HK1154591A - Anti-amyloid immunogenic compositions, methods and uses - Google Patents

Description

Anti-amyloid immunogenic compositions, methods and uses

Technical Field

The present invention relates to polypeptide constructs comprising a tandem array of Α β 42 peptide fragments and their use in the manufacture of antibodies and vaccines for the treatment of medical conditions such as alzheimer's disease.

Background

Amyloidogenic diseases, such as Alzheimer's Disease (AD), have been identified as the leading cause of dementia in the elderly. The decline in cognitive ability in AD is associated with histopathological changes in the brain, most relevant being the formation of amyloid plaques and neurofibrillary tangles.

Although amyloid plaques contain many proteins, they have amyloid- β (a β) peptide as their major component. A β peptide formation and thus a β amyloid plaque formation results from abnormal processing of Amyloid Precursor Protein (APP).

Currently, several pharmacological approaches have been developed to slow or reverse the progression of AD. While several approaches have been directed to inhibiting the metabolic production of a β peptide, others have been directed to preventing the aggregation of a β amyloid in the brain of AD-affected patients.

However, the most promising approach involves increasing the brain clearance rate of a β plaques by administering antigens capable of generating an immune response against a β (active immunization) or antibodies against a β (passive immunization).

An antigen or immunogen is generally a macromolecule that contains different antigenic sites or "epitopes" that are recognized and interact with different components of the immune system. They generally comprise a small molecule or "hapten", such as a short peptide, coupled to a suitable carrier, typically a high molecular weight protein.

In the immune response, B-lymphocytes, along with T-helper (TH) cells, produce and secrete antibodies. In most hapten-carrier systems, B cells produce antibodies specific for both hapten and carrier. In these cases, T lymphocytes have a specific binding domain on the carrier, but cannot recognize the hapten alone. In one type of synergy, B and T cells cooperate to induce a hapten-specific antibody response.

Therefore, in constructing effective antigens, the selection of suitable carriers and suitable haptens is critical to ensure a stable and selective immunogenic response. The safety of the antigen is also of critical importance. For example, the promising AN-1792 vaccine consisting of pre-aggregated A β 42 and the immunological adjuvant QS-21 given to Alzheimer's patients resulted in a severe meningoencephalitis incidence in about 6% of treated subjects (Steinberg, D.2002, Scientist 16: 22). Centrally activated cytotoxic T cells and autoimmune reactions are proposed as potential mechanisms of toxicity. The immune response to endogenous monomeric a β can be detrimental because unaggregated a β species have a physiological role in neuronal activity.

Therefore, the choice of hapten and carrier is of paramount importance to ensure antibody selectivity against harmful a β species and to prevent autoimmune toxicity.

WO2005/058940 proposes conjugating a peptide immunogen comprising an a β peptide or fragment thereof to a protein/polypeptide carrier. The immunogenic construct is produced by a chemical process which comprises derivatising functional groups of carrier amino acid residues, wherein any unconjugated derivatised amino acid residue functional groups are inactivated by capping to prevent them from reacting with other molecules. This approach yields immunogens in which the A.beta.fragment is conjugated to a carrier amino acid side chain. Although in WO2005/058940 several different carriers and haptens have been proposed, their histopathological efficacy in vivo has not been shown.

Anti-a β DNA vaccines consisting of 11-fold repeats of backbone-free a β 1-6 are proposed by Kim, h.d. et al in biochem, biophysis, res.commun.volume 336, pages 84-92. Such constructs produce antibodies that indiscriminately recognize the a β 42 species of monomers, oligomers, and fibrils.

WO 2007/096076 discloses promising immunogenic constructs based on a β 42 fragment incorporated into the active site of a bacterial thioredoxin carrier. The insertion of tandem multimers of a β 1-15 fragments at this site results in the production of polypeptides that are capable of eliciting antibodies, but not monomers, that selectively recognize a β 42 fibrils and oligomers. Best results were obtained using 4 copies of the a β 1-15 peptide in tandem.

The present invention provides alternative recombinant immunogenic constructs comprising tandem arrays of Α β 1-7 peptides that are safe and effective for prophylactic or therapeutic vaccination to prevent Α β amyloid aggregation in the brain of patients afflicted with alzheimer's disease or other amyloidogenic diseases such as down's syndrome.

