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WO2002034777A1 - Proteines de fusion utilisees comme traitements d'immunisation contre la maladie d'alzheimer - Google Patents

Proteines de fusion utilisees comme traitements d'immunisation contre la maladie d'alzheimer Download PDF

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Publication number
WO2002034777A1
WO2002034777A1 PCT/EP2001/012242 EP0112242W WO0234777A1 WO 2002034777 A1 WO2002034777 A1 WO 2002034777A1 EP 0112242 W EP0112242 W EP 0112242W WO 0234777 A1 WO0234777 A1 WO 0234777A1
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Prior art keywords
hsp
protein
amyloid
alzheimer
fusion protein
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PCT/EP2001/012242
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English (en)
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Silvia Ghirardi
Elisabetta Armani
Gabriele Amari
Paola Puccini
Bruno Imbimbo
Gino Villetti
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Chiesi Farmaceutici S.P.A.
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Priority to AU2002223640A priority Critical patent/AU2002223640A1/en
Publication of WO2002034777A1 publication Critical patent/WO2002034777A1/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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6043—Heat shock proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is related to fusion proteins derived from the condensation of ⁇ -amyloid protein or a fragment thereof and a heat shock protein, to the pharmaceutical preparations that contain them and to their use in the treatment or prophylaxis of disorders associated with the overproduction of ⁇ -amyloid, specifically, in patients with Alzheimer's disease.
  • Alzheimer's disease is a growing problem in developed countries. This disorder accounts for approximately 170,000 deaths in the United States every year, placing it in third place (7.1 % of total deaths) as cause of death (Ewbank DC, Am J Public Health 1999; 89: 90-92). The yearly cost of Alzheimer's disease in the United States is estimated to be 100 billion dollars (Schumock GT, Am J Health Syst Pharm 1998; 55, Suppl. 2, : S 17-S21). The cost of treating each patient is estimated to be 195,000 dollars, with a significant amount of this cost being attributed to the loss of productivity of the patient and caregiver and to costs sustained by the family.
  • the average annual cost for the care of patients with a mild, moderate or severe form of the disorder is approximately 19,000, 31 ,000 and 37,000 dollars, respectively.
  • the availability of drugs capable of slowing the development of the disease would lead to an estimated yearly savings of approximately 25,000 dollars per patient in medical assistance costs alone (Leon J et al, Health Aff 1998; 17: 206-216).
  • Alzheimer's disease is characterized by an atrophy of the cerebral cortex and by a massive loss of cortical neurons and cholinergic projections made by the nucleus basalis towards the cortex. From a histopathologic point of view there is a diffuse presence of extracellular and perivascular neuritic plaques and intracellular neurofibrillary tangles in the cerebral parenchyma of Alzheimer patients.
  • acetylcholine as well as other neurotransmitters such as serotonin, noradrenaline, dopamine, glutamate and substance P are degraded (Price DL et al, Ann NY Acad Sci 1985; 457: 35-51).
  • Acetylcholinesterase inhibitors have been employed in an attempt to increase acetylcholine levels in the brain, but this pharmacological approach to the problem has yielded modest cjinical results and has not had a significant impact on the natural history of the disorder (Davies P, JAMA 1999; 281 : 1433- 1434).
  • Neuritic plaques are composed mainly of aggregates of a protein with
  • ⁇ -amyloid 39-43 amino acid residues known as ⁇ -amyloid (Selkoe DJ, J Neiirochem 1986; 46: 1820-1834).
  • the ⁇ -amyloid protein is a derivative of a complex transmembrane glycoprotein (Kang J et al, Nature 1987; 325: 733-736) known as amyloid precursor protein (APP), and comprises a small part of the extracellular domain.
  • APP amyloid precursor protein
  • the initial step of the metabolic pathway of APP involves the ⁇ -secretase enzyme that cleaves out APP within the ⁇ -amyloid sequence (Esch FS et al, Science 1990; 248: 1 122- 1 124) allowing for the release of its transmembrane fragment.
  • This metabolic pathway is not amyloidogenic because it precludes the formation of ⁇ -amyloid and leads to the release of APP ⁇ that appears to exert neuroprotective activity (Mattson MP et al, Neuron 1993; 10: 243-254).
