitrile gradient.
  • Fractions containing pure rVCP SCR (1 ,2) were collected and lyophilised.
  • media was concentrated to approximately 50 ml using an Amicon concentrator (3 kDa cut-off) before being centrifuged at 20,000 * g for 1 hour and the pellet discarded.
  • the sample was exchanged into 5 mM sodium acetate (pH 4.0) using a PD-10 column and applied to a Mono-S cation exchange column (Amersham Pharmacia, Uppsala, Sweden), equilibrated in the same buffer. Protein was eluted with a NaCl gradient and fractions corresponding to pure rVCP SCR (2,3) were pooled and lyophilised. For purification of rVCP SCR (1-4), media was concentrated to a small volume by the same means as rVCP SCR (1 ,2). The protein was exchanged into 20 mM phosphate pH 6 using a PD-10 column and dried.
  • bovine serum albumin BSA
  • HBP heparin binding protein
  • MIP-1 ⁇ MIP-1 ⁇
  • rVCP SCR 1 ,2), rVCP SCR (2,3), rVCP SCR (3,4), rVCP or 10 ⁇ g of wild type VCP were each dissolved in 1 ml of ultrapure water and passed through separate 1 ml HiTRAP heparin columns and the unbound materials collected. After washing the column with 1 ml of ultrapure water, the proteins were eluted with sodium chloride concentrations ranging from 250 mM to 2.5 M. The fractions were then separated using SDS-PAGE and silver stained.
  • the biological activity of the proteins were determined by using the hemolysis assay, described earlier (Kotwal, G.J., et al. 1990. Science 250:827-830). In order to more specifically quantitate their activity, each protein was tested as a series of dilutions ranging from 0.5 to 5.0 ⁇ g.
  • VCP SCR 1 and VCP SCR 2 The structure of the two unknown VCP modules (VCP SCR 1 and VCP SCR 2) were modeled by homology with the four known VCP module structures: VCP SCR 3, VCP SCR 4, fH (factor H) SCR 15 and fH SCR 16, using the program MODELLER (Sali, A. and T.L. Blundell. 1993. J. Mol. Biol. 234:779-815). Five models of each module were created. The model which was closest to the average structure (as measured by r.m.s.d.) was used in the construction of the VCP SCR (1-4) model. Construction of the VCP SCR (1-4) was undertaken using the molecular visualization package,
  • VCP heparin binding by VCP
  • Flow cytometric analysis of HUVECs treated with mouse monoclonal antibodies to human MHC class I molecules - in the presence and absence of VCP - show that VCP is able to modulate antibody binding in a dose-dependent manner.
  • addition of 5 ⁇ l (2 ⁇ g) of VCP to antibody treated HUVECs reduced antibody binding from 91.5% down to 75.4%.
  • An additional 15 ⁇ l (5 ⁇ g total) of VCP reduced antibody binding from 91.5% to 61.0% - suggesting that it is blocking in a dose-dependent manner.
  • Lysozyme and MIP-1 ⁇ which bound heparin with moderate and equal affinity, were contained within the 500 mM and 750 mM sodium chloride fractions. HBP, showing the highest affinity for heparin, was eluted by a sodium chloride concentration of 2.0 M.
  • VCP, recombinant VCP, and various recombinant segments of VCP were passed through HiTRAP heparin columns and analyzed using SDS PAGE, shown in figure 3.
  • the amino acid sequences were analyzed using the MacVector software system. Putative heparin binding sites (K/R-X-K/R) were first identified - shown in figure 4 along with the sequence alignment of different pox virus VCP homologs - and predicted to be located on the surface of the protein. The amino acid sequences were then scanned to determine the total number of positive amino acids (K+R), the percentage made up of these positively charged amino acids (%K+R), and the overall pi of the protein - the results are summarized in figure 4. All of the proteins shown to bind heparin had an overall pi of greater than 7.0, and more importantly, were made up of greater than 9% positively charged amino acids.