Summary of The Invention

The present invention provides polypeptide molecules comprising a tandem array of peptide sequences, each peptide sequence (monomer) consisting of the N-terminal 7 amino acids of Abeta 42, i.e., DAEFRHD (SEQ ID NO: 1), orTo be interchangeably referred to as (A beta 1-7)_nWhere n is the number of peptide sequences (monomers) in the tandem array. Preferably the tandem array is coupled to a carrier molecule bacterial thioredoxin.

The present invention also provides polynucleotides encoding the polypeptides of the present invention and methods for preparing such polynucleotides. Preferably, the polynucleotide is a DNA expression vector.

In another aspect, the invention provides the use of a polypeptide of the invention in the preparation of a vaccine for the prevention or treatment of an amyloidogenic disease, such as alzheimer's disease.

In another aspect, the invention provides the use of a DNA expression vector encoding a polypeptide of the invention as a DNA vaccine for the prevention or treatment of amyloidogenic diseases such as Alzheimer's disease.

In another aspect, the invention provides antibodies, preferably monoclonal antibodies, raised against the polypeptides of the invention and their use in the prevention or treatment or diagnosis of amyloidogenic diseases.

In another aspect, the invention provides a pharmaceutical composition comprising a polypeptide or polynucleotide of the invention and one or more pharmaceutically acceptable excipients.

In another aspect, the invention provides a method of preventing or treating an amyloidogenic disease in a susceptible individual, comprising administering an effective amount of a polypeptide or polynucleotide of the invention or a therapeutic antibody.

Brief Description of Drawings

The FIGURE shows the results of an ELISA assay that measures all alum-adjuvanted Trx (A β 1-7) from immunization_nPolypeptides, Trx (thioredoxin) or reference antigen Trx (Abeta 1-15)₄Anti-a β 42 reactivity of mouse serum.

Description of The Preferred Embodiment

The present invention provides immunogenic constructs (or immunogens) comprising a multi-monomer tandem array of N-terminal fragments of Alzheimer's amyloid-beta peptide. The monomer is preferably located in a surface exposed region of the carrier polypeptide (active loop site or display site). It has now been demonstrated that antibodies raised against these constructs have a strong and specific affinity for the peptide a β 42.

The terms "peptide" and "polypeptide" as used herein mean a compound composed of single chains of which the amino acids are linked by peptide bonds.

The term "tandem array" means a set of multiple linear peptide sequence repeats (monomers) that are immediately adjacent, preferably spaced no more than 10 amino acids apart.

The term "immunogen" as used herein relates to a polypeptide or DNA vaccine which, when administered to a mammal, is capable of inducing an immune response to the polypeptide or polypeptides encoded by the DNA vaccine administered. An "immune response" may be defined as a humoral (antibody-mediated) and/or cellular (antigen-specific T cell or its secretory product-mediated) response that is directed against an antigen or immunogen.

The carrier polypeptide is preferably bacterial thioredoxin, most preferably E.coli thioredoxin, and the tandem array of peptides is preferably located at the well characterized residue Cys₃₃With Cys₃₆Within the active loop site of the vector (display site). However, the carrier polypeptide antigen is any carrier known to those skilled in the art having suitable surface exposed domains into which the monomers of the a β fragment can be inserted. The tandem array is fused in-frame to the carrier polypeptide.

Tandem arrays of peptide monomers are designated herein as (A β 1-7)_nWherein A β 1-7 is the monomer sequence DAEFRHD and "n" is the number of monomers in the tandem array, with "n" preferably being 2-15, more preferably 3-12. In particularly preferred embodiments, "n" is 3, 6, 9 or 12. In a more specific implementationIn one embodiment, "n" is 9, and in another specific embodiment "n" is 12.

The corresponding amino acid sequences of these preferred tandem arrays are listed in the tables.

The peptide monomers preferably have the same orientation, i.e., read DAEFRHD from N-to C-terminus. Optionally, one or more of the peptide monomers have a reverse orientation, i.e., N-to C-terminal reading of DHRFEAD (SEQ ID NO: 2).

Watch (A)

Synthetic peptide monomers and multimers can be synthesized by solid phase peptide synthesis or recombinant expression or can be obtained from natural sources. Peptide automated synthesizers are commercially available from a number of suppliers, such as Applied Biosystems, Foster City, California. Recombinant expression can be performed in bacteria, such as E.coli, yeast, insect cells, or mammalian cells. Recombinant expression methods were performed by Molecular Cloning, Sambrook et al: a Laboratory Manual (c.s.h.p.press, NY 2ded, 1989).

