A method for the treatment of Alzheimer's disease in a subject having or suspected of having Alzheimer's disease by administering to the subject a therapeutically effective amount of an antibody wherein the antibody is raised against a protofibril that contains an A.beta.-Arc peptide.

SUMMARY OF THE INVENTION

The present invention relates to an active immunisation against AD which will have a much more profound effect in the treatment of Alzheimer's disease, than using the wild-type peptide. Immunization according to the invention will yield antibodies directed to protofibrils, as the immunogen is a protofibril or compound(s) with greatly increased protofibril formation properties. These antibodies, generated in the periphery, will cross the blood brain barrier and mediate clearance of A.beta. in the brain in a protofibril state.

In present invention use is made of a pathogenic AD mutation at codon 693 (Glu693Gly), named the `Arctic mutation`, located within the A.beta. peptide domain of the APP gene, more closely position 22 of the A.beta.-Arc peptide. Carriers of this mutation develop progressive dementia with clinical features typical of AD without symptoms of cerebrovascular disease. Said AD is distinctly characterised by accelerated formation of protofibrils comprising mutated A.beta. peptides (40Arc and/or 42Arc) compared to protofibril formation of wild type A.beta. peptides.

Thus, in a first aspect the invention relates to use of a non-wild type protofibril or compound(s) with protofibril forming ability for immunisation for prevention or treatment of Alzheimer's disease (AD). Preferably, these protofibril or compound(s) have enhanced protofibril forming ability and/or enhanced immunogenicity compared to the wild-type counterparts. Protofibril chemistry has been described by, inter alia, Serpell (2000).

Preferably, the protofibril or compound(s) with protofibril forming ability comprises the following amino acid sequence KLVFFAEDV (SEQ ID NO:2). The A.beta. 1 42 fibrillisation process involves transitional conformation changes from .alpha.-helix via random coil to .beta.-sheet. The stable .alpha.-helix sequence of residues 16 24 (KLVFFAEDV (SEQ ID NO:2)) apparently plays an important role in this process.

The protofibril or compound(s) with protofibril forming ability may be mutated or modified in relation to corresponding wild-type counterparts. Changes in the KLVETAEDV (SEQ ID NO:2) sequence will affect the fibrillisation process. For example, changes of the charged amino acids Glu22 and Asp23 into neutral amino acids will induce a random coil structure in the A.beta. peptide. Furthermore, deprotonation of other amino acids such as Asp7, Glu11 and His 6, 13 and 14 in the N-terminal end, has been suggested to destabilize the .alpha.-helix, leading to initiation of the fibrillation process. Another example is mutations leading to increased irninunogenicity in man by using amino acids from mouse A.beta. at specific positions, e.g. Gly 5, Phe10 Arg13. Furthermore, amino acid 13 in A.beta. is known to be part of a heparan sulphate binding motif (13 16; His, His, Gln, Lys) in human, which has been speculated to be involved in AD disease mechanism (inflammation) (Giulian et al. (1998)). In mouse, His 16 is exchanged for Arg 13 destroying the heparan sulphate binding site. Interestingly, mice have never been observed to develop AD. Hence, the use of A.beta.-Arc/Arg13 as an immunogen would be a way to lower possible inflammatory side effects, elicited with A.beta. peptides with intact heparan sulphate binding motif.

Preferably, the protofibril or compound(s) with protofibril forming ability comprises an A.beta. peptide (.beta.-amyloid protein) and repeats thereof, such as dimeric, oligomeric or multimeric forms). In a preferred embodiment the protofibril or compound(s) with protofibril forming ability comprises a A.beta. peptide related to AD, In another embodiment the protofibril or compound(s) with protofibril forming ability comprises .alpha.-synuclein.

There exists a form of dementia characterised by patients having clusters in the brain of a structure called Lewy bodies. This form of dementia comprises about 20% of all dementia, Patients with Lewy bodies show, inter alia, Parkinson symptoms with progressive cognitive dysfunction. However, some patients also exhibit Alzheimer symptoms and this is called "Lewy variant of Alzheimer". The main component of the Lewy bodies is the protein .alpha.-synuclein. Two mutations in .alpha.-synuclein have been identified Ala53Thr and Ala30Pro. These mutations lead to dominant heritage of Parkinson's disease. These mutations affect the structure/solubility of .alpha.-synuclein and leads to formation of protofibrils. (Conway et al. (2000)).

The A.beta.-Arc as disclosed in SEQ ID NO:1. A.beta.-Arc comprises 39, 40 or 42 amino acids but may also be shorter as long as the protofibril forming ability is maintained.