  • the metabolic pathway, defined amyloidogenic, that leads to the formation of ⁇ -amyloid is due to the enzyme ⁇ -secretase that releases APP ⁇ plus a 12 kDa protein fragment, which in turn is cleaved by the ⁇ - secretase enzyme (Esler WP et al, Science 2001 ; 293 : 1449- 1454) giving way to soluble A ⁇ .
  • PS- 1 and PS-2 presinilins
  • ⁇ -amyloid protein derived by proteolytic cleavage from APP
  • a ⁇ 39 A ⁇ 40 , A ⁇ 42 and A ⁇ 43 .
  • the 40 and 42 aminoacid forms are found predominantly in the extracellular neuritic plaques, while the 39 residue peptide is the predominant component found in cerebrovascular deposits (Mori H et al, J Biol Chem 1992; 267: 17082-17086, Prelli F et al, Biochem Biophys Res Commun 1988; 151 : 1 150- 1 155).
  • the amino acid sequence (primary structure), characterized by the presence of some hydrophobic amino acids, is such that the amyloid protein may take on both an ⁇ -helical and ⁇ -sheet conformation. Even though the two structures are approximately isoenergetic in the diagram of Ramanchandran, the protein adopts the ⁇ -sheet conformation in Alzheimer's disease for reasons not yet clear.
  • ⁇ -amyloid fibrils are believed to be formed by two ⁇ -sheet filaments, derived from protein folding, and linked by an ⁇ -helix turn (Soto C et al, J Neurochem 1994; 63 : 1 19 1 - 1 198).
  • the deposition of ⁇ -amyloid aggregates is thus highly correlated with its secondary ⁇ -sheet structure (Soto C et al, Neurosci Lett 1995; 200: 105- 108).
  • the fibrillogenesis of the ⁇ -amyloid protein involves a two-stage polymerization process: an initial "nucleation” process followed by "polymer elongation” that gives origin to the protofibrils (Jarrett JC et al, Cell 1993; 73 : 1055- 1058).
  • the alignment of the protofibrils generates the ⁇ -amyloid fibrils.
  • Alzheimer's disease The correlation among histopathologic lesions, brain cell death and cognitive deficiency in Alzheimer's disease is the basis of different etiology theories for the pathogenesis of Alzheimer's disease.
  • Another approach involves the inhibition of ⁇ -amyloid aggregates with products that modify the secondary structure of the protein or that bind to specific regions of the protein itself (Soto C et al, Nat Med 1998; 4: 822-826, Poduslo JF et al, J Neurobiol 1999; 39: 371 -382, Biochemistry 1999; 38: 6791-6800).
  • Athena Neuroscience international patent application WO 99/27944 claimed the same ⁇ -amyloid protein or its active fragment, as an agent able to induce an immunogenic response to ⁇ -amyloid, in the prophylaxis and treatment of amyloidogenic disorders and in Alzheimer's disease in particular.
  • Schenk et al. have demonstrated that immunization using ⁇ -amyloid l -42 in transgenic mice harboring a mutant version of APP (PDAPP) inhibits the formation of amyloid plaques and histopathologic lesions (Schenk D et al, Nature 1999; 400: 173- 177).
  • the immunization of young animals results in the almost total absence of ⁇ -amyloid deposits in old age with a significant inhibition of neuritic dystrophy (a marker of neuronal dysfunction) and of astrogliosis (a marker of cerebral inflammation).
  • the immunization of aged animals ( 1 1-12 months) that had already developed ⁇ -amyloid plaques prevented additional histopathologic lesions and even led to their partial regression.
  • the residual amyloid plaques are actively metabolized by microglia cells, suggesting that the immunization may, in addition to preventing the deposit of new amyloid, lead to the elimination of already, existing deposits.
  • the antigen could indeed favor transconformation and/or denaturization processes that have a negative impact on the duration of the activity.
  • endogenous factors could indeed favor transconformation and/or denaturization processes that have a negative impact on the duration of the activity.
  • WO 99/27944 a generic awareness is reported about the conformational changes in the immunogen that could affect the qualitative form of the response, in the examples (p. 5, lines 27-30), the immune response in animal was tested by using the aggregated form of A ⁇ 42 , i.e. a mixture of oligomers in which the monomeric units are held together by noncovalent bonds (p.12, lines 32-34).