In a preferred embodiment, (A.beta.1-7)_nThe multimer is bound to the carrier polypeptide by a linker to prevent the formation of a conjugated epitope. The linker is a short amino acid sequence, preferably a linker consisting of 1-10 amino acids, more preferably 2-5 amino acids, more preferably 3 amino acids, most preferably glycine-proline (Gly-Pro). However, other linkers may also be used, such as glycine-proline-glycine (Gly-Pro-Gly) or serine-glycine-serine-glycine (Ser-Gly-Ser-Gly).

In a preferred class of polypeptides of the invention, each (A β 1-7) monomer unit is separated from adjacent units by a short linker peptide sequence, which may be the same or different, in each case as described above, preferably consisting of 1-10 amino acids, more preferably 2-5 amino acids, more preferably 3 amino acids, most preferably Gly-Gly-Pro.

However, optionally the peptide monomers are arranged in a tandem array such that the C-terminus of one peptide monomer is adjacent to the N-terminus of any peptide monomer fused to the C-terminus, i.e. there is no linker or other external amino acid sequence interposed between the (a β 1-7) monomers.

Construct structure can be determined by standard analytical techniques. Preferably Nuclear Magnetic Resonance (NMR) is used to obtain the 3D structural map and the conformation of the recombinant polypeptide.

Conventional cloning procedures such as those which afford restriction digestion and ligation of existing bacterial or other expression vectors may be used to construct vectors (plasmids) incorporating DNA sequences encoding polypeptides of the invention. An example of a commercially available vector is the pET series incorporating the T7 promoter for expression in E.coli.

US 5,270,181 describes in detail how to make and use constructs based on thioredoxin as a vector and this document is incorporated herein in its entirety by reference, including published or mentioned thioredoxin sequences. Coli thioredoxin sequences can be evaluated according to the EcoProDB online database, for example with the accession numbers P0AA25, P0A9P4 and P0AGG 4.

Construction (A.beta.1-7) by chemical synthesis and annealing of monomers encoding forward and reverse strand DNA oligomers and incorporating sequences optionally encoding linkers and by linking the monomers to the digestion vector in the presence of excess monomers_nA serial array. The number of monomer units in each recombinant vector can be confirmed by diagnostic methods such as PCR or restriction digestion and gel electrophoresis. The vector may then be transferred into a bacterium or another type of host cell (e.g., yeast) for expression and the recombinant polypeptide then purified by recombinant techniques.

The invention also relates to polynucleotides encoding polypeptides of the invention in prokaryotic or eukaryotic organisms, in particular polynucleotides encoding polypeptides of the invention having the sequence given in SEQ ID NO: 1-SEQ ID NO: 19. Preferably, the polynucleotide sequence is optimized for expression of the polypeptide in the selected host organism. For example, for E.coli expression and encoding the amino acid sequence of SEQ ID NO: 3 monomers suitable polynucleotides optimized suitable polynucleotides are SEQ ID NO: 20: ATG GAT GCG GAA TTT CGC CAT GATGGC GGT CCG (5 '-3').

The term "polynucleotide" as used herein means a polymer molecule having a backbone that supports bases capable of hydrogen bonding with a typical polynucleotide, wherein the polymer backbone provides the bases in a manner that allows such hydrogen bonding in a sequence-specific manner between the polymer molecule and the typical polynucleotide (e.g., single-stranded DNA). Such bases are typically inosine, adenosine, guanosine, uracil and thymidine. Polymer molecules include double-and single-stranded RNA and DNA and backbone modifications thereof, such as methylphosphonate linkages. Polynucleotides may be linear or circular and include plasmids, viruses, and other vectors. The polynucleotides of the invention comprise a promoter sequence to enable expression of the polypeptides in culture or in cells within a living multicellular organism (in situ).

The polynucleotides of the invention may also incorporate conventional vector elements such as origins of replication, polyadenylation sequences, translation termination sequences, enhancers, antibiotic resistance genes, and leader sequences.

For the purpose of preparing a DNA vaccine encoding a polypeptide of the present invention, a suitable vector (typically a plasmid) is selected in which endogenous polypeptide expression can be performed by using a suitable mammalian promoter sequence. The vector may optionally incorporate immune modulatory sequences such as CpG motifs.