The profibril or compound(s) with protofibril forming ability may be used in combination with A.beta. peptides having known mutations, such as the Dutch, Flemish, Italian mutation described above as well as the Iowa mutation (D694N) (Grabowski et al., 2001). The A.beta. peptide may comprise one or more of these and/or other mutations. Alternatively, a cocktail of different A.beta. peptides with different mutations is used.

In a second aspect, the invention relates to a peptide, A.beta.-Arc, having the amino acid sequence disclosed in SEQ ID NO 1 comprising a glycine at position 22 instead of glutamic acid compared to wild type A.beta. peptide. The peptide may be natural, synthetic or recombinantly produced. For the purposes of the invention the peptide may be used in monomeric, dimeric, oligomeric, protofibril or multimeric form.

The invention also relates to nucleic acid encoding the above peptide as well as a vector comprising the nucleic acid. The vectors for expressing the polypeptides of the invention require that the nucleic acid be "operatively linked." A nucleic acid is operatively linked when it is placed into a functional relationship with another nucleic acid sequence.

This vector may be inserted in a host cell. Such a host cell can be used to recombinantly produce the peptide of the invention for pharmaceutical or diagnostic use as well for research purposes. The peptide may also be produced synthetically and be purified by HPLC, RP-HPLC, SEC-HPLC.

In a further aspect, the invention relates to a transgenic non-human animal comprising the above vector. Furthermore, the invention relates to a transgenic non-human animal comprising a vector comprising the entire APP gene corresponding to NCBI database, accession no XM.sub.--009710, Homo sapiens amyoid .beta. (A4) precursor protein (protease nexin-II, Alzheimer's disease)(APP), mRNA. However, the APP gene for use in the invention comprises the Arctic mutation, i.e. nucleotide number 2225 is mutated from A to G leading to an amino acid substitution from Glutamic acid to Glycine. The transgenic animal may be used for modelling Alzheimer's disease and testing for therapeutic treatment efficacy. This transgenic animal will bear the entire APP gene comprising the Arctic mutation. This gene is preferably under control of a strong promoter, such as the prion-promoter. The APP gene may contain further mutations, besides the Arctic mutation.

The transgenic animal expresses a human APP or a fragment thereof which encodes glycine instead of glutamic acid at codon 693. Preferably, the animal expresses neuropathological characteristics of AD. Preferably, the mutated APP is expressed in cells which normally expresses the naturally-occurring endogenous APP gene (if present). Typically, the non-human animal is a mouse, Such transgenes typically comprises an Arctic mutation APP expression cassette, wherein a linked promoter and, preferably, an enhancer drive expression of structural sequences encoding a heterologous APP polypeptide comprising the Arctic mutation.

Such transgenic animals are usually produced by introducing the transgene or targeting construct into a fertilized egg or embryonic stem (ES) cell, typically by microinjection, electroporation, lipofection, or biolistics. The transgenic animals express the Arctic mutation APP gene of the transgene (or homologously recombined targeting construct), typically in brain tissue. Alzheimer phenotype and neuropathology is caused by protofibril formation. Such animals are suitable for use in a variety of disease models and drug screening uses, as well as other applications.

In yet a further aspect, the invention relates to antibodies against the A.beta. peptide of SEQ ID NO:1. The antibodies may be monoclonal or polyclonal or antibody fragments, Preferably the antibodies are humanized for use in passive immunisation for prevention of therapy against AD. Thus, antibodies which react with the unique epitope created by glycine at codon 693 are provided.

Another aspect of the invention relates to a pharmaceutical composition, comprising the above peptide and physiologically acceptable excipients for human and veterinary use. The preparation may comprise adjuvants for vaccination purposes. The administration route may be s.c., i.m., oral or nasal.

In a further aspect, the invention relates to use of the above A.beta. peptide for high throughput screening to find substances with anti-protofibrillar activity.

In a further aspect, the invention relates to a method for prevention or treatment of AD, comprising the step: decreasing the formation of A.beta. protofibrils and/or lower meric forms thereof in a subject having, or suspected of having, AD.

The decreasing step above may be by active immunisation with a profibril or compound(s) with protofibril forming ability for prevention or treatment of Alzheimer's disease (AD). wherein said protofibril or compound(s) have enhanced protofibril forming ability and/or enhanced immunogenicity compared to the wild-type counterparts.

Alternatively, the decreasing step above is by passive immunisation with antibodies against protofibrils or compound(s) with protofibril forming ability, such as A.beta.-Arc, The passive immunisation may be in combination with antibodies against other A.beta. peptides with mutations/modifications leading to increased protofibril formation and/or immunogenicity, preferably AD related mutations.