  • Heat shock proteins also known as stress proteins, were described for the first time in Drosophila as proteins that are released following heat shock. Later it was found that their release is also related to stress or traumas of other types (hypoxemia, chemical, etc.).
  • Hsp are classified according to at least four categories based on size: Hsp90 (90 kDa), Hsp70 (70 kDa), Hsp60 (60 kDa) and small Hsp ( ⁇ 20 kDa). Hsp act as an intracellular chaperone to proteins, facilitating the transition among different compartments, modifying the secondary and tertiary structure and assembly of multimers (Beckmann RP et al, Science 1990; 248: 850-854). Recently, an important humoral and cellular immunogenic role has also been attributed to Hsp (Mizzen L, Biotherapy 1998; 10: 173- 189). Their strong immunostimulant properties have made them potentially significant therapeutic anti-infection and cancer agents. Hsp 90 and Hsp70 have recently been assessed as anti-cancer vaccines.
  • Hsp 70 the neurotoxicity of ⁇ -amyloid and ⁇ hyperphosphorylate proteins induces an increase in Hsp, especially Hsp 70 in the brain of Alzheimer patients (Perez N et al, Mol Brain Res 1991 ; 1 1 : 249- 254, Johnson G et al, Ann N Y Acad Sci 1993; 695: 194- 197, Yoo BC et al, J Neural Transm Suppl 1999; 57: 315-322). It has also been shown that the expression levels of Hsp 27 increase in degenerative astrocytes in brain areas rich in senile plaques during the inflammatory process (Renkawek K et al, Ada Neuropathol 1994; 87: 5 1 1 -519).
  • fusion proteins (Ag-Hsp) deriving from a covalent bonding of an antigen protein
  • the compounds of the present invention are fusion proteins (A ⁇ -Hsp) (III) derived from the covalent bonding of protein ⁇ -amyloid or fragment thereof (A ⁇ ) (I) and a heat shock protein (Hsp) (II). Fusion proteins A ⁇ -Hsp (III) exert immunization activity against the ⁇ -amyloid protein and are thus useful in the treatment of all pathologic conditions that are characterized by an overproduction of ⁇ -amyloid and by ⁇ -amyloid deposits, in particular in Alzheimer's disease.
  • the products (III), and the pharmaceutical preparations containing them induce an immune response to the ⁇ -amyloid protein and inhibit and prevent neuropathologic symptoms induced by the aggregates of the protein, and as such, are useful in the treatment and in the prophylaxis of Alzheimer's disease.
  • the specific heat shock protein that is bonded to the peptidic fraction of the ⁇ -amyloid protein may be appropriately modulated in order to stabilize the seco-ndary and tertiary structure of the peptidic fragment of the ⁇ -amyloid protein, to increase the immunogenic potential and to favor cellular transition.
  • the fusion protein obtained by coupling A ⁇ 42 with Hsp70 is able to induce an immune response comparable to that of A ⁇ 42 in the presence of the Freund's adjuvant, suggesting that compositions containing said agents as vaccine could be used for inducing a therapeutic immune response in patients affected by Alzheimer's disease, without the need of an adjuvant.
  • EP 526,51 1 proposes administration of homeopathic dosages (less than or equal to 10 "" mg/day) of A ⁇ to patients with pre-established Alzheimer's disease.
  • a ⁇ . peptides can be linked to suitable carriers such as serum albumin, choleric toxin, immunoglobulin or attenuated diphtherotoxin CRM 197 (p. 20, lines 1-7) which may favor release and/or immune response; it is also generically stated that immunogenic peptides can also be expressed as fusion proteins with carriers (p.
  • WO 94/29459 refers to the use of stress proteins for modulating the immune response.
  • the invention is also directed to fusion protein comprising a stress protein fused to a protein against which an immune response is desired but fusion proteins between A ⁇ and Hsp are nor mentioned, neither the less exemplified.
  • the ⁇ A-fusion protein (III) can include any of the naturally occurring ⁇ -amyloid proteins or a peptidic fragments thereof, and any Hsp chosen among the heat shock proteins with a low (Hsp25, Hsp27, Hsp28, etc.) or those with a high molecular weight (Hsp60, Hp70, Hsp90, etc.).
