CN100562338C - beta-amyloid-analogue-T-cell epitope vaccine - Google Patents

Abstract

Novel methods of combating diseases characterized by amyloid deposition are invented. Broadly, the method is based on immunization against Amyloid Precursor Protein (APP) or beta amyloid (a β). Preferably the immunization is effected by administration of an analogue of autologous APP or a β, said analogue being capable of inducing the production of antibodies against the autologous amyloid polypeptide. Particularly preferred as immunogen is autologous a β modified to introduce one or several foreign immunodominant promiscuous T cell epitopes. In addition, nucleic acid immunizations against APP or a β and immunizations using live vaccines are also invented, as well as methods and means used in immunizations. These methods and means include methods for preparing analogs and pharmaceutical preparations as well as nucleic acid fragments, vectors, transformed cells, polypeptides, and pharmaceutical compositions.

Description

β-amyloid-analogue-T-cell epitope vaccine

field of invention

The present invention relates to improving the treatment and prevention of Alzheimer's disease (AD) and other diseases characterized by amyloid deposition, for example in the central nervous system (CNS). More specifically, the present invention provides a method for down-regulating (without requiring amyloid deposition) by producing antibodies against related proteins (APP or Aβ) or components thereof in patients suffering from or at risk of suffering from diseases with pathological features involving amyloid deposition. method of amyloid deposition. The invention also provides methods of producing polypeptides used in such methods and polypeptides so modified. The present invention also includes nucleic acid fragments encoding modified polypeptides, vectors incorporating these nucleic acid fragments, and host cells and cell lines transfected therewith. Finally, the present invention also provides a novel conjugate peptide immunogen.

Background of the invention

Amyloidosis is the extracellular deposition of insoluble protein fibrils leading to tissue damage and disease (Pepys, 1996; Tan et al., 1995; Kelly, 1996). Fibrils form when normally soluble proteins and peptides self-crosslink in an abnormal manner (Kelly, 1997).

Amyloid is associated with serious diseases, including systemic amyloidosis, AD, maturity onset diabetes, Parkinson's disease, Huntington's disease, fronto-temporal dementia, and prion-associated infectivity Spongiform encephalopathies (Kuru and Creutzfeldt-Jakob diseases in humans and scrapie in sheep and cattle) are related, and the formation of amyloid plaques in, for example, Alzheimer's disease appears to be most relevant to disease development in humans. Overexpression or expression of modified forms of proteins found in deposits, such as β-amyloid, in animal models has been shown to induce various symptoms of disease, such as Alzheimer's disease-like symptoms. There is currently no specific treatment for amyloid deposits, and the disease is often fatal.

对刚果红表现出特征性染色(Pepys，1996)。它们特征在于交叉的β-结构(Pauling&Corey，1951)，其中多肽链组织成β-折叠形式。虽然淀粉样蛋白具有非常不同的前体结构，但它们都可经历一种结构转换，或许沿类似的途径，转换成β-折叠螺旋原纤维构件的错折叠形式。Subunits of amyloid fibrils can be wild-type, mutant, or truncated proteins, and similar fibrils can be formed in vitro from oligopeptides and denatured proteins (Bradbury et al., 1960; Filshie et al., 1964; Burke & Rougvie, 1972 ). The nature of fibril polypeptide components characterizes amyloidosis. Although amyloids vary widely in size, native structure, and function, all amyloid fibrils are variable-length, unbranched, 70-diameter

Shows characteristic staining for Congo red (Pepys, 1996). They are characterized by an intersecting β-structure (Pauling & Corey, 1951), in which the polypeptide chains are organized into β-sheets. Although amyloid proteins have very different precursor structures, they can all undergo a structural transformation, perhaps along a similar pathway, into a misfolded form of the β-sheet helical fibril building block.

This unique pattern of fibers leads to an amyloidosis called β-fibrillar, (Glenner, 1980a,b), and the fibrillar proteins of AD were called β-proteins before their secondary structure was discovered (Glenner & Wong, 1984 ). The characteristic cross-linked β-diffraction pattern, together with the appearance and staining properties of fibrils, is now an accepted diagnostic marker for amyloid and suggests that although fibrils form from very different protein precursors, regardless of their precursor protein However, they share structural similarities to a certain extent and comprise a structural superfamily (SundeM, Serpell LC, Bartlam M, Fraser PE, Pepys MB, Blake CCFJ Mol Biol 1997Oct 31; 273(3): 729- 739).

AD is one of the most widespread and well-known diseases in which amyloid deposits present in the central nervous system imply a central role in disease development.

AD

Alzheimer's disease (AD) is an irreversible progressive brain disease that occurs gradually and leads to memory loss, behavioral and personality changes, and mental decline. These losses were associated with the death of brain cells and a decline in the connections between them. The causes of the disease vary from person to person, as does the rate of decline. On average, AD patients live only 8 to 10 years after diagnosis, although the disease can last up to 20 years.

AD develops in stages, from early mild amnesia to severe loss of mental function. This loss is known as dementia. In most people with AD, symptoms first manifest after age 60, but earlier onsets are not uncommon. The earliest symptoms often include loss of recent memory, errors in judgment, and personality changes. Often, people in the early stages of AD think less clearly and forget the names of familiar people and common objects. In advanced stages of the disease, they may even forget how to do simple things. Eventually, a person with AD loses all reasoning abilities and becomes dependent on others for their daily care. Eventually, the disease becomes so advanced that the patient becomes bedridden and may develop other diseases and infections. Most commonly, people with AD die from pneumonia.

Although the risk of developing AD increases with age, AD and dementia symptoms are not a normal part of aging. AD and other dementias are caused by diseases that affect the brain. In normal aging, nerve cells in the brain are not lost in large numbers. Instead, AD disrupts three key processes: nerve cell communication, metabolism and repair. This damage eventually causes many nerve cells to stop functioning, lose their connections with other nerve cells, and die.

First, AD destroys neurons in the parts of the brain that control memory, specifically the hippocampus and related structures. As the nerve cells in the hippocampus completely shut down, short-term memory is lost and often a person's ability to do easy and familiar things begins to decline. AD also attacks the cerebral cortex, especially the area responsible for language and reasoning. Finally, many other brain regions are involved, all of which atrophy (shrink), and AD patients become bedridden, incontinent, completely helpless, and unresponsive to the outside world (source: National Institute on Aging Progress Report on Alzheimer's Disease, 1999).

AD Shock

AD is the most common cause of dementia in people 65 and older. AD has become a major health problem due to its enormous impact on individuals, families, health care systems, and society at large. Scientists estimate that more than 4 million people currently suffer from the disease, and the trend is doubling every 5 years among people over the age of 65. It is also estimated that about 360,000 new cases (incidence) occur each year, but this number increases with the age of the population (Brookmeyer et al., 1998).

AD imposes a severe economic burden on society. A recent study conducted in the United States estimated that the annual cost of caring for an AD patient was $18,408 for a patient with mild AD, $30,096 for a patient with moderate AD, and $30,096 for a patient with moderate AD. Patients with severe AD were $36,132. In the United States, it is estimated that the annual national cost of caring for AD patients is slightly over $50 billion (Leon et al., 1998).

About 4 million Americans are 85 or older, and in most industrialized countries, this age group is the fastest growing segment of the population. In the United States, the group is estimated to number nearly 8.5 million by 2030; some experts who study population trends suggest the number could be even greater. As more people live longer, the number of people affected by diseases of aging, including AD, will continue to grow. For example, some studies suggest that about half of all people aged 85 and older have some form of dementia (National Institute on Aging Progress Report on Alzheimer's Disease, 1999).

The main features of AD

The hallmark of AD are two abnormal structures in the brain: amyloid plaques and neurofibrillary tangles (NFTs). Plaques are dense, massive insoluble deposits of protein and cellular material on the outside of and around brain neurons. Tangles are insoluble tangles of fibers that form inside neurons.

There are two types of AD: familial AD (FAD), which follows a defined pattern of inheritance, and sporadic AD, where no clear pattern of inheritance is observed. Due to differences in age of onset, AD can further be described as early-onset (occurring in persons younger than 65 years) or late-onset (occurring in persons 65 years and older). Early-onset AD is rare (approximately 10% of cases) and usually affects people between the ages of 30-60. Some forms of early-onset AD are inherited and run in families. Early-onset AD also often develops more rapidly than the more prevalent late-onset form.

All FADs known to date have an early onset, and 50% of FAD cases are now known to be caused by defects in three genes located on three different chromosomes. They are mutations in the APP gene on chromosome 21; a mutation in a gene called presenilin 1 on chromosome 14; and a mutation in a gene called presenilin 2 on chromosome 1. However, there is no evidence that any of these mutations also play a major role in the more prevalent sporadic or nonfamilial late-onset AD (National Institute on Aging Progress Report on Alzheimer's Disease, 1999).

amyloid plaque

In AD, amyloid plaques first occur in brain regions used for memory and other cognitive functions. They contain large insoluble deposits of β-amyloid protein (hereafter named Aβ), which is a relatively small protein called amyloid precursor protein (APP, the amino acid sequence of which is listed in SEQ ID NO: 2). Protein fragments of large proteins - mixed with some neurons and non-neuronal cells such as microglia and astrocytes. It is currently unknown whether amyloid plaques themselves constitute the primary cause of AD or whether they are a by-product of the AD process. Of course, changes in the APP protein can lead to AD, as in the hereditary form of AD caused by mutations in the APP gene, and the formation of Aβ plaques appears to be closely related to the development of the human disease (Lippa C.F. et al., 1998).

app

APP is one of many proteins associated with cell membranes. When it is synthesized, APP is embedded in the cell membrane of nerve cells, partly inside and partly outside the cell. Recent studies using transgenic mice indicate that APP appears to play an important role in the growth and survival of neurons. For example, certain forms and amounts of APP can protect neurons from short-term and long-term damage, enable damaged neurons to better repair themselves, and help some neurons grow after brain injury.

When APP is embedded in the cell membrane, proteases act on specific sites of APP and cut it into protein fragments. One protease helps cleave APP to form Aβ, and the other protease cleaves APP in the middle of the amyloid fragment so that Aβ cannot be formed. Aβ is formed in two different lengths, the shorter 40 (or 41) amino acid Aβ, which is relatively soluble and aggregates slowly, and the slightly longer, 42 amino acid "sticky" Aβ, which can rapidly An insoluble mass formed. When Aβ is formed, it's not known exactly how it moves through or around nerve cells. In the final stage of the process, "sticky" Aβ aggregates into filaments extracellularly and forms the plaques that are characteristic of AD in brain tissue with dead and dying neuronal fragments and microglia and astrocytes.

Some evidence exists that mutations in APP confer greater potential for splicing Aβ from the APP precursor, resulting in more total Aβ or a relatively more "sticky" form. It also showed that mutations in the presenilin gene can cause neuronal degeneration in at least two ways: by modifying Aβ production or more directly by triggering cell death. Other researchers have suggested that mutated presenilin 1 and 2 may be involved in accelerating the rate of apoptosis.

It is expected that as the disease progresses, more and more lesions will develop, filling more and more brain areas. Studies suggest that Aβ may aggregate and disaggregate simultaneously in a dynamic equilibrium. This gives us hope that it may even be possible to degrade plaques after they have formed (National Institute on Aging Progress Report on Alzheimer's Disease, 1999).

A[beta] is thought to be toxic to neurons. In tissue culture studies, investigators found increased cell death in hippocampal neurons engineered to overexpress a mutant form of human APP compared to neurons overexpressing normal human APP (Luo et al., 1999).

In addition, it has been shown in animal models that overexpression or expression of modified forms of the Aβ protein induces Alzheimer's disease symptoms (Hsiao K. et al., 1998).

Given that increased A[beta] production, its aggregation into plaques and the resulting neurotoxicity may contribute to AD, it would be of therapeutic interest to investigate conditions under which A[beta] aggregation into plaques could be reduced or even blocked.