In another aspect, the invention relates to antibodies raised against the polypeptides of the invention. They may be polyclonal or monoclonal antibodies and derivatives thereof (e.g. humanized antibodies). Methods for making such antibodies are well known in the art. These may have diagnostic applications (e.g. in the diagnosis of alzheimer's disease by selective recognition of neurotoxic oligomer species of a β amyloid) and prophylactic or therapeutic applications (passive vaccination) by administration to patients at risk of or suffering from amyloidogenic disease.

Pharmaceutical compositions comprising a polypeptide, polynucleotide (carrier) or antibody of the invention may further comprise one or more pharmaceutically acceptable excipients well known in the art, such as carriers, diluents, wetting agents, emulsifiers, binders, coatings, fillers, glidants, lubricants, disintegrants, preservatives, surfactants, pH buffering substances and the like. Examples of Excipients and their use are provided in Handbook of Pharmaceutical Excipients, 4 th edition (2003), ed.rowe et al, Pharmaceutical Press.

Examples of suitable diluents for liquid dosage forms include distilled water, physiological phosphate buffered saline, ringer's solution, dextrose solution, and Hank's solution.

For vaccination, the pharmaceutical composition of the invention is advantageously administered together with an adjuvant.

The choice of adjuvant and/or carrier depends on the stability of the vaccine containing the adjuvant, the route of administration, the administration protocol, the adjuvant used to vaccinate the species, and in humans, a pharmaceutically acceptable adjuvant is one that is approved or approvable by the appropriate regulatory agency for human administration.

Suitable adjuvants include 3 de-O-acylated monophosphoryl lipid A (MPL), muramyl-di-peptides, saponins such as QS21 and Quil A, squalene, oil-based adjuvants, virosomes, dsRNA and other immunostimulatory oligonucleotides, lipopolysaccharides and CpG motifs.

A preferred type of adjuvant is an aluminium salt (alum), such as aluminium hydroxide, aluminium phosphate and aluminium sulphate.

Other adjuvants include cytokines such as interleukins (IL-1, IL-2 and IL-12), macrophage colony stimulating factor (M-CSF) and Tumor Necrosis Factor (TNF). The adjuvant may be administered with the immunogen as a single composition, or may be administered prior to, concurrently with, or after the immunogen is administered. Optionally, two or more different adjuvants may be used simultaneously.

The immunogen and adjuvant may be packaged and provided in the same vial or may be packaged in separate vials and mixed prior to use. In one embodiment, the invention relates to a kit of parts comprising an immunogen of the invention and an adjuvant for separate, simultaneous or sequential administration.

The compositions of the present invention may be prepared as injectable formulations, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in a liquid vehicle prior to injection may also be prepared. For parenteral administration, the immunogenic constructs of the invention may be administered as a solution or suspension of an injectable dose of the substance in a physiologically acceptable diluent comprising a pharmaceutically acceptable carrier, which may be a sterile liquid such as water, oil, saline, glycerol or ethanol.

DNA vaccines are typically formulated for parenteral administration, for example, by injection or by using a gene gun or aerosol. Pharmaceutical formulations are thus employed. DNA vaccines are typically administered by the Intramuscular (IM) or Intradermal (ID) routes and typically comprise saline or another diluent and optionally other ingredients such as microparticles, liposomes and viral DNA.

The immunogenic constructs of the invention may be administered in the form of long-acting injectable or implantable formulations, which may be formulated in such a way as to allow sustained release of the active ingredient.

Other formulations suitable for other modes of administration include oral, intranasal, inhalation/pulmonary formulations, suppositories, topical and transdermal applications. The dosage form can be tablet, capsule, patch, powder, spray, etc.

The pharmaceutical compositions of the invention may comprise one or more other immunogens (in the case of multivalent vaccines) or therapeutic antibodies.

One skilled in the art can readily determine how to construct a therapeutically effective amount of an immunogen or therapeutic antibody of the present invention. In general, the dose may be between 0.1ng and 10mg of immunogen or antibody, preferably between 10ng and 1mg, more preferably between 100ng and 100. mu.g.

In some cases, a single dose of the therapeutic antibody vaccine is sufficient to reverse or alleviate or prevent symptoms of the amyloidogenic state disease. However, it may be necessary to administer one or more booster injections or doses. Suitable dosing regimens are readily determined by those skilled in the art.