  • a ⁇ protein i.e. A ⁇ 39 , A ⁇ 40 , A ⁇ 41 , A ⁇ 42 and A ⁇ 43 .
  • Both the A ⁇ peptidic fragment and the heat shock protein can be appropriately chosen to obtain: i) the highest immunogenic response possible; improved tolerability; the most stable structure.
  • This invention also includes all the possible isomers of the compounds having the general formula (III) and their mixtures.
  • the present invention also includes the salts of those compounds having the general formula (III) that possess groups that can be salified, in particular the carboxylic and amino groups.
  • the physiologically tolerated salts are for example, in the case of molecules containing acid groups, salts of alkaline metals or alkaline-earth, such as sodium, potassium, lithium, calcium, magnesium, or salts formed with organic or amino acid amines, such as arginine.
  • the salts can comprise inorganic acids, such as hydrochloric acid and sulfuric acid, and mono and dicarboxylic organic salts, such as acetic acid, tartaric acid and methansulfonic acid.
  • the present invention also relates to preparations containing the fusion protein of general formula (III) and immune adjuvants such as that of Freund (complete or incomplete), Jung, Gerbu, QS-21.
  • a further embodiment of the invention concerns the nucleic acid sequence encoding the fusion protein .
  • the fusion proteins according to the invention can be prepared with the aid of appropriate recombinant vectors in host cells.
  • the techniques of constructing vectors, transforming cells, bringing about expression of the fusion proteins in the transformed cells and isolating and purifying the expressed proteins are known per se to the person skilled in the art.
  • the A ⁇ peptide can be linked to the heat shock protein by suitable linker amino acids.
  • Expression can take place not only in bacteria but also in a large number of host systems such as, for example, in mammalian, yeast and insect cells.
  • the DNA constructs suitable for the various host cells are synthesized by known methods and incorporated in the genome of the host cells with appropriate control sequences in a conventional way.
  • Effective doses of the compositions of the present invention, for the treatment of the above described conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, other medicament administered and whether treatment is prophylactic or therapeutic.
  • the amount of fusion protein for administration can vary from 10 ⁇ g to 20 mg per injection, preferably from 100 ⁇ g to 10 mg.
  • a typical regimen consists of an immunization (priming induction) followed by booster injections at suitable intervals.
  • the fusion proteins of the invention can be administered by any route for prophylactic and/or therapeutic treatment.
  • the most typical route of administration are subcutaneous and intramuscular injections.
  • the pharmaceutical compositions containing the fusion proteins of the invention can also include a variety of other pharmaceutically acceptable components depending on the intended mode of administration.
  • the following examples illustrate in detail the invention.
  • Example 1 PREPARATION OF THE TEST MATERIALS, NAMELY THE FUSION PROTEIN A ⁇ 42 -HSP70 AND THE A ⁇ 42 PEPTIDE Materials and Methods Expression Vector Constructs
  • the DNA fragment containing the Mycobaterium tuberculosis Hsp70 sequence was synthesized by PCR technique using the genomic DNA extracted from the M. tuberculosis HR37Rv strain as template.
  • the upstream primer overlapping the AUG start codon (Hsp70-up) contains the BamHI and Ncol sites, while the downstream primer overlapping the UGA stop codon (Hsp70-dw) contains the Xhol site.
  • the amplified DNA was then digested with the BamHI and Xhol enzymes and cloned in a pGEMl 1 Z(f+) (Promega). A positive clone (p l l [Hsp70J) was isolated and fully sequenced.
  • the DNA fragment encoding for the Homo sapiens amyloid beta peptide 1 -42 was synthesised by the annealing of the oligos Beta- 1 and Beta-2.
  • the Beta l/2 double strand DNA fragment contains: the Hindlll site in the 5 'of the untraslated sequence, the Ndel site that overlap the AUG start codon and the BamHI site coding for the linking aminoacids (Gly-Ser) between the A ⁇ 4 and the Hsp70 sequence.
  • the Betal/2 DNA fragment was digested with Hindlll and BamHI enzymes, and then cloned in the p l l [Hsp70] vector.
  • a positive clone (p i l [A ⁇ 42 -Hsp70]) was isolated and fully sequenced.