Presenilin

Mutations in presenilin-1 (S-180) account for approximately 50% of all early-onset familial AD (FAD) cases. Approximately 30 AD-causing mutations have been identified. The onset of AD varies with the mutation. Mutations in presenilin-2 account for a much smaller fraction of FAD cases, but are still an important factor. It is not known whether presenilins are involved in sporadic non-familial AD. The function of presenilins is unknown, but since Aβ-42 levels are increased in AD patients with presenilin mutations, they appear to be involved in the processing of APP to produce Aβ-42 (longer stickier form of peptide, SEQ ID NO: 2, residues 673-714). It is unclear whether presenilins also play a role in the production of NFT's. Some suggest that presenilins have a more direct role in neuronal degeneration and neuronal death. Presenilin-1 is located on chromosome 14, while presenilin-2 is on chromosome 1. If a person has a mutated form of only one of these genes, he or she will almost certainly develop early-onset AD.

There remains some uncertainty as to whether presenilin-1 is consistent with the putative gamma-secretase involved in APP processing (Naruse et al., 1998).

apolipoprotein E

Apolipoprotein E is normally associated with cholesterol, but is also found in lesions and tangles of AD brains. Although alleles 1-3 do not appear to be involved in AD, the presence of the APOE-ε4 allele is significantly associated with the development of late AD (Strittmatter et al., 1993). However, it is a risk factor, not a direct cause, as is the case with presenilin and APP mutations, and it is not restricted to familial AD.

The pathways by which the APOE ε4 protein increases the likelihood of developing AD are not known exactly, but one possible theory is that it promotes the construction of Aβ, which contributes to a lower age of AD onset, or that the presence or absence of the APOE allele may affect neuronal response to injury Mode of response (Buttini et al., 1999).

It has also been shown that Apo Al is amyloidogenic (amyloigenic). In vitro, intact apo A1 itself forms Congo red-positive amyloid-like fibrils (Am J Pathol 147(2): 238-244 (Aug 1995), Wisniewski T, Golabek AA, Kida E, Wisniewski KE, Frangione B).

There are some conflicting results that the APOE-ε4 allele has a positive effect in reducing symptoms of intellectual loss relative to other alleles (Stern, Brandt, 1997, Annals of Neurology 41).

neurofibrillary tangles

A second AD hallmark includes abnormal collections of tangled filaments found inside nerve cells. The main component of the tangle is a form of a protein called tau (τ). In the central nervous system, tau protein is known to bind to and stabilize microtubules, building blocks of the cell's internal support structure or skeleton. In AD, however, tau undergoes a chemical change that no longer stabilizes microtubules but causes their breakdown. Disruption of this transport system first leads to a breakdown in communication between nerve cells, which can subsequently lead to neuronal death.

In AD, chemically altered tau tangles into paired helical filaments - two strands of tau intertwined. These filaments are the main substance found in neurofibrillary tangles. In a recent study, researchers found that in a specific part of the hippocampus of the healthy brain, fewer than 6 percent of neurons undergo neurofibrillary changes, while those neurons in those who died from mild AD More than 43% of these neurons were present in more than 71% of those who died of severe AD. A similar progression is found in studies of neuronal loss. This evidence supports the notion that there is simultaneous tangle formation and loss of neuronal development as AD progresses (National Institute on Aging Progress Report on Alzheimer's Disease, 1999).

Tauopathies and Tangles

In addition to AD, several neurodegenerative diseases are characterized by the aggregation of tau into insoluble filaments in neurons and glia, leading to dysfunction and death. Recently, several groups of investigators studying families with multiple genetic forms of dementia other than AD found that the first tau mutation was on chromosome 17 (Clark et al., 1998; Hutton et al., 1998; Poorkaj et al., 1998; Spillantini et al., 1998). In these families, mutations in the tau gene lead to neuronal cell death and dementia. These disorders, which share some features with AD but differ in several major ways, are collectively referred to as "frontotemporal dementia and parkinsonism associated with chromosome 17" (FTDP-17). These diseases are, for example, Parkinson's disease, certain forms of amyotrophic (spinal) lateral sclerosis (ALS), corticobasal degeneration, progressive supranuclear palsy, and Pick's disease, all marked by abnormal aggregation of tau protein as a feature.

Other AD-like neurological disorders

There are important parallels between AD and other neurological diseases, including prion diseases (such as Kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalitis), Parkinson's disease, Huntington's disease, and frontotemporal dementia. All of these diseases involve the deposition of abnormal proteins in the brain. AD and prion diseases, which lead to dementia and death, are both associated with the formation of insoluble amyloid fibrils, but differ from each other in terms of membrane proteins.

Scientists studying Parkinson's disease, the second most prevalent neurodegenerative disease after AD, have discovered the first gene associated with the disease. This gene encodes a protein called synuclein which, interestingly, is also found in amyloid plaques in the brains of AD patients (Lavedan C, 1998, Genome Res. 8(9):871-80). Researchers have discovered that a genetic defect in Huntington's disease, another progressive neurodegenerative disease that causes dementia, leads to the formation of Huntington protein very similar to the insolubility of Aβ fibrils in AD and protein fibrils in prion diseases Fibrils (Scherzinger E et al., 1999, PNAS U.S.A. 96(8):4604-9).

Scientists have also identified a new gene that, when mutated, leads to familial British dementia (FBD), a rare genetic disorder that causes severe mobility impairment and progressive dementia similar to that seen in AD . In the biochemical analysis of amyloid plaques found in FBD lesions, a unique peptide called ABri was discovered (Vidal et al., 1999). Mutations at specific points in this gene result in a longer-than-normal Bri protein.

ABri peptides obtained by cleavage of the mutant ends of the Bri protein were deposited like amyloid fibrils. These plaques are thought to contribute to the neuronal dysfunction and dementia that characterizes FBD.

Immunized with Aβ

The immune system is normally involved in the removal of foreign proteins and protein particles in the body, but the deposits associated with the above diseases mainly consist of self proteins, thus making the role of the immune system in the control of these diseases less obvious. In addition, deposits are often located in a compartment separate from the immune system (CNS), both of which suggest that any vaccine or immunotherapy approach will be ineffective.

However, scientists have recently attempted to immunize mice with a vaccine comprising heterologous human Aβ and substances known to stimulate the immune system (Schenk et al., 1999 and WO 99/27944). The vaccine was tested in a partially transgenic AD mouse model in which the human APP mutant gene was inserted into the mouse DNA. The mice produced the modified APP protein and developed amyloid plaques as they grew up. This mouse model was used to test whether vaccination against modified transgenic human APP plays a role in plaque formation. In the first experiment, a group of genetically modified mice were given monthly injections of the vaccine, starting at 6 weeks of age and ending in November. The second group of transgenic mice was not injected as a control group. By 13 months of age, control mice had lesions covering 2-6% of the brain. In contrast, virtually no lesions developed in the immunized mice.

In the second experiment, the researchers started the injections at 11 months when some lesions had developed. Over a seven-month period, the number of lesions in the brains of control transgenic mice increased 17-fold, while those mice that received the vaccine had a 99 percent decrease compared with 18-month-old control transgenic mice. In some mice, some pre-existing lesion deposits appeared to be removed by the treatment. It was also found that other lesion-related injuries, such as inflammation and abnormal neuronal processes, were reduced as a result of immunity.

So these are preliminary studies in mice, for example, and scientists need to know whether the vaccinated mice remained healthy in other ways, and whether memory in those vaccinated remained normal. Also, because the mouse model is not a complete representation of AD (animals do not develop neurofibrillary tangles and most of their neurons do not die), additional studies are needed to determine whether humans have similar or different neurofibrillary tangles than mice. reaction. Another consideration is that the approach may "cure" the amyloid deposits, but may not halt the progression of dementia.

Technical issues were also majorly challenging. For example, it is not possible to use this technology to make a vaccine that makes a person develop antibodies against their own proteins. Therefore many questions of safety and efficacy need to be resolved before any experiments in humans can be performed.

Thus, the work of Schenk et al. showed that if it were possible to mount a strong immune response to self-proteins in protein deposits in the central nervous system, such as the lesions that form in AD, it would be possible to both prevent the formation of deposits and to Remove formed lesions.

Recently, clinical trials using the Aβ vaccines discussed above have been discontinued due to the side effect of a large number of immunized subjects developing chronic encephalitis, possibly due to uncontrolled autoimmunity against Aβ in the CNS.

purpose of invention

It is an object of the present invention to provide novel therapeutic methods against diseases characterized by amyloid deposition, such as AD. A further aim is to develop an autovaccine against amyloid for a novel treatment of AD and other pathological diseases involving amyloid deposition.

Summary of the invention

Described here is the application of an autoimmune technique to generate a robust immune response against otherwise non-immunogenic APP and A[beta]. Also described are formulations of such vaccines for preventing, possibly curing or alleviating the symptoms of such diseases associated with amyloid deposition.

Accordingly, in its broadest and most general scope, the present invention relates to a method of down-regulating amyloid precursor protein (APP) or beta-amyloid (Aβ) in animals, including humans, comprising the The animal's immune system effectively presents an immunologically effective amount of at least one APP or Aβ analog that integrates at least one APP and/or Aβ B cell epitope and at least one exogenous T helper cell epitope in the same molecule ( _TH epitope), so that immunization of animals with this analog can induce the production of antibodies against the animal's own APP or Aβ. the analogues

a) is a polyamino acid consisting of at least one copy of a subsequence of residues 672-714 of SEQ ID NO: 2, wherein the exogenous _TH epitope is added and/or inserted and/or deleted and/or substituted by amino acids wherein the subsequence is selected from residues 1-42, residues 1-40, residues 1-39, residues 1-35 of the amino acid sequence consisting of amino acid residues 673-714 in SEQ ID NO: 2 , residues 1-34, residues 1-28, residues 1-12, residues 1-5, residues 13-28, residues 13-35, residues 17-28, residues 25-35, residues bases 35-40, residues 36-42 and residues 35-42; and/or

b) is a polyamino acid comprising an exogenous T _H epitope and an interrupted APP or Aβ sequence, so that the analog does not include any of the molecules in SEQ ID NO: 2 that can effectively bind to MHC class II molecules that trigger a T cell response A subsequence of ; and/or

c) is a polyamino acid comprising an exogenous _TH epitope and an amino acid derived from APP or Aβ, and contains a single methionine residue at the C-terminus of the analog, wherein APP or Aβ contains an exogenous _TH epitope Other methionine residues in position have been substituted or deleted, preferably by leucine or isoleucine; and/or

d) is a conjugate comprising a polyhydroxy polymer backbone on which the polyamino acids defined in a) and/or the polyamino acids defined in b) and/or the polyamino acids defined in c) are coupled to the backbone, respectively. polyamino acids; and/or

e) is a conjugate comprising a polyhydroxypolymer backbone to which 1) an exogenous _TH epitope and 2) a polyamino acid selected from the subsequences defined in a) are coupled, respectively , the interrupted sequence of APP or Aβ as defined in b), and an amino acid sequence derived from APP or Aβ, the C-terminus comprising a single methionine residue, wherein APP or Aβ neutralizes the exogenous _TH epitope The other methionine residues have been substituted or deleted, preferably by leucine or isoleucine.

The attorney has previously filed an international patent application concerning safe vaccination strategies for anti-amyloidogenic polypeptides such as APP and Aβ, see WO 01/62284. This application was not published at the filing date of the present application and does not contain detailed information on the useful APP and A[beta] analogs mentioned above.

The invention also relates to APP and A[beta] analogs and nucleic acid fragments encoding subsets of these. Immunogenic compositions comprising analogs or nucleic acid fragments are also part of the invention.

Figure legend

Figure 1: Schematic depiction of the antibody response against the Aβ protein Aβ-43 (or C-100) derived from autoimmune variants of the amyloid precursor protein. APP is shown at the top of the figure, and the rest of the schematic building blocks indicate that the patterned epitopes P2 and P30 are substituted or inserted into various spliced forms of APP. In the figure, the black part represents the signal sequence of APP, the bidirectional cross-hatching is the extracellular part of APP, the dark vertical shading is the transmembrane domain of APP, the light vertical shading is the intracellular domain of APP, and the thick cross-hatching Shading indicates P30 epitopes and thin cross-hatching indicates P2 epitopes. The solid line box represents Aβ-42/43, and the solid line box and dotted line box together represent C-100. "Abeta" means Aβ.