According to a preferred embodiment, the vaccination method of the invention is effective in generating an immune response, characterized by a serum titer of at least 1: 1000 relative to the amyloid protein against which the immunogenic peptide is directed. In another preferred embodiment, the serum titer is at least 1: 5000 relative to the amyloid content. According to a related embodiment, the immune response is characterized by an immunoreactive serum mass that is equivalent to more than about 4-fold higher than the immunoreactive serum level measured in a pretreated control serum sample. This latter feature is particularly suitable when determining serum immunoreactivity by ELISA techniques, but may be applied to any relevant or absolute measure of serum immunoreactivity. According to a preferred embodiment, immunoreactivity is measured at a serum dilution of about 1: 100 to 1: 10,000 to determine antibody titer.

The present invention provides means for the prevention and treatment of all diseases of the amyloidogenic state, in particular neurodegenerative diseases such as alzheimer's disease, dementia associated with down's syndrome, Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy. The vaccines of the present invention may be used to prevent individuals predisposed to developing amyloidogenic disease (e.g., as determined based on a genetic map) or to treat patients who have shown signs and symptoms of the disease.

The ability of the polypeptides, DNA vaccines and antibodies of the invention to have a therapeutic or prophylactic effect on amyloidogenic disease can be verified and confirmed by various in vitro and in vivo protocols well known to those skilled in the art (see also example 3). Some suitable techniques are described in Moretto et al (2007), j.biol.chem.282 (15): 11436-: 1580, 1586. One such immunohistochemical assay relies on the increased ability of antisera directed against polypeptides to bind to amyloid plaques in brain sections of alzheimer's humans. Antisera from mice were added to sequential formalin-fixed brain sections previously treated with formic acid (80%, 15 min). Suitable immunolabeling systems are used to reveal the brain regions to which the antibodies bind. Another assay uses transgenic mice with the Swedish APP mutation and relies on injection of antisera into the brain, followed by visualization of tissue sections. Spleen cell cultures from immunized mice can be analyzed for T cell proliferation and also tested for cytokine production. Cognitive performance improvement in mice or other mammals can be averaged by standard tests such as the Morris water maze test for memory function in mice.

Examples

Example 1: preparation of plasmid constructs

Starting from the amino acid sequence of the human Α β 1-7 peptide (DAEFRHD), two codon-optimized (e.coli) oligonucleotides encoding this peptide were designed:

a β 1-7-forward SEQ ID NO: 21

5′-GTCCGATGGATGCGGAATTTCGCCATGATCG-3′(33nt)

A β 1-7-reverse SEQ ID NO: 22:

5′-GACCGATCATGGCGAAATTCCGCATCCATCG-3′(33nt)

the incomplete (5' -overhanging) CpoI restriction site is located within the oligonucleotide (underlined). The Cpo-I site formed when the oligonucleotide was ligated to the Cpo-I digested pT7Kan-Trx vector (Moretto et al 2007, J.biol.chem.282, 11436) encodes the "spacer" amino acids Gly (G) and Pro (P). The third G residue was added to this "spacer" by incorporating an A.beta.1-7 oligonucleotide of two additional GG/CC nucleotides upstream of the distal CpoI site (in italics).

Two oligonucleotides (A.beta.1-7 forward/reverse) bearing phosphate groups on the 5' -ends were annealed under standard conditions to generate the corresponding A.beta.1-7 double stranded (ds) DNA.

The resulting Abeta 1-7ds-DNA fragment (33bp) was ligated with the CpoI-digested pT7Kan-Trx vector (pET28 vector variant) at a vector/Abeta 1-7 insert ratio of 1/100. After transfer into E.coli cells (BL21Codon Plus, DE3 lysogenic strain; genotype: F-ompthsdSB (rB-mB-) gal dcm rne 131; Stratagene) and selection of antibiotics, colony-PCR analysis was carried out on 100 randomly selected transformants. The resulting amplicons (corresponding to the inserted DNA fragment contained between the two Cpo I sites) were analyzed by agarose gel electrophoresis to identify pT7Kan-Trx (A.beta.1-7) in diversity spanning the desired range of A.beta.1-7_nA subset of bacterial transformants contained in the plasmid.