  • the p i l [A ⁇ 42 -Hsp70] was digested with the enzymes Ndel and Xhol, and the 2 Kb band coding for the chimeric protein A ⁇ -Hsp70 was then cloned in the pET24a plasmid (Novagen).
  • a positive clone (p24[A ⁇ 42 -Hsp70]) was isolated and fully sequenced.
  • the Escherichia coli BL21/DE3 strain was transformed with the p24[A ⁇ -Hsp70] vector expressing the chimera A ⁇ 42 -Hsp70 and with the pET24a as control.
  • Overnight cultures of the transformants were diluted 1/100 in LB medium containing 30 ⁇ g/ml of Kanamycin.
  • the cultures were grown to an OD 6 oo nm of 1 and recombinant protein production was induced by addition to the cultures of isopropylthiogalactoside (IPTG) at the final concentration of 1 mM, for a total of four hours.
  • IPTG isopropylthiogalactoside
  • the A ⁇ 42 -Hsp70 protein formed inclusion bodies and was then purified as follows.
  • the cells were harvested by centrifugation at 10,000 rpm in a JA10 Beckman rotor.
  • the cell pellet was resuspended in lysis buffer A and incubated at 30°C for 15' .
  • an equal volume of lysis buffer B was added and incubated at 37°C for 15'.
  • the cells were sonicated and then pelleted at 12,000 rpm in a JA17 Beckman rotor at 4°C for 15' .
  • the pellet were resuspended and pelleted three times with the buffer C, D and E, respectively.
  • the final pellet was resuspended in buffer F and then centrifuged as previously described the supernatant were frozen at - 20°C.
  • Proteins were refolded by dilution 1 : 100 in the refolding buffer for 16 hours at 4°C in agitation.
  • the ultra-filtered solution was buffer exchanged using a G25 HiPrep 26/ 10 column (Amersham Pharmacia Biotech) using the buffer G, the protein fraction was used for the ATP affinity chromatography.
  • a column of ATP- agarose (Sigma) was equilibrated in buffer G plus 0.2% TritonXlOO and 100 mM NaCl. Protein was loaded on the ATP column and subsequently the column was rinsed with: buffer G plus 0.2%o Triton X 100 and 600 mM NaCl; buffer G plus 100 mM NaCl; buffer G plus 100 mM NaCl and 5 mM ATP.
  • the protein A ⁇ 2 -Hsp70 was eluted with buffer G plus 100 mM NaCl and 25 mM ATP.
  • a ⁇ 2 ⁇ Hsp70 protein was polished by gel filtration using a HiLoad
  • Buffer D 50 mM Tris-HCl, 1 M NaCl.
  • Buffer E 50 mM Tris-HCl, 1 M urea.
  • Refolding buffer 500 mM L-Arginina: 105.335 g/litro; 100 mM Tris-HCl pH -
  • Mycobaterium tuberculosis Hsp70 protein was achieved.
  • the synthetic nucleotide sequence of the A ⁇ 2 peptide was fused with Hsp70 gene of the ⁇ l tuberculosis ( Figure 1 ) and then cloned into the pET24a vector.
  • This vector allows the expression of high levels of recombinant protein using the T7 expression system (Studier FW et aL Methods Enzymol 1990, 1 85 ; 60-89).
  • the A ⁇ 42 -Hsp70 fusion protein w as found to be expressed at very high levels in E . coli ( Figure 2).
  • the ⁇ 2 ⁇ Flsp70 protein was purified as inclusion bodies and subsequently refolded.
  • the protein was further purified by ATP affinity chromatograpln and gel filtration using a Superdex 200 prep grade column. The purity of the recombinam protein was assessed by SDS- PAGE (Figure 2): lanes 1. 2. 3 are cell 1 ⁇ ates of the BL21 /DE3 cells transformed w ith the empt ⁇ vector. p24
  • the A ⁇ 42 -Hsp70 fusion protein was prepared according to the Example 1.
  • European guide lines for the use of animals in in vivo experimentation The animals were housed under controlled conditions in group of ten per cage. On arrival to laboratory, each animal was assigned an identifying code. Periodic examinations of health were performed using microbiological, parasitological and serological techniques aimed at identifying undesirable and pathogenic organisms.