Figure 2: Schematic depiction of an example of the synthesis of commonly used immunogenic conjugates. Peptide A (any antigenic sequence, eg, the A[beta] sequence described herein) and Peptide B (an amino acid sequence including an exogenous T helper epitope) are synthesized and mixed. They are then brought into contact with a suitable activated polyol, and peptides A and B are linked via activating groups in an amount corresponding to the initial ratio of these two substances in the peptide mixture. See embodiment 4 for details.

Detailed description of the invention

definition

A number of terms used in the specification and claims are defined and explained in detail below to clarify the metes and bounds of the present invention.

的范围变动。The terms "amyloid" and "amyloid protein" are used interchangeably herein to denote a class of protein unbranched fibrils of indeterminate length. Amyloid fibrils exhibit characteristic staining for Congo red with crossed β-structures in which the polypeptide chains are organized as β-sheets. Amyloids are generally derived from amyloid proteins that have very different precursor structures but all undergo a structural transformation into misfolded forms, the building blocks of beta-sheet helical protofilaments. Typically, amyloid fibrils range in diameter from about 70 to about

range changes.

The term "amyloidogenic protein" is intended to mean a polypeptide that participates in the formation of amyloid deposits by being part of the deposits as such or by being part of the biosynthetic pathway leading to the formation of the deposits. Thus, examples of amyloidogenic proteins are APP and Aβ. But the proteins involved in these metabolisms may also be amyloidogenic proteins.

"Amyloid polypeptide" herein is intended to mean a polypeptide comprising the above-discussed amino acid sequence of an amyloidogenic protein derived from a human or other mammal (or a cleavage thereof, which is the same as intact amyloid -Amyloidogenic proteins share many B-cell epitopes), -amyloidogenic polypeptides may thus, for example, comprise substantial parts of amyloidogenic polypeptide precursors (substantial parts for Aβ, a possible The amyloid polypeptide may be derived from APP). Non-glycosylated forms of amyloidogenic polypeptides produced from prokaryotic systems are also included within the scope of the term, as are forms with various glycosylation patterns resulting from the use of, for example, yeast or other non-mammalian eukaryotic expression systems. It should be noted, however, that when the term "amyloidogenic polypeptide" is used, it is meant that said polypeptide is generally not immunogenic when administered to the animal to be treated. In other words, amyloidogenic polypeptides are self-proteins or analogs of such self-proteins, which generally do not elicit an immune response against the amyloidogenicity of said animal.

"Analog" refers to a molecule derived from APP or Aβ that incorporates one or several changes in its molecular structure. Such alterations may be, for example, fusions of APP or Aβ polyamino acids with suitable fusion partners (i.e. alterations involving only C- and/or N-terminal amino acid residue additions in the primary structure), and/ Or it may be in the form of insertions and/or deletions and/or substitutions in the amino acid sequence of the polypeptide. The term also includes derivatized APP or A[beta]-derived molecules, see Modifications of APP or A[beta] discussed below. In some cases, analogs can be constructed so that little or no antibody is elicited against the normal amyloid precursor protein, thereby avoiding the production of (physiologically normal) non-aggregated forms of the polypeptide that is an amyloid precursor No need to interfere.

It should be noted that it is conceivable to use human APP or Aβ xenologs (eg, canine or porcine analogs) as vaccines in humans, potentially generating the desired immunity against APP or Aβ. The use of such xenologs for immunization is therefore also part of the invention.

The term "polypeptide" herein is intended to mean short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. In addition, the term is also intended to include proteins, ie, functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may be linked covalently or non-covalently to form a complex. Polypeptides in proteins may be glycosylated and/or lipidated and/or contain prosthetic groups. Also the term "polyamino acid" is the equivalent of the term "polypeptide".

The terms "T lymphocyte" and "T cell" are used interchangeably to denote thymus-derived lymphocytes responsible for various cell-mediated immune responses as well as assistance in humoral immune responses. Likewise, the terms "B lymphocyte" and "B cell" are used interchangeably to refer to antibody-producing lymphocytes.

The term "subsequence" refers to any contiguous sequence of at least 3 amino acids or at least 3 nucleotides when corresponding, directly derived from the naturally occurring amyloid amino acid sequence or nucleic acid sequence, respectively.

The term "animal" herein is generally intended to refer to an animal species (preferably a mammal), such as a human, Canis domesticus, etc., rather than to refer to a single animal. However, the term also refers to a population of this animal species, since it is important that the individuals immunized according to the methods of the invention do all have the same APP or Aβ, thus allowing the animals to be immunized with the same immunogen. It will be clear to the skilled person that an animal herein refers to an organism having an immune system. Preferably the animal is a vertebrate, such as a mammal.

The term "in vivo down-regulation of APP or A[beta]" here refers to a reduction in the total amount of deposited amyloid of the relevant type (or amyloid as such) in a living organism. This downregulation can be achieved through several mechanisms: simple intervention of amyloid through antibody binding to prevent misaggregation is the simplest. However, it is also within the scope of the invention that antibody binding results in clearance of amyloid by scavenging cells such as macrophages and other phagocytes, as well as antibody interference with other amyloidogenic polypeptides that lead to amyloid formation. A further possibility is that the antibody binds Aβ outside the CNS, thereby efficiently removing Aβ from the CNS by a simple mass action principle.

The expression "effectively presented to the immune system" is intended to mean that the animal's immune system is subjected to an immunogenic challenge in a controlled manner. As demonstrated below, this challenge of the immune system can be achieved in a number of ways, the most important of which is the use of polypeptides comprising "pharmaccines" (i.e., administered to treat or ameliorate progressive disease Vaccination) or nucleic acid "drug vaccination". The precise manner in which the important result to be achieved is that of immunocompetent cells in the animal against the antigen in an immunologically effective manner is immaterial to the inventive concept underpinning the present invention.

The term "immunologically effective amount" has its usual meaning in this field, that is, a certain amount of immunogen that can elicit an immune response that can effectively kill the pathogenic agent having the same immunological properties as the immunogen.

When the expression APP or Aβ has been "modified" is used, it means herein that a chemical modification of the polypeptide has been carried out on APP or Aβ. For example, such modification may be derivatization (eg, alkylation) of specific amino acid residues in the sequence, however, as understood from the description below, preferred modifications include changes in the primary structure of the amino acid sequence.

When discussing "self-tolerance to APP or Aβ", it is understood that since the polypeptide is an own protein of the population to be vaccinated, normal individuals in the population do not have an immune response against the polypeptide; although this cannot be ruled out in animal populations Individual individuals may develop antibodies against the native polypeptide, for example, as part of an autoimmune disease. In any case, animals are usually only self-tolerant to their own APP or Aβ, but it is not excluded that analogues with different phenotypes derived from other animal species or populations may also be tolerated by the animal.

A "foreign T cell epitope" (or "foreign T lymphocyte epitope") is a peptide capable of binding to MHC molecules and stimulating T cells in animal species. Preferred exogenous T cell epitopes in the present invention are "promiscuous" epitopes, ie, epitopes that bind the majority of a particular class of MHC molecules in an animal species or population. Only a limited number of such promiscuous T cell epitopes are known and they are discussed in detail below. Promiscuous T-cell epitopes are also referred to as "universal" T-cell epitopes. It should be noted that for the immunogens used according to the invention to be effective on as large a population of animals as possible, it is necessary to either 1) insert several foreign T-cell epitopes in the same analog, or 2) prepare several analogs, Each of these analogs has a different promiscuous epitope inserted. It should also be noted that the concept of exogenous T-cell epitopes also includes the use of cryptic T-cell epitopes, i.e., epitopes derived from self-proteins that exert immunity only when present in isolated form, rather than as part of said self-proteins. Behavior.

"Exogenous T helper lymphocyte epitope" (exogenous T _H epitope) is an exogenous T cell epitope that binds to MHC class II molecules and can be presented to antigen presenting cells bound to MHC class II molecules ( APC) surface.

A "functional part" of a (bio)molecule is here intended to mean the part of a molecule responsible for at least one biochemical or physiological effect exerted by the molecule. It is well known in the art that many enzymes and other effector molecules have an active site responsible for the molecule's effect. Other parts of the molecule may serve the purpose of enhancing stability or solubility and therefore may be omitted if these purposes are not relevant in the context of a particular embodiment of the invention. For example, it is possible to use specific cytokines as modifying moieties in APP or Aβ (see detailed discussion below), in which case stability issues may arise as conjugation to APP or Aβ provides the necessary stability. It doesn't matter.

The term "adjuvant" has the usual meaning in the field of vaccine technology, that is, a substance or combination of substances that 1) cannot elicit a specific immune response against a vaccine immunogen by itself, but 2) nonetheless can Enhances the immune response against the immunogen. Or in other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen, vaccination with the immunogen may or may not elicit an immune response against the immunogen, but vaccination with the combination of the immunogen and the adjuvant induces a greater A stronger immune response against the immunogen.

"Targeting" of a molecule herein is intended to mean a location at which a molecule introduced into an animal will preferentially occur in a particular tissue or be preferentially associated with a particular cell or cell type. This effect can be achieved in a number of ways, including formulating the molecule in a targeting-promoting composition or by introducing targeting-friendly groups into the molecule, which are discussed in detail below.

"Stimulation of the immune system" means a substance or combination of substances that exhibits a generalized, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants, such as certain cytokines, have the ability to stimulate the immune system. A consequence of the use of immunostimulants is an increased "vigilance" of the immune system, ie, simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response than the immunogen alone.

By "effectively binding" is meant that the peptide binds to an MHC molecule (class I or II) so as to be able to stimulate T cells to kill cells presenting the peptide bound to the MHC molecule. For example, a peptide bound to an MHC class II molecule on the surface of an APC is said to be efficiently bound if the APC will stimulate _TH cells that bind to the presented peptide-MHC class II molecule complex.

Preferred embodiments for downregulation of amyloid

Preferred analogues for use as immunogens in the methods of the invention are modified APP or Aβ molecules in which there is at least one change in the amino acid sequence of APP or Aβ, since that greatly facilitates the acquisition of all important blocking autoantigens. Chance of tolerance - This is evident, for example, from the results obtained in Example 2 herein, where immunization with wild type A[beta] was compared with immunization with A[beta] variant molecules. It has been shown (in Dalum I et al., 1996, J. Immunol. 157:4796-4804) that in normal individuals, potentially self-reactive B lymphocytes that recognize self-proteins are physiologically present. However, in order to induce these B lymphocytes to actually produce antibodies reactive with the relevant self proteins, the help of cytokine producing T helper lymphocytes ( _TH cells and _TH lymphocytes) is required. Typically, this assistance is not provided because T lymphocytes generally do not recognize T cell epitopes derived from self-proteins when presented by antigen presenting cells (APCs). But by providing an "foreign" element in the self-protein (i.e., by introducing an immunologically important modification), the foreign-recognizing T cell. Polyclonal B lymphocytes (also APCs), which recognize self-epitopes on modified self-proteins, also internalize antigens and subsequently present their exogenous T-cell epitopes, and activated T-lymphocytes then provide cytokines to Help these autoreactive polyclonal B lymphocytes. Because the antibodies produced by these polyclonal B lymphocytes react with different epitopes on the modified polypeptide, including those also present in the native polypeptide, antibodies that cross-react with the non-modified self protein are induced. Overall, T lymphocytes can be caused to act as if the polyclonal population of B lymphocytes had recognized the entire foreign antigen, when in fact only the inserted epitope was foreign to the host. In this way, antibodies capable of cross-reacting with unmodified self-antigens are induced.

Several ways are known in the art to modify peptide self-antigens to block self-tolerance. Preferably however, analogues according to the invention include

- introducing at least one first moiety enabling localization of the modified molecule to antigen-presenting cells (APC), and/or

- introduction of at least a second part that stimulates the immune system, and/or

- Introducing at least one optimized analogue for presentation to the third part of the immune system.

However, all these modifications should be done while maintaining a substantial portion of the native B-lymphocyte epitopes in APP or A[beta], since recognition of self-molecules by B-lymphocytes is thereby enhanced.