The same set of bacterial transformants was grown in small cultures, induced with Isopropylthiogalactoside (IPTG), examined for recombinant Trx (A β 1-7) of the expected size by denaturing SDS-polyacrylamide (11%) gel electrophoresis_nAnd (3) expressing the polypeptide. Selecting Trx (A.beta.1-7) based on the above assay_nCloning (where n is 3, 6, 9 and 12 respectively), sequence verification for the corresponding Trx (a β 1-7)_nLarge-scale production of polypeptides.

Example 2: recombinant of A beta 1-7 peptide copies with 3, 6, 9 or 12 GGP-spacers TrxAβ(1-7) _n Production of polypeptides

By metal (Ni) in a low voltage FPLC system²⁺) -affinity chromatography purityTrx (Abeta 1-7) polypeptides are converted. The protein was eluted with a 100-. Fractions with estimated purity ≥ 95% (by SDS-PAGE) were collected. Imidazole was removed and the collected fractions were transferred to Phosphate Buffered Saline (PBS), 137mM NaCl, 2.7mM KCl, 10mM Na by dialysis/ultrafiltration (Amicon, Millipore; Vivaspin, Sartorius; cut-off: 5kDa), sterile filtration (cellulose acetate, 0.22 μm pore size; Sartorius)₂HPO₄，2mM KH₂PO₄pH7, pH 7.0). The protein concentration in the final product was determined by Bradford assay (Coomassie Brilliant blue R-250; BioRad) using bovine serum globulin as standard and by UV spectrophotometry using the calculated extinction coefficient.

The final concentration was adjusted to 100 μ M in PBS. 5ml of each Trx (A beta 1-7)_nPolypeptide (500 nmole ea.) and 4 plasmid DNA samples for recombinant protein expression (named pT7Kan-Trx (A. beta.1-7)₃、pT7Kan-Trx(Aβ1-7)₆、pT7Kan-Trx(Aβ1-7)₉、pT7Kan-Trx(Aβ1-7)₁₂) For further experiments.

Example 3: trx (A.beta.1-7) by ELISA _n Immunological properties of polypeptides

Before use, fixed amount of 4 kinds of Trx (A beta 1-7)_nPolypeptides (10 nmol/100. mu.l) with 50. mu.l of aluminium gel 2.0%, aluminium hydroxide (AlOH) based approved for human use₃) The immunoadjuvant of (Brenntag Biosector A/S) was mixed well immediately and injected subcutaneously into BALB/c mice (Charles River Laboratories). Prime (day 1) was followed by 3 booster injections on days 15, 30 and 60. The same immunization protocol was applied to PBS, AlOH injection respectively₃And 3 negative control animals with empty Trx vector. Trx (A.beta.1-15) verified as described above₄Antigen (Moretto et al 2007, J.biol. chem.282, 11436; Ottonello S, Moretto N, Imbimbo BP, Villetti G, WO2007096076) was used as a positive control. 7 animals were injected with 4 Trx (A.beta.1-7) independently_nConstructs (n ═ 3, 6, 9, 12) and controls (PBS, AlOH) cited above₃Trx Carrier + AlOH₃、Trx(Aβ1-15)₄). Sera were collected 2 weeks after the last booster injection and used in enzyme-linked immunosorbent assay (ELISA).

ELISA was performed in duplicate at fixed 1/200 serum dilution ratio using pre-activated 96-well plates (Sigma-Aldrich) and aggregated a β 42(1 μ g/well) in PBS as target antigen. After incubation, washing and addition of Alkaline Phosphatase (AP) -conjugated anti-mouse immunoglobulin (1/5000; Sigma-Aldrich) and the chromogenic substrate 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich), the plates were read spectrophotometrically at 405 nm.

The figure shows the indicated Trx (A beta 1-7) from immunization, all adjuvated with alum_nPolypeptides, Trx or reference antigen Trx (Abeta 1-15)₄Serum anti-a β 42 reactivity of the mouse of (a); mice injected with PBS or aluminum hydroxide alone were used as negative controls. The serum was diluted 1: 200 with PBS and ELISA was performed on aggregated synthetic A.beta.42 as the target antigen. Data are mean ± s.d. (standard deviation) of 7 biological replicates, each in duplicate.

As shown in the figure, significant and statistically significant immune responses were observed using all Trx (a β 1-7) n polypeptides. Just Trx (A beta 1-7)₉And Trx (A beta 1-7)₁₂The immune response is obviously higher (P is less than or equal to 0.05) than that of the Trx (A beta 1-15)₄Antigen assay (about 2-fold).