  • VCP The ability of VCP to bind heparin sulfate proteoglycan molecules adds a new dimension to its role in modulating the host immune response.
  • the ability of VCP to bind heparin was found to permit uptake by mast cells - possibly allowing for tissue persistence over extended periods of time.
  • earlier studies have shown that binding to heparin-like molecules on the surface of endothelial cells can block chemokine adherence, resulting in reduced chemotactic signaling.
  • flow cytometry was used to demonstrate that VCP can interfere with antibody binding to endothelial cells. More importantly, this study demonstrates that addition of VCP significantly decreases the amount of antibody able to attach to HUVECs in a dose-dependent manner.
  • VCP binding to heparin-like molecules on the surface of endothelial cells, prevents antibody adherence. This binding may therefore block antibody attachment through stearic interference, and block both molecular and cellular interactions - which are dependent on target cell adhesion molecules binding to cytotoxic cells. Furthermore, this novel mechanism can enable virus infected cells or the antigen presenting cell from destruction by host defense mechanism.
  • VCP enables orthopoxviruses, all of which encode homologs of VCP, to evade the immune system by multiple mechanisms, e.g. by blocking complement pathways, blocking antibody-dependent cytotoxic cell activity (ADCC) or by interfering with attachment of cytotoxic T cells to virus infected cells.
  • ADCC antibody-dependent cytotoxic cell activity
  • VCP can inhibit alternative pathway activation in a manner similar to factor H, another heparin binding protein.
  • VCP Due to the apparent importance heparin binding has in many of the biological activities exhibited by VCP, we became interested in better characterizing the molecular reasons for this interaction.
  • Recombinant segments of VCP - each representing two of the four SCRs - were generated by genetic engineering vaccinia virus DNA, encoding VCP, into the Pichia pastoris expression system. The ability of these recombinant segments to bind heparin was then tested. It was determined that recombinant proteins rVCP SCR (1 ,2) and rVCP SCR (3,4) retained the same binding affinity as the full-length native protein. While recombinant protein rVCP SCR (2,3) retained no heparin binding ability at all.
  • VCP SCR 3 VCP SCR 4
  • fH factor H
  • fH SCR 15 fH SCR 16 - see figure 6.
  • heparin binding exists primarily at the ends - SCRs one and four. The middle of the molecule therefore does not contribute any to the binding (hence VCP SCR (2,3) showed no ability to bind). It is clear that the ability to bind polyanions, such as heparin, is crucial for the function of many immune-regulating proteins.
  • fH Factor H
  • VCP vascular endothelial growth factor
  • fH is made up of SCRs, two of which have been shown to have heparin binding abilities (Blackmore, T.K., et al. 1998. J. Immunol. 160:3342-3348). It is postulated that these heparin-binding domains are responsible for binding sialic acid, thereby preventing alternative pathway activation.
  • the heparin binding regions of many other proteins exhibiting heparin binding activity have been mapped (Cardin, A.D., et al. 1991. Methods Enzymol.
  • VCP heparin-binding ability