Antibodies generated against the human A.beta. sequence containing the Arctic mutation are directed towards A.beta. protofibrils and therefore are of therapeutic value in the treatment of Alzheimer's disease. Because the A.beta. peptide is in a protofibril conformation when used as an immunogen, antibodies against A.beta. protofibrils are generated. Availability of such antibodies opens Lip possibilities for the development of an efficient and lasting vaccination for the prevention and treatment of Alzheimer's disease.

In another alternative the decreasing step of the method according to the invention is by administration of agents with anti-protofibrillar activity.

In yet a further aspect of the invention, a combination of the vaccine or passive immunization with monoclonal antibodies or compounds with anti-fibrillar activity with one or several other AD treatments such as, acetylcholinesterase inhibitors, nootropics, anti-inflammatory drugs, estrogen, neurotrophic factor agonists, .beta.-secretase inhibitors, .gamma.-secretase inhibitors and .alpha.-secretase agonists, can improve AD treatment efficacy. The rational is that these substances/treatments work with completely different mechanisms of action and hence can be combined to the benefit for the AD patient.

DETAILED DESCRIPTION OF THE INVENTION

The basis of the present invention is a pathogenic amyloid precursor protein (APP) mutation located within the A.beta. sequence at codon 693 (E693G), causing AD in a family from northern Sweden. Surprisingly, carriers of this "Arctic" mutation show decreased A.beta.42 and A.beta.40 levels in plasma. This finding is corroborated in vitro, where the A.beta.42 concentration was low in conditioned media from cells transfected with APP.sub.E693G. Fibrillization studies demonstrate that A.beta. peptides with the Arctic mutation (A.beta.40Arc) form protofibrils at a much higher rate and in larger quantities than wild-type (wt) A.beta. (A.beta.40wt). The unique Ending of decreased A.beta. plasma levels in the Arctic AD family highlights the complexity of the disease and is likely to reflect a novel pathogenic mechanism. The mechanism disclosed in the present invention involves a rapid A.beta. protofibril formation leading to accelerated build-up of insoluble A.beta. intra- and/or extracellularly.

In the present invention, the single amino acid substitution Glu to Gly at position 22 in the A.beta.4040Arc molecule was found to cause a dramatic increase in rate and capacity to form protofibrils compared to the A.beta.40wt peptide. Thus, when A.beta.42Arc and A.beta.40Arc are formed in the brain it is likely that they are more prone to be retained by cellular systems since the accelerated drive to form protofibrils enhances both A.beta. bulk and insolubility. Thus, factors promoting protofibril formation should be considered in the pathogenesis of sporadic AD. Increased protofibril formation is probably also operating in these more common forms of the disease. Indeed, the findings of the present invention open new avenues for possible therapeutic intervention using drugs targeted at preventing protofibril formation.

Studies on the Arctic mutation of the present invention have demonstrated a previously not described pathogenic mechanism for Alzheimer's disease through increased formation of A.beta. protofibrils. A.beta. with the Arctic mutation formed more stable protofibrils and at a much higher rate and in larger quantities than wild-type A.beta., even in the presence of equimolar amounts of wild-type A.beta.. The formation is accelerated at least 2 10 times compared to protofibrill formation of wild type A.beta. peptides. The implication of this finding is that the dangerous species in the amyloid forming pathway that eventually leads to Alzheimer's disease is not the A.beta. fibrils, but a form of the peptide that appears earlier in the fibril maturation process, the protofibrils. One implication of the findings realted to the present invention is that it is important to prevent the formation of protofibrils in order to be able to prevent and treat Alzheimer's disease.

Non-human animals comprising transgenes which encode Arctic mutation APP can be used commercially to screen for agents having the effect of lowering the formation of A.beta. protofibrils. Such agents can be developed as pharmaceuticals for treating abnormal APP processing and/or Alzheimer's disease amongst other neurodegenerative conditions in humans and animals, such as dogs. The transgenic animals of the present invention exhibit abnormal APP processing and expression, and can be used for pharmaceutical screening and as disease models for neurodegenerative diseases and APP biochemistry.

Claim 1 of 18 Claims

1. A method for the treatment of Alzheimer's disease (AD) in a subject having or suspected of having AD, comprising administering to said subject a therapeutically effective amount of an antibody wherein said antibody is raised against a protofibril comprising an A.beta.-Arc peptide selected from the group consisting of A.beta.39-Arc (Amino Acids 1 39 of SEQ ID NO:1), A.beta.40-Arc (Amino Acids 1 40 of SEQ ID NO:1), and A.beta.42-Arc (SEQ ID NO:1), wherein said antibodies bind to arctic and wild-type A.beta. peptides in protofibril conformation.

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