In a preferred embodiment, side chain groups (in the form of exogenous T cell epitopes or the first, second and third moieties described above) are introduced covalently or non-covalently. That is, a stretch of amino acid residues derived from APP or Aβ is derivatized without altering the primary amino acid sequence, or at least without introducing changes in the peptide bonds between individual amino acids in the chain.

An alternative and preferred embodiment utilizes amino acid substitutions and/or deletions and/or insertions and/or additions (which can be accomplished recombinantly or with the aid of peptide synthesis; modifications involving amino acids over longer stretches can produce fusions peptides). A particularly preferred form of this embodiment is the technique described in WO 95/05849, which discloses a method for the downregulation of self proteins by immunization with self protein analogs in which a number of amino acid sequences have been A corresponding number of amino acid sequences each comprising an exogenous immunodominant T cell epitope is substituted, while at the same time maintaining the entire tertiary structure of the self protein in the analog. However, for the present invention, it is sufficient if the modification (which may be insertion, addition, deletion or substitution) can generate exogenous T cell epitopes while preserving a large number of B cell epitopes in APP or Aβ. However, for maximum efficiency of the induced immune response, it is preferred that the entire tertiary structure of APP or A[beta] is maintained unchanged in the modified molecule.

In some cases it is preferred that APP or A[beta] or fragments thereof are mutated. Particularly preferred are substitution variants in which the methionine at position 35 of A[beta]-43 has been substituted, preferably with leucine or isoleucine, or simply deleted. It is particularly preferred that the analogue comprises a single C-terminal methionine, either because it is naturally present in the amyloidogenic polypeptide or in the exogenous _TH epitope, or because it has been inserted or increased. Accordingly, it is also preferred that the moiety of the analogue comprising the exogenous _TH epitope is free of methionine, with the possible exception of a methionine at the C-terminal position.

The main reason for deleting all but one methionine is that it is possible to make polymeric analogs recombinantly, which can then be cleaved by cyanogen bromide to give single analogs. The advantage is that this facilitates recombinant production.

Indeed, it is generally preferred that all APP or Aβ analogues used according to the invention are characterized in that they comprise only a single methionine as the C-terminal amino acid in the analogue, whereas in the amyloidogenic polypeptide or All other methionines in the exogenous _TH epitope were deleted or substituted with another amino acid.

A further mutation of interest is the deletion or substitution of phenylalanine at position 19 of Aβ-43, particularly preferably the mutation is the substitution of this phenylalanine residue with proline.

Other polyamino acids of interest for use in analogs are truncated portions of the Aβ-43 protein. These can also be used in the immunogenic analogues according to the invention. Particularly preferred are truncated forms Aβ(1-42), Aβ(1-40), Aβ(1-39), Aβ(1-35), Aβ(1-34), Aβ(1-34), Aβ (1-28), Aβ(1-12), Aβ(1-5), Aβ(13-28), Aβ(13-35), Aβ(17-28), Aβ(25-35), Aβ( 35-40), Aβ(36-42) and Aβ(35-42) (where the numbers in brackets indicate the amino acid sequence of Aβ-43 constituting the relevant fragment - for example, Aβ(35-40) with SEQ ID NO: 2 706-711 amino acids are the same). All these truncated Aβ-43 variants can be prepared using the Aβ fragments described here, in particular variants 9, 10, 11, 12 and 13 mentioned in Example 1.

The following formulas describe the molecular constructs generally encompassed by the invention:

(MOD ₁ ) _s1 (amyloid _e1 ) _n1 (MOD ₂ ) _s2 (amyloid _e2 ) _n2 ... (MOD _x ) _sx (amyloid _ex ) _nx (I)

- where amyloid _e1 -amyloid _ex is xB cell epitope comprising APP or Aβ subsequence, which are the same or different, respectively, and may or may not contain exogenous side chain groups, x is ≥3 Integer, nl-nx are x integers ≥ 0 (at least one ≥ 1), MOD ₁ -MOD _x are x modifications introduced between reserved B cell epitopes, s ₁ -s _x are x ≥ 0 Integer of (at least one ≥ 1 if no side chain groups are introduced in the amyloid _ex sequence). Thus, the present invention allows all kinds of permutations of the APP or A[beta] original sequence and all kinds of modifications therein, taking into account the general functional constraints of the constructs on immunogenicity. Thus, included in the present invention are modified APP or Aβ obtained by deleting parts of the sequence, for example parts that exhibit side effects in vivo, or parts that are usually intracellular and thus can generate an undesired immune response.

When used, a preferred form of the constructs set forth above are those in which the B cell epitope comprising the amyloid subsequence is not extracellularly exposed in the precursor polypeptide from which the amyloid is derived. By selecting such epitopes, it is ensured that no antibodies reactive with precursor-producing cells are produced, thereby limiting the resulting immune response to undesired amyloid deposits. For example, in this case it is feasible to induce an immune response against epitopes of APP or A[beta] that are only exposed in the extracellular phase when not coupled to any cells that produce them.

A substantial fraction of B cell epitopes as described herein, or even the entire tertiary structure of the modified protein, is maintained by several methods. One approach is to simply prepare a polyclonal antiserum directed against the polypeptide (e.g., in rabbits) and then use this antiserum as a detection reagent against the modified protein produced (e.g., in a competitive ELISA ). Modified forms (analogues) that react with antisera to the same extent as APP or Aβ must be considered to have the same overall tertiary structure as APP or Aβ, while exhibiting a restrictive (but still major) and specific) reactive analogs are believed to have retained a substantial portion of the original B cell epitope.

Alternatively, selected monoclonal antibodies reactive with unique epitopes on APP or A[beta] can be prepared and used as assay panels. The advantage of this method is to obtain 1) a map of the epitopes of APP or A[beta] and 2) a map of the epitopes that are retained in the analogs produced.

Of course, a third method resolves the three-dimensional structure of APP or A[beta] or a biologically active truncation thereof (see above) and compares this structure with the resolved three-dimensional structure of an analog prepared. The three-dimensional structure can be resolved by X-ray diffraction studies and NMR spectroscopic analysis. Further information on three-dimensional structure can be obtained to some extent from circular dichroism studies, which have the advantage that only pure polypeptides are required (whereas X-ray diffraction provides crystallized polypeptides and NMR provides isotopic variants of polypeptides) to provide Useful information about the three-dimensional structure of a given molecule. Ultimately, however, X-ray diffraction and/or NMR are required for conclusive data, since circular dichroism can only provide indirect evidence of correct three-dimensional structure through information on secondary structural elements.

A preferred embodiment of the invention utilizes multiple presentation of B-lymphocyte epitopes of APP or A[beta] (ie, at least one B-cell epitope is present in two positions in Formula I). This effect can be accomplished by various methods, for example, by simply preparing a fusion polypeptide comprising the structure (APP or Aβ-derived polypeptide) _m , where m is an integer ≥ 2, and then introducing this in at least one of the APP or Aβ sequences Modifications discussed here. Preferably the introduced modification comprises at least one copy of a B-lymphocyte epitope and/or the introduction of a hapten. These embodiments involving multiple presentation of selected epitopes are particularly preferred when only minor fractions of APP or A[beta] are used as components in vaccine agents.

Introduction of exogenous T cell epitopes can be accomplished by introducing at least one amino acid insertion, addition, deletion or substitution, as described above. Of course, it is normal to introduce more than one change in the amino acid sequence (e.g., insertion or substitution with a complete T-cell epitope), but the important goal to be achieved is that when processed by antigen-presenting cells (APCs), similar This exogenous immunodominant T cell epitope is presented by MHC class II molecules on the surface of the APC. Thus, if the amino acid sequence of APP or Aβ contains many of the amino acid residues also found in the exogenous _TH epitope at the appropriate positions, the remaining amino acids of the exogenous epitope can be provided by amino acid insertions, additions, deletions and substitutions. , so as to complete the introduction of exogenous _TH epitope. In other words, it is not necessary to introduce an entire _TH epitope by insertion or substitution to achieve the object of the invention.

Preferably the number of amino acid insertions, deletions, substitutions or additions is at least 2, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 25 insertions, substitutions, additions or deletions. In addition, it is preferred that the number of amino acids inserted, substituted, added or deleted is not more than 150, for example at most 100, at most 90, at most 80 and at most 70. It is particularly preferred that the number of substitutions, insertions, deletions or additions does not exceed 60, in particular this number does not exceed 50 or even 40. Most preferably this number does not exceed 30. With regard to amino acid additions, it should be noted that these are often considered higher than 150 when the resulting construct is in the form of a fusion polypeptide.

A preferred embodiment of the invention comprises modification by introduction of at least one exogenous immunodominant T-cell epitope. It will be appreciated that the question of immunodominance of T cell epitopes is dependent on the animal species in question. As used herein, the term "immunodominant" refers only to epitopes that generate an important immune response in a vaccinated individual/population, but it is a well-known fact that T cell epitopes that are immunodominant in an individual/population do not necessarily have to be present in Immunodominant in another individual of the same species even though it may be able to bind MHC class II molecules in the latter individual. Thus, for the purposes of the present invention, an immunodominant T cell epitope is a T cell epitope that is effective in providing T cell help in the presence of antigen. Typically, immunodominant T cell epitopes have the intrinsic feature that they are substantially always presented for binding to MHC class II regardless of the polypeptide in which they are presented.

Another important issue is the MHC restriction of T cell epitopes. Typically, naturally occurring T-cell epitopes are MHC-restricted, i.e., certain peptides that make up the T-cell epitope bind efficiently to only a subset of MHC class II molecules. This in turn has the effect that in most cases application of a specific T cell epitope will result in vaccine components being effective against only a fraction of the population and, depending on the size of this fraction, must be in the same molecule More T cell epitopes are included, or alternatively a multi-component vaccine is prepared in which the components are variants of APP or A[beta] that differ from each other according to the nature of the T cell epitopes introduced.

If the MHC restriction of the T cells used is completely unknown (such as in the case of vaccinated animals with a poor defined MHC composition), the fraction covered by a particular vaccine composition can be approximated by

-where _pi is the frequency in the population of responders to the ith exogenous T-cell epitope present in the vaccine composition and n is the total number of exogenous T-cell epitopes in the vaccine composition. Thus, a vaccine composition comprising 3 exogenous T cell epitopes with response frequencies in the population of 0.8, 0.7 and 0.6, respectively, will give

1-0.2 x 0.3x 0.4＝0.976

- ie 97.6% of the population produced a statistically significant MHC-II mediated response to the vaccine.

The above formula cannot be applied in cases where the more or less precise MHC restriction map of the polypeptide used is already known. If, for example, a certain peptide binds only to human MHC-II molecules encoded by the HLA-DR alleles DR1, DR3, DR5 and DR7, then use this peptide and another simultaneously with the rest encoded by the HLA-DR alleles 100% coverage of the population will be achieved with peptides bound by MHC-II molecules. Likewise, if the second peptide only binds to DR3 and DR5, adding this peptide will not increase coverage at all. If the calculation of population response is based purely on the MHC restriction of T cell epitopes in the vaccine, the minimum fraction of the population covered by a particular vaccine composition can be determined by the following formula:

是编码与疫苗中任何一种T细胞表位结合、并属于三个已知HLA位点(DP、DR和DQ)的第j个的MHC分子的等位基因单元型在群体中频率的总和；实际上，首先要确定哪些MHC分子可识别疫苗中的每个T细胞表位，其后并以类型(DP，DR和DQ)列出，然后不同的列出的等位基因单元型的单个频率以每种类型进行加和，从而产生

和

-in

is the sum of allelic haplotype frequencies in the population that encode the MHC molecule that binds to any T cell epitope in the vaccine and belongs to the jth of the three known HLA loci (DP, DR, and DQ); In practice, it is first determined which MHC molecules recognize each T-cell epitope in the vaccine, then listed by type (DP, DR, and DQ), and then the individual frequencies of the different listed allelic haplotypes Adding in each type yields

and

The value of p _i in formula II may exceed the corresponding theoretical value ∏ _i :

${\mathrm{<span\; class="notranslate">\; \&pi;</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; -</span>\underset{\mathrm{<span\; class="notranslate">\; j</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\overset{\mathrm{<span\; class="notranslate">\; 3</span>}}{\mathrm{<span\; class="notranslate">\; \&Pi;</span>}}}{<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; v</span>}}_{\mathrm{<span\; class="notranslate">\; j</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; IV</span>}<span\; class="notranslate">\; )</span>$

- where _vj is the allelic haplotype encoding the MHC molecule that binds to the ith T-cell epitope in the vaccine and belongs to the jth of the three known HLA loci (DP, DR, and DQ) in the population sum of frequencies. This shows that in the 1-Π _i population, the frequency of the respondent is _{fresidual_i} = (p _i - Π _i )/(1 - Π _i ). Equation III can therefore be adjusted to obtain Equation V:

- where term 1-f _{remaining -i} is set to 0 in the negative case. It should be noted that Formula V requires that all epitopes have been haplotype-mapped for the same set of haplotypes.