Claims (30)

1. Comprising the peptide sequence SEQ ID NO: 1 or SEQ ID NO: 2, wherein n is the number of peptide sequences in the array and n.gtoreq.2, and adjacent peptide sequences are separated from each other by 10 or fewer amino acid residues.

2. The polypeptide according to claim 1 comprising bacterial thioredoxin incorporating said tandem array within its active loop site.

3. A polypeptide according to claim 1 or claim 2 wherein n is any number between 3 and 12.

4. The polypeptide according to claim 3, wherein n is 3, 6, 9 or 12.

5. The polypeptide according to claim 4, wherein n is 9 or 12.

6. A polypeptide according to any one of the preceding claims wherein adjacent peptide sequences are separated from each other by an amino acid linker.

7. The polypeptide according to claim 6, wherein the linker is Gly-Gly-Pro.

8. The polypeptide according to any one of the preceding claims, wherein said tandem array is coupled to a thioredoxin carrier via an amino acid linker.

9. The polypeptide according to claim 8, wherein the amino acid linker is Gly-Pro.

10. A polypeptide according to any preceding claim, which comprises SEQ ID NO: 3 to SEQ ID NO: 19, respectively.

11. A polypeptide according to any one of the preceding claims, which, when used to immunise a mammalian organism, causes the production of antibodies capable of specifically recognizing human Α β 42 peptide.

12. A polynucleotide comprising a DNA sequence encoding the expression of the polypeptide of any one of claims 1-11 under the control of a promoter.

13. A polynucleotide according to claim 12 which is a vector capable of expressing said polypeptide in prokaryotic or eukaryotic cells in culture.

14. A polynucleotide according to claim 12 comprising a promoter suitable for expression of the polypeptide in situ in a mammal.

15. A polynucleotide according to any one of claims 12 to 14 comprising SEQ ID NO: 20, or a fragment thereof.

16. A pharmaceutical composition comprising the polypeptide of any one of claims 1-11 or the polynucleotide of claim 14 and further comprising one or more pharmaceutically acceptable excipients.

17. A pharmaceutical composition according to claim 16, comprising an adjuvant.

18. The pharmaceutical composition according to claim 17, wherein the adjuvant is selected from the group consisting of 3 de-O-acylated monophosphoryl lipid a (mpl), saponin QS21, muramyl-di-peptides, aluminum salts, and CpG motifs.

19. The pharmaceutical composition according to claim 18, wherein the adjuvant is an aluminium salt selected from the group consisting of aluminium hydroxide, aluminium phosphate and aluminium sulphate.

20. An antibody raised against a polypeptide according to any one of claims 1 to 11 and capable of specifically recognizing the a β 42 peptide.

21. An antibody according to claim 20 raised against a polypeptide of claim 5.

22. A monoclonal antibody that specifically recognizes the polypeptide of any one of claims 1-11.

23. A therapeutic agent for preventing or treating an amyloidogenic disease, comprising the monoclonal antibody of claim 22.

24. The therapeutic agent of claim 23, wherein the amyloidogenic disease is alzheimer's disease.

25. Use of an antibody according to any one of claims 20 to 22 for the manufacture of a diagnostic agent for the diagnosis of alzheimer's disease or other amyloidogenic diseases in a human patient.

26. A polypeptide according to claims 1-11 or a polynucleotide according to claim 14 or an antibody according to claims 20-22 for use as a medicament.

27. Use of a polypeptide according to claims 1-11 or a polynucleotide according to claim 14 or an antibody according to claims 20-22 for the manufacture of a medicament for the prevention or treatment of an amyloidogenic disease or condition, including alzheimer's disease, dementia associated with down's syndrome, Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy.

28. A polypeptide according to claims 1-11 or a polynucleotide according to claim 14 or an antibody according to claims 20-22 for use in the prevention or treatment of an amyloidogenic disease or disorder.

29. A method of preventing or treating an amyloidogenic disease or disorder in a susceptible individual, comprising administering an effective amount of a polypeptide of claims 1-11 or a polynucleotide of claim 14 or an antibody of claims 20-22.

30. A method for the preparation of a polynucleotide according to any one of claims 12-15, comprising the steps of: (i) preparing a DNA insert encoding (A.beta.1-7); and ii) ligating a molar excess of said DNA insert encoding (A β 1-7) with a restriction digested vector comprising an expression cassette.