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Abstract

La présente invention concerne un nouveau traitement de la démence sénile (type Alzheimer) consistant à administrer une protéine anti-complément à un patient nécessitant un tel traitement, dans une quantité suffisante pour inhiber la cascade du complément et, de ce fait, inhiber la formation ou l'agrandissement de plaques amyloïdes dans son cerveau. La présente invention concerne également des compositions pharmaceutiques contenant la protéine anti-complément, des dérivés de cette protéine, et/ou des sels de cette protéine, acceptables d'un point de vue pharmaceutique, sous diverses formes posologiques uniques.
PCT/US2000/001115 1999-01-19 2000-01-19 Utilisation d'une proteine inhibitrice du complement viral dans le traitement et le diagnostic de la maladie d'alzheimer WO2000043027A1 (fr)

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Cited By (11)

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WO2002065977A2 (fr) * 2001-02-20 2002-08-29 Israel Institute For Biological Research Composes co-induisant une regulation positive et une regulation negative de l'inflammation cholinergiques et des utilisations de ceux-ci
WO2005023296A1 (fr) * 2003-09-05 2005-03-17 University Of Louisville Research Foundation, Inc. Traitement de maladies rhumatismales
EP2035585A2 (fr) * 2006-06-15 2009-03-18 Eastern Virginia Medical School Procedes de regulation de proteines de la cascade du complement en utilisant une proteine d'enveloppe astrovirale et ses derives
US7897561B2 (en) * 2005-06-06 2011-03-01 Girish J. Kotwal Methods for treatment or prophylaxis of atherosclerosis and reperfusion injury
US8834409B2 (en) 2008-07-29 2014-09-16 Covidien Lp Method for ablation volume determination and geometric reconstruction
US8906845B2 (en) 2010-07-21 2014-12-09 Eastern Virginia Medical School Peptide compounds to regulate the complement system
US10005818B2 (en) 2010-07-21 2018-06-26 Realta Holdings, Llc Derivative peptide compounds and methods of use
US10933116B2 (en) 2015-06-26 2021-03-02 Realta Holdings, Llc Synthetic peptide compounds and methods of use
US10947279B2 (en) 2015-06-26 2021-03-16 Realta Holdings, Llc Synthetic peptide compounds and methods of use
US11020460B2 (en) 2018-01-09 2021-06-01 Realta Holdings, Llc PIC1 inhibition of myeloperoxidase oxidative activity in an animal model
EP3733205A4 (fr) * 2017-12-28 2021-12-08 Hyogo College Of Medicine Agent d'accélération de la production de prostaglandine d2 synthase de type lipocaline

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002065977A2 (fr) * 2001-02-20 2002-08-29 Israel Institute For Biological Research Composes co-induisant une regulation positive et une regulation negative de l'inflammation cholinergiques et des utilisations de ceux-ci
WO2002065977A3 (fr) * 2001-02-20 2003-12-04 Israel Inst Biolog Res Composes co-induisant une regulation positive et une regulation negative de l'inflammation cholinergiques et des utilisations de ceux-ci
WO2005023296A1 (fr) * 2003-09-05 2005-03-17 University Of Louisville Research Foundation, Inc. Traitement de maladies rhumatismales
US7897561B2 (en) * 2005-06-06 2011-03-01 Girish J. Kotwal Methods for treatment or prophylaxis of atherosclerosis and reperfusion injury
EP2035585A2 (fr) * 2006-06-15 2009-03-18 Eastern Virginia Medical School Procedes de regulation de proteines de la cascade du complement en utilisant une proteine d'enveloppe astrovirale et ses derives
EP2035585A4 (fr) * 2006-06-15 2010-10-13 Eastern Virginia Med School Procedes de regulation de proteines de la cascade du complement en utilisant une proteine d'enveloppe astrovirale et ses derives
US8241843B2 (en) 2006-06-15 2012-08-14 Eastern Virginia Medical School Methods for regulating complement cascade proteins using astrovirus coat protein and derivatives thereof
US8834409B2 (en) 2008-07-29 2014-09-16 Covidien Lp Method for ablation volume determination and geometric reconstruction
US9914753B2 (en) 2010-07-21 2018-03-13 Eastern Virginia Medical School Peptide compounds to regulate the complement system
US9422337B2 (en) 2010-07-21 2016-08-23 Eastern Virginia Medical School Peptide compounds to regulate the complement system
US8906845B2 (en) 2010-07-21 2014-12-09 Eastern Virginia Medical School Peptide compounds to regulate the complement system
US10005818B2 (en) 2010-07-21 2018-06-26 Realta Holdings, Llc Derivative peptide compounds and methods of use
US10414799B2 (en) 2010-07-21 2019-09-17 Realta Holdings, Llc Peptide compounds to regulate the complement system
US10844093B2 (en) 2010-07-21 2020-11-24 Realta Holdings, Llc Peptide compounds to regulate the complement system
US10933116B2 (en) 2015-06-26 2021-03-02 Realta Holdings, Llc Synthetic peptide compounds and methods of use
US10947279B2 (en) 2015-06-26 2021-03-16 Realta Holdings, Llc Synthetic peptide compounds and methods of use
US11814414B2 (en) 2015-06-26 2023-11-14 Realta Holdings, Llc Synthetic peptide compounds and methods of use
EP3733205A4 (fr) * 2017-12-28 2021-12-08 Hyogo College Of Medicine Agent d'accélération de la production de prostaglandine d2 synthase de type lipocaline
US11020460B2 (en) 2018-01-09 2021-06-01 Realta Holdings, Llc PIC1 inhibition of myeloperoxidase oxidative activity in an animal model
US11135272B2 (en) 2018-01-09 2021-10-05 Realta Holdings, Llc PIC1 inhibition of myeloperoxidase oxidative activity in an animal model
US11712462B2 (en) 2018-01-09 2023-08-01 Realta Holdings, Llc PIC1 inhibition of myeloperoxidase oxidative activity in an animal model

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