Therefore, when selecting a T cell epitope to introduce into an analog, it is very important to include all available knowledge about the epitope: 1) the frequency of responders to each epitope in the population, 2) MHC restriction data and 3) frequency of associated haplotypes in the population.

There are many naturally occurring "promiscuous" T-cell epitopes that are active in a majority of individuals in an animal species or animal population, and these epitopes are preferably introduced into vaccines to reduce the need for a large number of different T-cell epitopes in the same vaccine. Analog needs.

According to the invention, promiscuous epitopes may be naturally occurring human T-cell epitopes, for example, from tetanus toxoid (e.g. P2 and P30 epitopes), diphtheria toxoid, influenza virus hemaggluttinin (HA) and epitopes of the P. falciparum CS antigen.

In recent years, a large number of other promiscuous T cell epitopes have been identified. In particular peptides have been identified which are able to bind most of the HLA-DR molecules encoded by the different HLA-DR alleles, which are all possible T-cell epitopes to be introduced into the analogues used according to the invention. See the epitopes discussed in the following references, which are hereby incorporated by reference: WO98/23635 (Frazer IH et al., assigned to The University of Queensland); Southwood S et al., 1998, J. Immunol.160:3363-3373 ; Sinigaglia F et al., 1988, Nature 336:778-780; Chicz RM et al., 1993, J.Exp.Med 178:27-47; Hammer J et al., 1993, Cell 74:197-203; and Falk K et al., 1994 , Immunogenetics 39: 230-242. The latter references also refer to HLA-DQ and HLA-DP ligands. Since all of the epitopes listed in these five references share the same motif, they are accordingly candidate natural epitopes for use in the present invention.

Alternatively, the epitope can be any artificial T cell epitope, which is capable of binding most MHC class II molecules. The pan DR epitope peptide ("PADRE") described in WO 95/07707 and in related literature Alexander J et al., 1994, Immunity 1: 751-761 (both contents are incorporated herein by reference) is based on this Candidates for interesting epitopes for use in the invention. It should be noted that the most potent PADRE peptides disclosed in these articles have D-amino acids at the C- and N-termini to increase stability upon administration. However, the main purpose of the present invention is to integrate the relevant epitope as part of the analogue, which is then enzymatically degraded within the lysosomal compartment of APCs for subsequent presentation in the context of the MHC-II molecule, thus in the present Integrating D-amino acids in epitopes used in the invention is not advantageous.

A particularly preferred PADRE peptide is one having the amino acid sequence AKFVAAWTLKAAA (SEQ ID NO: 17) or an immunogenically effective subsequence thereof. This peptide and other epitopes with the same MHC restriction deficiency are preferred T cell epitopes that should be present in analogs used in the inventive methods. Such hyper-promiscuous epitopes allow for the simplest embodiment of the invention, in which only a single analog is presented to the immune system of the vaccinated animal.

As noted above, modification of APP or A[beta] may also include the introduction of a first moiety that localizes the modified amyloidogenic polypeptide to APCs or B lymphocytes. For example, the first moiety may be a specific binding partner for a B-lymphocyte-specific surface antigen or an APC-specific surface antigen. Many such specific surface antigens are known in the art. For example, the moiety can be a sugar and have its receptors on B lymphocytes or APCs (eg mannan or mannose). Alternatively, the second moiety may be a hapten. Moreover, antibody fragments that specifically recognize surface molecules on APCs or lymphocytes can also be used as the first part (surface molecules can be, for example, FCγ receptors of macrophages and monocytes, such as FCγRI or any other specific surface marker of choice substances, such as CD40 or CTLA-4). It should be noted that all of these exemplified targeting molecules may also be used as part of an adjuvant, see below.

As an alternative or in addition to targeting the analog to specific cell types to achieve an enhanced immune response, the level of immune system response can be increased by including a second moiety that stimulates the immune system as described above. Typical examples of such second moieties are cytokines, heat shock proteins or molecular chaperones, and active parts thereof.

Suitable cytokines to be used according to the invention are those cytokines which usually also function as adjuvants in vaccine compositions, i.e. for example interferon gamma (IFN-γ), interleukin 1 (IL-1), interleukin 2 (IL -2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15) and granulocyte-macrophage Colony stimulating factor (GM-CSF); alternatively, a functional part of the cytokine molecule is sufficient as the second part. For the use of such cytokines as adjuvant substances, see the discussion below.

Suitable heat shock proteins or chaperones for use as the second part according to the invention may be HSP70, HSP90, HSC70, GRP94 (also known as gp96, see, Wearsch PA et al., 1998, Biochemistry 37:5709-19) and CRT (calreticulin).

Alternatively, the second moiety may be a toxin such as listeriolycin (LLO), lipid A and heat labile enterotoxin. Also, many mycobacterial derivatives such as MDP (muramoyl dipeptide), CFA (complete Freund's adjuvant) and trehalose diesters TDM and TDE are also interesting possible options.

Likewise, the introduction of a third component that enhances presentation of the analog to the immune system is an important embodiment of the invention. Several examples of this principle have been demonstrated in the art. For example, it is known that the palmitoyl lipidation anchor in Borrelia burgdorferi protein A can be used to provide self-assisted polypeptides (see e.g. WO 96/40718) - it appears that the lipidated protein forms micelles structure, with a core consisting of a lipidated anchor portion of the polypeptide and the rest of the molecule protruding from it, resulting in multiple presentation of the epitope. Therefore, this method and the use of different lipidation anchors (for example, myristyl, myristyl, farnesyl, geranyl-geranyl groups, GPI anchors and N-acyl diglyceride groups) The use of related methods is a preferred embodiment of the invention, especially since the provision of such lipidation anchors in recombinantly produced proteins is quite straightforward, requiring only the use of, for example, naturally occurring signal sequences as fusion partners for analogues. Another possibility is to use the C3d fragment of complement factor C3 or C3 itself (see, Dempsey et al., 1996, Science 271, 348-350 and Lou & Kohler, 1998, Nature Biotechnology 16, 458-462).

Another alternative embodiment of the invention that results in the preferential presentation of multiple (eg, at least 2) copies of important epitope regions of APP or Aβ to the immune system is the covalent coupling of analogs to specific molecules, Namely, variants d and e above. For example, polymers can be used, such as sugars such as dextran, see, e.g., Lees A et al., 1994, Vaccine 12:1160-1166; Lees A et al., 1990, J Immunol.145:3594-3600, and mannose and mannose Glycans are useful candidates. Integral membrane proteins from eg E. coli and other bacteria are also useful binding partners. Traditional carrier molecules such as keyhole limpet hemocyanin (KLH), tetanus toxoid, diphtheria toxoid and bovine serum albumin (BSA) are also preferred and useful binding partners.

A preferred embodiment for the covalent coupling of an APP or Aβ-derived substance to a polyhydroxy polymer such as a sugar involves the use of at least one APP or Aβ-derived peptide and at least one exogenous T helper epitope, which are respectively coupled to On the polyol (ie, the exogenous T helper epitope and the APP or A[beta]-derived amino acid sequence are not fused to each other, but linked to the polyol which then serves as the backbone of the carrier). Moreover, such an embodiment is most preferred when suitable B-cell epitopes with APP or Aβ-derived peptide regions are constructed by short peptide sequences - this is because this method is one that can be realized in the resulting immunological reagents. Very convenient route for multiple presentation of epitopes of choice. However, it is also possible to simply couple the analogues already described here to the polyhydroxy polymer backbone, ie the APP or A[beta]-derived species are not attached to the backbone separately from the exogenous _TH epitope.

It is particularly preferred to couple the exogenous T helper epitope to the APP or A[beta]-derived (poly)peptide via an amide bond which is cleavable by a peptidase. The effect of this strategy is that APCs are able to take up the conjugate and simultaneously process it and subsequently present foreign T cell epitopes in the context of MHC class II.

One way to accomplish peptide conjugation (of interest are APP or Aβ-derived peptides and exogenous epitopes) is with a tresyl (trifluoroethylsulfonyl) group or other suitable activating group e.g. Amine groups (maleimido), p-Nitrophenyl cloroformate (activating OH groups, forming peptide bonds between peptides and polyhydroxy polymers) and tosyl groups (p-toluenesulfonyl) activate suitable polyhydroxy polymers. For example, it is possible to prepare activated polysaccharides as described in WO 00/05316 and US 5,874,469 (both incorporated herein by reference) and to couple them to APP or Aβ-derived peptides or polymeric amino acids and to On T cell epitopes prepared by solid-phase or solution-phase peptide synthesis techniques. The resulting product comprises a polyhydroxypolymer backbone (e.g., a dextran backbone) to which is linked either via its N-terminus or via other available nitrogen moieties derived from APP or Aβ and derived from exogenous T cell epitopes. poly amino acid. If desired, it is possible to synthesize APP or Aβ peptides that have protected all available amino groups except the one at the N-terminus, and subsequently link the resulting protected peptides to trifluoroethylsulfonylated dextran moieties, and finally, The resulting conjugate is deprotected. Specific examples of this method are described in the Examples below.

In addition to using water-soluble polysaccharide molecules as taught in WO 00/05316 and US 5,874,469, cross-linked polysaccharide molecules can also be used to obtain particulate conjugates between polypeptide and polysaccharide - this is believed to lead to improved presentation of the polypeptide to the immune system , because both goals were achieved, namely to obtain a local deposition effect when injecting the conjugate, and to obtain particles that are attractive targets for APCs. Methods using such microparticle systems are also detailed in the examples.

Considerations for introducing modifications into preferential regions of APP or Aβ are to a) preserve known and predicted B-cell epitopes, b) preserve tertiary structure, c) avoid B-cell epitopes on 'producer cells' etc. Presenting, however, that it is easy to screen a panel of analogs that have introduced T cell epitopes at different positions, as described above.

Since the most preferred embodiment of the present invention is the down-regulation of human Aβ, preferably the above-mentioned APP or Aβ polypeptide is human APP or Aβ polypeptide. In this embodiment, it is particularly preferred that the APP or Aβ polypeptide has been modified by substituting at least one amino acid sequence of SEQ ID NO: 2 with at least one amino acid sequence of equal or different length and comprising an exogenous _TH epitope. Preferred examples of modified amyloidogenic APP and A[beta] are shown schematically in Figure 1 with the P2 and P30 epitopes as examples. The rationale for this construct is discussed in detail in the Examples.

More specifically, _TH comprising (or all of) the amino acid sequence introduced in SEQ ID NO:2 may be introduced in any one amino acid of SEQ ID NO:2. That is, the introduction may be after any of amino acids 1-770, but preferably at 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, After any one of amino acids 707, 708, 709, 710, 711, 712, 713 and 714. Combinations can be made to delete any or all of amino acids 1-671, or any or all of amino acids 715-770. In addition, amino acids 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684 in SEQ ID NO: 2 can be deleted in combination with the introduction when the substitution technique is applied , 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709 , 710, 711, 712, 713 and 714 in any one.

Another embodiment of the invention is the presentation of analogs that do not include any subsequence of SEQ ID NO: 2 that operatively binds to an MHC class II molecule that initiates a T cell response.

The rationale for this strategy of designing immunogens so that the immune system induces an immune response against Aβ is as follows: It should be noted that when immunized with a large amount of a self-protein such as Aβ formulated in an adjuvant strong enough to break tolerance to self-protein in vivo , there is a risk that in some vaccinated individuals the immune response cannot be terminated simply by cessation of immunization. This is because the immune response induced in such individuals is most likely due to natural _TH epitopes of self-proteins, with the side effect that the vaccinated individual's own proteins will themselves function as immune agents: a autoimmune conditions.