  • test materials were used as lyophilised powders. Each test material was suspended at a concentration of 2 mg in 0.9 mL of sterile water, vortexed and added with 100 ⁇ L of 10 X phosphate buffered saline (PBS), where 1 X
  • PBS phosphate buffered saline
  • PBS is 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5. Suspensions were vortexed again and incubated overnight at 37°C for use the next day.
  • Suspensions were prepared and codified by an independent operator in order to perform the study following a double blind procedure. Before administration, the A ⁇ 42 peptide was mixed with complete ⁇ priming induction) or incomplete ⁇ subsequent challenges) Freund's adjuvant in a ratio of 1 : 1
  • mice (20-25 g, Balb/c, Charles-River, Lecco, Italy).
  • the choice of the administration route and animal species is based on the fact that: a) the subcutaneous route of administration is widely used in immunisation studies as the presence of specialised antigen-presenting cells (e.g. macrophages and dendritic cells) in the subcutaneous district induce a strong immunologic response; b) the mouse is a widely used animal species for studies on induced immunological responses.
  • all transgenic animal models of Alzheimer's disease currently available are in the mouse.
  • mice were administered 10 or 100 ⁇ g of test material, according to the experimental scheme reported below. Each group was formed by 3-5 animals. Animals received, subcutaneously and in a dose corresponding to the assigned group, the A ⁇ 42 peptide mixed with complete Freund's adjuvant in a ratio of 1 : 1 (v/v) or alternatively the A ⁇ 2 -Hsp70 fusion protein alone ⁇ priming induction). Fifteen and 30 days after priming, animals received the first and second boost, respectively. Each time they were injected subcutaneously with A ⁇ - 2 peptide solution mixed with incomplete Freund's adjuvant in ratio of 1 : 1 (v/v) or with the A ⁇ 42 -Hsp70 fusion protein solution ⁇ subsequent challenges following priming induction).
  • Antibody formation was initially evaluated in the sera prepared from the collected blood samples by immuno-blot analyses. In the case of positive results, antibody titres in the sera of the different animals were assessed via
  • mice injected on day 1 , day 15 and day 30 with 10 ⁇ g of A ⁇ 2 peptide (2 nmoles) or 100 ⁇ g of A ⁇ 42 -Hsp70 ( 1 nmole) fusion protein are plotted in Figure 3.
  • mice treated with the A ⁇ 42 -Hsp70 fusion protein did not receive Freund's adjuvant.
  • the average titre ( ⁇ standard error) is shown. Titres were defined as the reciprocal of the dilution of serum giving one half the maximal optical density.
  • the A ⁇ 42 -Hsp70 fusion protein obtained by coupling A ⁇ 42 with Hsp70, is able to induce an immune response comparable to that of A ⁇ 42 in the presence of the Freund's adjuvant, suggesting that compositions containing the fusion protein as vaccine could be used for inducing a therapeutic immune response in patients affected by Alzheimer's disease, without the need of an adjuvant.

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Abstract

L'invention concerne des protéines de fusion (Aβ-Hsp) (III) ainsi que leur utilisation dans le traitement ou la prophylaxie de troubles associés à une accumulation de β-amyloïde, notamment chez les patients atteints de la maladie d'Alzheimer. Ces protéines de fusion (III) proviennent de la condensation de la protéine β-amyloïde ou des fragments (Aβ) (I) avec une protéine de choc thermique (Hsp) (II), selon le mécanisme suivant : Aβ + Hsp → Aβ - Hsp
PCT/EP2001/012242 2000-10-24 2001-10-23 Proteines de fusion utilisees comme traitements d'immunisation contre la maladie d'alzheimer WO2002034777A1 (fr)

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Cited By (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6743427B1 (en) 1997-12-02 2004-06-01 Neuralab Limited Prevention and treatment of amyloidogenic disease
US6750324B1 (en) 1997-12-02 2004-06-15 Neuralab Limited Humanized and chimeric N-terminal amyloid beta-antibodies
US6761888B1 (en) 2000-05-26 2004-07-13 Neuralab Limited Passive immunization treatment of Alzheimer's disease
US6787144B1 (en) 1997-12-02 2004-09-07 Neuralab Limited Prevention and treatment of amyloidogenic disease
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