The preferred method, which involves the application of exogenous _TH epitopes must be the best known to the inventors, this effect has not been observed since anti-autoimmune responses are induced by exogenous _TH epitopes and the inventors have It has been demonstrated repeatedly that the induction elicited by the preferred technique actually decreases after cessation of immunization. However, it could theoretically occur in a small number of individuals, i.e. the individual is immunized, and an immune response could also be elicited by a self-derived _TH epitope of the relevant self-protein - this is more relevant when the self-protein under consideration is relatively abundant, such as Aβ , while other therapeutically relevant autoproteins are present only locally or in such low numbers in the body that an "autoimmune effect" cannot occur. Therefore, a simple way of avoiding this effect is to avoid use of _TH epitopes in immunogens altogether (since peptides shorter than 9 amino acids cannot be used as _TH epitopes, using shorter fragments is an easy way method) inclusion body of the peptide sequence. Thus, this embodiment of the invention also serves to ensure _that the immunogen does not include the peptide sequence of the target APP or Aβ as an "autostimulatory _TH epitope" comprising only the peptide sequence contained in the target protein. Conservatively substituted sequences in the sequence of , allowing it to be additionally used as a _TH epitope.

A preferred embodiment of the immune system presenting an APP or A[beta] analog comprises the use of a mimetic peptide comprising at least one APP or A[beta]-derived peptide that does not operatively bind to MHC secondary molecules, and at least one exogenous T helper cell epitope. In addition, preferably, the APP or Aβ-derived peptide has a B-cell epitope. It is advantageous if the immunological analogue comprises one or more B cell epitopes represented as a continuous sequence or an inserted sequence comprising an exogenous T helper epitope.

Moreover, this embodiment is most preferred when a suitable B-cell epitope carrying APP or Aβ region is constructed by short peptide sequences such that it never binds efficiently to MHC class II molecules. Accordingly, the selected B-cell epitope or epitope of an amyloidogenic polypeptide should include at most 9 contiguous amino acids of SEQ ID NO:2. Shorter peptides are preferred, such as those having up to 8, 7, 6, 5, 4 or 3 contiguous amino acids of the amyloidogenic polypeptide amino acid sequence.

Preferably, the analog comprises at least one subsequence of SEQ ID NO: 2 such that each such at least one subsequence is independently selected from the group consisting of 9 consecutive amino acids, 8 consecutive amino acids, 7 consecutive amino acids, 6 consecutive amino acids , 5 consecutive amino acids, 4 consecutive amino acids and 3 consecutive amino acids of APP or Aβ amino acid sequence composition.

Particularly preferably, consecutive amino acids are selected from residues 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, Amino acid residues 713 and 714 begin.

Protein/peptide vaccination; formulation and administration of analogs

When administering the analog to the animal's immune system by administering the analog to the animal, formulation of the polypeptide follows principles recognized in the art.

The preparation of vaccines comprising peptide sequences as active ingredients is well known in the art, as exemplified in US Patent Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; Typically, such vaccines are prepared in injectable form, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The formulation may also be emulsified. Active immunological components are usually mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or similar substances, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents or adjuvants which enhance the efficacy of the vaccine; see, below, for a detailed discussion of adjuvants.

Vaccines are traditionally administered parenterally by injection, eg subcutaneously, intradermally or intramuscularly. Additional formulations suitable for other modes of administration include suppositories, and in some cases, oral, buccal, sublingual, intraperitoneal, intravaginal, anal, epidural, spinal, intracranial preparation. For suppositories, traditional binders and carriers may include, for example, poly(alkylene) glycols (polyalkalene glycols) or triglycerides; such suppositories may be composed of active ingredient containing 0.5%-10%, preferably 1-2%. A mixture forms. Oral formulations include commonly used excipients, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% active ingredient, preferably 25-70%. For oral formulations, cholera toxin is an interesting formulation partner (and also a possible conjugate partner).

Polypeptides can be formulated in vaccines in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) which are formed with inorganic acids such as hydrochloric or phosphoric, or organic acids such as acetic, oxalic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be obtained from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol , histidine, procaine, etc. derived.

Vaccines are administered in a manner consistent with dosage formulations and in doses that are therapeutically effective and immunogenic. The amount administered depends on the subject to be treated, including, for example, the ability of the individual's immune system to elicit an immune response and the degree of protection desired. Suitable dosage ranges are on the order of several hundred micrograms of active ingredient per inoculation, preferably in the range of about 1 μg-2,000 μg (even though higher amounts in the range of 1-10 mg are contemplated), for example, in the range of about 0.5 μg-1,000 μg, preferably It is in the range of about 1 μg-500 μg, especially in the range of about 10 μg-100 μg. Suitable regimens for the initial dose and booster vaccination also vary, but typically follow the initial dose, followed by vaccination or other doses.

The manner of application can vary widely. Any conventional method of administering vaccines can be used. These include oral administration on solid physiologically acceptable substrates or in the form of physiologically acceptable dispersions, parenteral injection or the like. Vaccine dosage depends on the route of administration and varies according to the age of the recipient and the formulation of the antigen.

In vaccines, the polypeptides of some vaccines are sufficiently immunogenic, while for others the immune response will be enhanced if the vaccine further comprises adjuvant substances.

Various methods of achieving an adjuvant effect on vaccines are known. Rationale and methods in "The Theory and Practical Application of Adjuvants", 1995, Duncan E.S. Stewart-Tull (editor), John Wiley & Sons Ltd, ISBN 0-471-95170-6 and "Vaccines: New Generationn Immunological Adjuvants", 1995, Gregoriadis G et al., (eds.), Plenum Press, New York, are described in detail in ISBN 0-306-45283-9, both of which are hereby incorporated by reference.

Particular preference is given to the use of adjuvants which have been shown to facilitate the breaking of self-tolerance to self-antigens; indeed, this is very necessary when using non-modified triterpenoid polypeptides as active ingredients in self-vaccines. Non-limiting examples of suitable adjuvants are selected from immune-directing adjuvants; immunomodulatory adjuvants such as toxins, cytokines and branched bacterial derivatives; oil preparations; polymers; adjuvants in micellar form; saponins; immunostimulatory complexes Matrix (ISCOM matrix); particles; DDA; aluminum adjuvant; DNA adjuvant; gamma-inulin and encapsulating adjuvant. In general it should be noted that the foregoing in the second and third parts of the analogues refer to the compounds and agents used first, mutatis mutandis also to their use in the adjuvants of the vaccines of the invention.

The use of adjuvants involves the use of agents such as aluminum hydroxide or aluminum phosphate, usually at 0.05-0.1% solution in buffered saline, in mixture with sugar synthetic polymers (e.g., ), the dosage is 0.25% solution, and the protein polymer in the vaccine obtained by heat treatment at 70-101° C. for 30 seconds to 2 minutes respectively, may also be a polymer obtained by a cross-linking agent. Polymers obtained by reactivation of anti-albumin antibodies (Fab fragments) by pepsin treatment can also be used in mixtures with bacterial cells such as C. parvum or endotoxin or lipopolysaccharide fractions of Gram-negative bacteria in Emulsion in a physiologically acceptable oil vehicle such as mannide monooleate (Aracel A) or with 20% perfluorocarbon (Fluosol-DA) used as a block substitute Emulsion of solution. Mixtures with oils such as squalene and IFA are also preferred.

According to the present invention, DDA (dimethyl dioctadecyl ammonium bromide) is an interesting adjuvant candidate, along with DNA and γ-inulin, and Freund's complete and incomplete adjuvant Agents and Quillaja saponins such as QuilA and QS21 are of interest as well as RIBI. Further possibilities are monophosphoryl lipid A (MPL), the aforementioned C3 and C3d and muramyl dipeptide (MDP).

Liposomal formulations are also known to have adjuvant effects, therefore liposomal adjuvants are preferred according to the invention.

基质由来源于皂树皂苷的皂苷(三萜类化合物)、胆固醇和磷脂组成(可选择性地馏分)。当与免疫原性蛋白混合时，所得到的颗粒制剂即为已知为ISCOM颗粒，其中皂苷占60-70％重量/重量，胆固醇和磷脂为10-15％重量/重量，蛋白为10-15％重量/重量。关于免疫刺激复合物的组成及应用的详细资料可在例如上述关于佐剂的教材中找到，而且，Morein B等.，1995，Clin.Immunother.3：461-475和Barr IG和Mitchell GF，1996，Immunol.and Cell Biol.74：8-25(二者均结合于此作为参考)提供了制备完全的免疫刺激复合物的指导。According to the present invention, immunostimulatory complex matrix type ( stromal) adjuvants are also a preferred choice, especially since this type of adjuvant has been shown to be able to upregulate the expression of MHC class II molecules by APCs.

The matrix consists of saponins (triterpenoids) derived from Quillaja saponins, cholesterol and phospholipids (optionally fractionated). When mixed with an immunogenic protein, the resulting granular formulation is known as ISCOM particles, with 60-70% w/w saponins, 10-15% w/w cholesterol and phospholipids, and 10-15% w/w protein. % w/w. Detailed information on the composition and use of immunostimulatory complexes can be found, for example, in the aforementioned text on adjuvants, but also, Morein B et al., 1995, Clin. Immunother.3: 461-475 and Barr IG and Mitchell GF, 1996 , Immunol. and Cell Biol. 74:8-25 (both incorporated herein by reference) provide guidance for preparing complete immunostimulatory complexes.

Another very interesting (and therefore preferred) possibility of achieving an adjuvant effect is to apply the technique described in Gosselin et al., 1992 (which is hereby incorporated by reference). Briefly, presentation of relevant antigens, such as those of the present invention, can be enhanced by linking the antigen to antibodies (or antigen-binding antibody fragments) against Fcγ receptors on monocytes/macrophages. In particular the combination of antigen and anti-Fc[gamma]RI has been shown to enhance immunogenicity for vaccination.

Other possibilities involve the use of the above-mentioned targeting and immunomodulatory substances, especially cytokines, as candidates for the first and second moiety in the modified form of the amyloidogenic polypeptide. As such, synthetic cytokine inducers, such as poly I:C, are also a possibility.

Suitable mycobacterial derivatives are selected from muramyl dipeptide, complete Freund's adjuvant, RIBI and diesters of trehalose such as TDM and TDE.

Suitable immune targeting adjuvants are selected from CD40 ligands and CD40 antibodies or specific binding fragments thereof (see discussion above), mannose, Fab fragments and CTLA-4.

Suitable polymeric adjuvants are selected from sugars such as dextran, PEG, starch, mannan and mannose; plastic polymers such as; and latexes such as latex beads.

Yet another interesting way of modulating the immune response is to include the immunogen (optional together with adjuvants and pharmaceutically acceptable carriers and vehicles). The VLN (a thin, tubular device) mimics the structure and function of a lymph node. Subcutaneous insertion of a VLN creates a site of sterile inflammation where cytokines and chemokines are elevated. T and B cells and APCs quickly respond to danger signals, seek out the site of inflammation and accumulate in the contents of the VLN pore-like matrix. It has been shown that the necessary dose of antigen required to elicit an immune response to the antigen is reduced when VLN is used, and that vaccination with VLN achieves immune protection that exceeds that achieved by conventional immunization with Ribi as an adjuvant. This technique is also used in Gelber C et al., 1998, "Elicitation of Robust Cellular and Humoral Immune Responses to Small Amounts of Immunogens Using a Novel Medical Device Designated the Virtual LymphNode", in: "From the Laboratory to the Clinic, Book, October Abstracts Briefly described in 12-15 1998, Seascape Resort, Aptos, Calfornia".

Microparticle formulations of vaccines have been shown to increase the immunogenicity of protein antigens in many cases and are therefore another preferred embodiment of the invention. Microparticles can be prepared as co-formulations of the antigen with polymers, lipids, sugars or other molecules suitable for making particles, or the microparticles can be homogeneous particles containing only the antigen itself.

Examples of polymer-based microparticles are PLGA and PVP-based particles (Gupta, R.K. et al., 1998), in which polymer and antigen are condensed into solid particles. Lipid-based microparticles can be prepared as lipid micelles (also known as liposomes) in which antigens are encapsulated (Pietrobon, P. J. 1995). Sugar-based microparticles are typically prepared from suitable degradable sugars such as starch or chitosan. Sugar and antigen are mixed and condensed into particles in a process similar to that of polymer particles (Kas, H.S. et al. 1997).

Antigen-only particles can be prepared by various nebulization and freeze-drying techniques. Particularly suitable for the purposes of the present invention is supercritical fluid technology, which can be used to prepare very uniform particles of controlled size (York, P. 1999 & Shekunov, B. et al. 1999).

It is contemplated that the vaccine should be administered 1-6 times per year, eg 1, 2, 3, 4, 5 or 6 times a year to an individual in need thereof. It has previously been shown that the anamnestic immunity induced using the preferred autovaccine according to the invention is not permanent and therefore the immune system requires periodic stimulation with amyloidogenic polypeptides or modified amyloidogenic polypeptides.

Due to genetic differences, different individuals may have different strengths of immune response to the same polypeptide. Therefore, the vaccine of the present invention may contain several different polypeptides to enhance the immune response, see above for the option of introducing exogenous T cell epitopes. A vaccine may comprise two or more polypeptides, wherein all such polypeptides are as defined above.

Thus the vaccine may comprise 3-20 different modified or non-modified polypeptides, eg 3-10 different polypeptides.

nucleic acid inoculation

As an alternative to traditional peptide-based vaccine delivery methods, nucleic acid vaccination techniques (also known as "nucleic acid immunization", "genetic immunization" and "genetic immunization") offer a number of attractive features.

First, in contrast to traditional vaccine approaches, nucleic acid vaccination does not require the resource-consuming large-scale production of immunogenic agents (eg, factory-scale fermentation of microorganisms that produce modified amyloidogenic polypeptides). Additionally, no equipment is required to purify and refold the immunogen. Finally, because nucleic acid vaccination relies on the biochemical equipment of the vaccinated individual to produce the expression product of the introduced nucleic acid, it is desirable that optimal post-translational processing of the expression product occur; As mentioned above, an effective part of the original B-cell epitope should be retained in the modified molecule, and because in principle a B-cell epitope can be constructed from parts of any (bio)molecule (eg sugars, lipids, proteins, etc.). Therefore, the natural glycosylation and lipidation patterns of the immunogen are very important for the overall immunogenicity, and this is best ensured by having a host in which the immunogen is produced.

Thus, a preferred embodiment of the variant a-c of the invention comprises introducing the nucleic acid encoding the analogue into animal cells, so as to obtain in vivo expression of the cells into which the nucleic acid is introduced, thereby enabling the presentation of the analogue to the immune system.

In this embodiment, the nucleic acid introduced is preferably DNA, which can be in the form of naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, contained in viral DNA in a vector, DNA formulated with a protein or polypeptide that facilitates transfection, DNA formulated with a targeting protein or polypeptide, DNA formulated with a calcium deposition reagent, DNA coupled to an inert carrier molecule, encapsulated in a polymer such as PLGA ( See the technique described in WO 98/31398) or DNA in chitin or chitosan, and DNA formulated with an adjuvant. It should be noted here that virtually all considerations regarding adjuvants suitable for use in traditional vaccine formulations are also applicable to DNA vaccine formulations. Thus, everything herein regarding the use of adjuvants in polypeptide-based vaccines applies mutatis mutandis to use in nucleic acid vaccination techniques.

Regarding the routes of administration and regimens of the polypeptide-based vaccines that have been described in detail above, they can also be applied to the nucleic acid vaccines of the present invention, and all discussions above about the routes of administration and regimens suitable for polypeptides are modified Applies to nucleic acids. To this it should be added that nucleic acid vaccines are suitable for intravenous and intraarterial administration. In addition, it is well known in the art that nucleic acid vaccines can be administered by using so-called gene guns, so this and equivalent methods of administration are also part of the present invention. Finally, good results have been reported for the use of VLNs in the administration of nucleic acids, so this particular mode of administration is particularly preferred.

In addition, nucleic acids that are immunological agents may include regions encoding first, second and/or third moieties, such as the aforementioned immunomodulatory substances such as cytokines discussed as useful adjuvants. A preferred version of this embodiment involves having the coding region of the analog and the coding region of the immune modulator in different reading frames or at least under the control of different promoters. This avoids the creation of analogs or epitopes as fusion partners for immune modulators. Alternatively, two different nucleotide fragments can be used, but this is not preferred since it is advantageous to have the coding regions of both in the same molecule to ensure their co-expression.

Therefore, the present invention also relates to a composition for inducing anti-APP or Aβ antibody, the composition comprising

- a nucleic acid fragment or vector of the invention (see discussion of vectors below), and

- Pharmaceutically and immunologically acceptable vehicles and/or carriers and/or adjuvants as discussed above.

Under normal circumstances, the nucleic acid encoding the variant is introduced in the form of a vector in which expression takes place under the control of a viral promoter. For a more detailed discussion of vectors of the invention see the Discussion below. Further detailed descriptions of nucleic acid vaccine formulations and applications are available, see, Donnelly JJ et al., 1997, Annu. Rev. Immunol. 15:617-648 and Donnelly JJ et al., 1997, Life Sciences 60:163-172. These references are hereby incorporated by reference.

live vaccine

A third alternative for efficient presentation of analogues as defined in variants a-c to the immune system is the application of live vaccine technology. In in vivo vaccination, presentation to the immune system is achieved by administering to the animal a non-pathogenic microorganism transformed with a nucleic acid fragment encoding an analog or a vector integrating such a nucleic acid fragment. The non-pathogenic microorganism can be any suitable attenuated bacterial strain (attenuated by passage or removal of pathogenic expression products by recombinant DNA techniques), for example, Mycobacterium bovis BCG., non-pathogenic Streptococcus species, Escherichia coli, Salmonella, Vibrio cholerae, Shigella, etc. A review of the state of the art for the preparation of live vaccines can be found in, eg, Saliou P, 1995, Rev. Prat. 45:1492-1496 and Walker PD, 1992, Vaccine 10:977-990, both of which are incorporated herein by reference.

For details on nucleic acid fragments and vectors used in such live vaccines, see the discussion below.

As an alternative to live bacterial vaccines, the nucleic acid fragments of the invention discussed below may be incorporated into an avirulent viral vaccine vector, such as a vaccinia strain or any other suitable poxvirus.

Typically, a non-pathogenic microorganism or virus is administered to an animal only once, but in certain circumstances it may be necessary to administer the microorganism more than once during a lifetime to maintain protective immunity. It is even contemplated that the above detailed immunization schemes for polypeptide vaccination can also be used in live or viral vaccines.

Alternatively, live or virus vaccination is combined with prior or subsequent polypeptide and/or nucleic acid vaccination. For example, live or viral vaccines can be used to generate primary immunization, followed by booster immunization using polypeptide or nucleic acid methods.

Microorganisms or viruses can be transformed with nucleic acids comprising regions encoding the first, second and/or third part, for example in the form of the aforementioned immunomodulatory substances such as cytokines as useful adjuvants. A preferred version of this embodiment involves having the coding region of the analog and the coding region of the immune modulator in different reading frames or at least under the control of different promoters. The generation of analogues or epitopes as fusion partners for immune modulators is thereby avoided. Alternatively, two different nucleotide fragments can be used as transformation reagents. Of course, it is possible to provide an analogue of the invention having the first and/or the second and/or the third part in the same reading frame as the expression product, and this embodiment is particularly preferred according to the invention.

Application of the method of the present invention in disease treatment

As understood from the above discussion, the methods provided by the present invention allow for the management of diseases characterized by amyloid deposits. In this context, AD is the primary target of the method of the invention, but other diseases characterized by A[beta] containing amyloid deposits are possible targets. Therefore, an important embodiment of the method of the present invention for down-regulating amyloid activity comprises treating and/or preventing and/or ameliorating AD or other diseases characterized by amyloid deposits, the method according to the present invention comprising APP or Aβ was downregulated to such an extent that the amount of amyloid decreased significantly.

It is particularly preferred that the reduction in amyloid results in an inversion of the balance between amyloid formation and amyloid degradation/clearance, ie the rate of amyloid degradation/clearance exceeds the rate of amyloid formation. By carefully controlling the amount and effect of immunization in individuals in need, it is possible to achieve a balance over time that results in a net decline in amyloid deposits without additional side effects.

Alternatively, if the methods of the invention fail to clear or reduce pre-existing amyloid deposits in an individual, the methods of the invention may be used to achieve a clinically significant reduction in neo-amyloid formation, thereby significantly prolonging the disease There is no time for symptoms to worsen. Should be scanned by measuring serum concentrations of amyloid (which is thought to be in equilibrium with deposited material), or by using positron-emission tomography (PET), see Small GW, et al., 1996, Ann N Y Acad Sci 802:70-78, to monitor the rate of amyloid deposition.

Other diseases and conditions that the present means and methods can be used to treat or ameliorate in a similar manner are mentioned in the "Background of the Invention" or listed below in the section entitled "Other Amyloid Diseases and Proteins Associated Therewith".

Invented peptides, polypeptides and compositions

As is evident from the foregoing, the present invention is based on the concept of immunizing individuals against APP or A[beta] antigens in order to achieve a reduction in the number of pathogenicity-associated amyloid deposits. A preferred way to achieve this immunity is to use analogs as described herein, thereby providing molecules not previously disclosed in the art.

The analogs discussed herein are considered inventive in their own right, and therefore an important part of the invention applies to analogs as described above. Accordingly, any reference herein to modified APP or A[beta] is relevant to the amyloidogenic analogs described herein, and any such reference is adapted to the description of these analogs mutatis mutandis.

It should be noted that preferred modified APP or Aβ molecules comprise modifications such that the polypeptide has at least 70% sequence homology to APP or Aβ or a subsequence thereof at least 10 amino acids in length. Higher sequence homology is preferred, eg at least 75% or even at least 80, 85, 90 or 95%. The sequence homology of proteins and nucleic acids can be calculated by (N _ref -N _dif )*100/N _ref , where N _dif is the total number of different residues in the two sequences when aligned, and N _ref is the number of residues in a sequence number of residues. Thus, the DNA sequence AGTCAGTC has 75% sequence homology to the sequence AATCAATC (N _dif =2 and N _ref =8).

The invention is also suitable for use in assaying compositions of the methods of the invention. Therefore, the present invention also relates to an immunological composition comprising an immunologically effective amount of the above-mentioned analogs, said composition further comprising pharmaceutically and immunologically acceptable diluents and/or vehicles and/or carriers and/or excipients excipients and optional adjuvants. In other words, the invention relates in part to formulations of analogues, essentially as described above. When formulations involving modified and non-modified amyloidogenic polypeptides are used in the methods of the invention to downregulate APP or Aβ, the choice of adjuvant, carrier and vehicle corresponds accordingly to what has been discussed above.

Polypeptides are prepared according to methods well known in the art. Longer polypeptides are usually prepared by recombinant gene technology, including introducing a nucleic acid sequence encoding an analog into a suitable vector, transforming a suitable host cell with the vector, expressing the nucleic acid sequence through the host cell, and obtaining the nucleic acid sequence from the host cell or its culture supernatant. Recovery of the expression product, and subsequent purification and optionally further modification, eg, refolding or derivatization.

Preferably shorter peptides are prepared by well known solid or solution phase peptide synthesis techniques. However, recent advances in the technology have made it possible to produce full-length polypeptides and proteins by these methods, so it is also within the scope of the invention to make long constructs synthetically.

Nucleic acid fragments and vectors of the invention

It can be understood from the above that polyamino acid analogs can be prepared by recombinant gene technology, or by chemical synthesis or semi-synthesis; when the modification includes coupling to protein carriers (for example, KLH, diphtheria toxoid, tetanus toxoid The latter two options are particularly relevant when the modification involves the addition of side chains or side chain groups on APP or Aβ-derived peptide chains, as well as non-protein molecules such as carbohydrate polymers.

Nucleic acid fragments encoding analogues are very important chemical products for recombinant gene technology and, of course, for nucleic acid immunization. Thus, an important part of the invention is suitable for nucleic acid fragments encoding analogues of the invention, i.e. the analogues are APP or Aβ-derived polypeptides comprising native sequences to which fusion partners have been added or inserted, or preferably An APP or Aβ-derived polypeptide wherein it has introduced exogenous T-cell epitopes by insertion and/or addition, preferably by substitution and/or deletion. The nucleic acid fragments of the present invention are DNA or RNA fragments.

The nucleic acid fragments of the invention are typically inserted into suitable vectors to form cloning or expression vectors carrying the nucleic acid fragments of the invention; such novel vectors are also part of the invention. A detailed description of the construction of these vectors of the invention is discussed below in relation to transformed cells and microorganisms. According to the purpose and type of application, the vector can be in the form of plasmid, phage, cosmid, minichromosome or virus, and naked DNA transiently expressed in specific cells is also an important vector. Preferred cloning and expression vectors of the invention are autonomously replicable so that high copy numbers can be achieved for high level expression or subsequent high level replication of clones.

The general profile of the vector of the present invention includes the following features in terms of 5'→3' orientation and operable linkage: a promoter for promoting the expression of the nucleic acid fragment of the present invention, optionally encoding the secretion of the polypeptide fragment (to the extracellular phase, or Applicable, into the periplasm) or the nucleic acid sequence of the leader peptide integrated into the membrane, the nucleic acid fragment of the present invention, and optionally the nucleic acid sequence encoding the terminator. When operating an expression vector in a production strain or cell line, it is preferable that the vector integrates into the host cell genome when introduced into the host cell, for the genetic stability of the transformed cell. In contrast, when vectors are used to achieve in vivo expression in animals (i.e., when vectors are used in DNA vaccination), it is preferred for safety reasons that the vector cannot integrate into the host cell genome; typically, naked DNA or non-integrating viruses are used Vectors, the selection of which is well known to those skilled in the art.

The vectors of the invention are used to transform host cells to produce analogs of the invention. Such transformed cells, which are also part of the present invention, may be cultured cells or cell lines used for the propagation of the nucleic acid fragments and vectors of the present invention, or for the recombinant production of analogs of the present invention. Alternatively, the transformed cell may be a suitable live vaccine strain into which the nucleic acid fragment (single or multiple copies) has been inserted to effect secretion or integration of the analog into the bacterial cell membrane or cell wall.

Preferred transformed cells of the invention are microorganisms, e.g., bacteria (e.g., Escherichia sp. [e.g. E. coli], Bacillus [e.g. Bacillus subtilis], Salmonella or Mycobacterium [preferably non- bovis BCG]), yeasts (eg Saccharomyces cerevisiae) and protozoa. Alternatively, transformed cells are derived from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells. Most preferred are cells of human origin, see discussion of cell lines and vectors below. Recent results have shown promising prospects for recombinant production of polypeptides in the Applicant's laboratory using the commercially available Drosophila melanogaster cell line (Schneider 2 ( _S2 ) cell line and vector system available from Invitrogen), therefore, This expression system is particularly preferred.

For cloning and/or optimized expression, preferably transformed cells are capable of replicating the nucleic acid fragments of the invention. Cells expressing nucleic acid fragments are a preferred useful embodiment of the invention; they can be used for small-scale or large-scale production of analogs of the invention, or in the case of non-pathogenic bacteria, as vaccine components of live vaccines.

When the analogs of the invention are produced by transformed cells, it is very convenient, but far from essential, that the expression product be delivered to the culture medium or displayed on the surface of the transformed cells.

When an effective producer cell has been identified, it is preferably used as a basis to establish a stable cell line carrying the vector of the present invention and expressing the nucleic acid fragment encoding the modified amyloidogenic polypeptide. Preferably, the stable cell line secretes or carries the analog of the invention, thereby facilitating its purification.

Generally, a plasmid vector derived from a species compatible with the host cell and containing the replicon and regulatory sequences is used in conjunction with the host. Such vectors usually carry a replication site, as well as a marker sequence that provides for phenotypic selection in transformed cells. For example, E. coli is typically transformed with pBR322, a plasmid derived from E. coli species (see, eg, Bolivar et al., 1977). The pBR322 plasmid contains ampicillin and tetracycline resistance genes, thereby providing an easy way to identify transformed cells. The pBR plasmid or other microbial plasmid or phage must also include, or be modified to include, a promoter for prokaryotic expression.

Those most commonly used in recombinant DNA construction include the β-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) and the tryptophan (trp) promoter system (Goeddel et al., 1979; EP-A-0 036 776). Although these promoters are the most commonly used, other microbial promoters have been discovered and used, and detailed information about their nucleotide sequences has been reported, allowing the skilled artisan to functionally link them to plasmid vectors (Siebwenlist et al., 1980). Certain genes of prokaryotic origin can be efficiently expressed in E. coli from their own promoter sequence, thereby obviating the need to artificially add another promoter.

In addition to prokaryotes, eukaryotic microorganisms such as yeast cultures can also be used, where the promoter should be able to initiate expression. Saccharomyces cerevisiae, or S. commonis, is the most commonly used among eukaryotic microorganisms, although a large number of other strains are generally available. For example, in S. cerevisiae expression, the plasmid YRp7 is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmid already contains the trp1 gene, which provides a selectable marker for mutant strains of yeast lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, 1977). Trp1 impairment, a hallmark of the yeast host genome, subsequently provides an efficient environment for detection of transformation by growth in the absence of tryptophan.

Suitable promoter sequences in yeast vectors include 3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate Dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase Enzyme and glucokinase promoters. During construction of appropriate expression plasmids, termination sequences associated with these genes are also ligated to the 3' end of the desired expression sequence in the expression vector to provide for polyadenylation and termination of the mRNA.

Other promoters with the added advantage of growth-condition-regulated transcription are alcohol dehydrogenase 2, isocytochrome c, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and the promoter regions for the enzymes responsible for maltose and galactose utilization. Any plasmid vector that includes a yeast-compatible promoter, origin of replication, and termination sequences is suitable.

In addition to microorganisms, cell cultures derived from multicellular organisms can also be used as hosts. In principle, any such cell culture is usable, whether from vertebrate or invertebrate culture. However, the greatest interest has been in vertebrate cells, and the propagation of vertebrates in culture (tissue culture) has become routine in recent years (Tissue Culture, 1973). Examples of such useful host cell lines are VERO and Hela cells, the Chinese Hamster Ovary (CHO) cell line and W138, BHK, COS-7 293, Spodoptera frugiperda (SF) cells (commercially available as complete expression systems Obtained from, mutatis mutandis (ia) Protein Sciences, 1000 Research Parkway, Meriden, CT 06450, USA and from Invitrogen), and the MDCK cell line. In the present invention, a particularly preferred cell line is _S2 obtained from Invitrogen, PO Box 2312, 9704 CH Groningen, The Netherlands.

Expression vectors for such cells typically include (if necessary) an origin of replication, a promoter in front of the gene to be expressed, and any necessary ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequence.

For use in mammalian cells, the regulatory functions on expression vectors are usually provided by viral material. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, and more often simian virus 40 (SV40). Both the early and late promoters of the SV40 virus are particularly useful since they are readily obtained from the virus as fragments that also include the SV40 viral origin of replication (Fiers et al., 1978). Smaller or larger SV40 fragments can also be used as long as they include a sequence extending approximately 250 bp from the Hind III site to the Bgl I site at the viral origin of replication. Additionally, it is also possible, and often desirable, to utilize promoters or regulatory sequences normally associated with the desired gene sequence to provide regulatory sequences compatible with the host cell system.

The origin of replication may be provided by vector construction to include an exogenous origin, eg, may be derived from SV40 or other viruses (eg, polyoma, adenovirus, VSV, BSV), or by the host cell chromosomal replication machinery. The latter is usually sufficient if the vector integrates into the host cell chromosome.

Identification of useful analogs

It is clear to the skilled person that not all possible variations or modifications of naturally occurring APP or A[beta] are capable of eliciting antibodies in animals that cross-react with the native form. However, it is not difficult to establish an effective standard screen for modified amyloidogenic molecules meeting the minimum requirements for immunoreactivity described herein. Therefore, it is possible to use a method to identify modified amyloidogenic polypeptides capable of inducing the production of antibodies against non-modified amyloidogenic polypeptides in animal species, wherein the non-modified amyloidogenic polypeptides are ( non-immunogenic) self protein, the method comprising

- preparation of a group of mutually distinct analogues of the invention, wherein amino acids have been added, inserted, deleted or substituted into the amino acid sequence of APP or Aβ of an animal species, by peptide synthesis or genetic engineering techniques, thereby producing a A group comprising amino acid sequences of T-cell epitopes that are foreign to the animal species, or preparing a group of nucleic acid fragments encoding a group of analogs that are different from each other,

- detecting the ability of a member of the group of analogs or nucleic acid fragments to induce anti-unmodified APP or Aβ antibodies by an animal species, and

- identify and optionally isolate members of the group of analogs that significantly induce anti-unmodified APP or Aβ antibody production in species, or identify and optionally isolate members of the group of nucleic acid fragments that induce significantly in animal species Polypeptide expression products produced by anti-unmodified APP or Aβ antibodies.

In this context, a "mutually distinct set of modified amyloidogenic polypeptides" is a collection of analogs that are not identical, e.g., it can be selected according to the criteria discussed above (e.g., in combination with circular dichroism, NMR spectroscopy studies and/or X-ray diffraction pattern analysis). The group consists of only a few members, but it is contemplated that the group may include several hundred members.

Detection of membership in a panel is ultimately performed in vivo, but a number of in vitro assays can be used to narrow down the number of modified molecules suitable for the purposes of the present invention.

Since the purpose of introducing exogenous T-cell epitopes is to support B-cell responses through T-cell help, a prerequisite is that T-cell proliferation is induced by the analog. T cell proliferation can be detected by standard in vitro proliferation assays. Briefly, a T cell-enriched sample is obtained from a subject and subsequently maintained in culture. Cultured T cells are contacted with the subject's APCs, which have pre-adsorbed the modified molecule and are processed to present their T cell epitopes. Proliferation of T cells is monitored and compared to appropriate controls (eg, T cells in culture contacted with APCs treated with intact native amyloidogenic polypeptide). Alternatively, proliferation can be measured by measuring the concentration of relevant cytokines released by T cells in response to their recognition by foreign T cells.

Having conferred a high probability that at least one analog of each type group is capable of inducing the production of antibodies against APP or Aβ, it is possible to prepare an immune composition comprising at least one analogue capable of inducing in animal species against non-modified Antibody analogs of APP or Aβ, wherein the non-modified APP or Aβ is a self-protein, the method comprising combining a member of the group that significantly induces the production of antibodies reactive with APP or Aβ in an animal species with a pharmaceutically and immunologically acceptable Acceptable carriers and/or vehicles and/or diluents and/or excipients are mixed, optionally combined with at least one pharmaceutically and immunologically acceptable adjuvant.

The detection of the above-mentioned polypeptide group can be easily accomplished by the following method. First, a large number of mutually different nucleic acid sequences or vectors of the present invention are prepared, these are inserted into a suitable expression vector, and a suitable host cell (or host animal) is transformed with the vector. , and realize the expression of the nucleic acid sequence of the present invention. These steps are followed by isolation of the expression product. Preferably, the nucleic acid sequences and/or vectors are prepared by methods comprising performing molecular amplification techniques, such as PCR or by nucleic acid synthesis.

Specific amyloidogenic targets

In addition to the proteins most commonly associated with Alzheimer's disease, APP, ApoE4, and Tau, others are characterized by their direct presence in the lesions or tangles of the AD brain or by their apparent genetic association with an increased risk of developing AD. There is a long list of proteins that are associated in some way with AD. Most, if not all of these antigens, along with A[beta], APP, Presenilin and ApoE4 described above, are putative target proteins in certain embodiments of the invention. These putative target proteins have been discussed in detail in WO01/62284. Accordingly, these putative targets are only briefly mentioned here, while a more detailed background discussion can be found in WO 01/62282, which is hereby incorporated by reference:

α1-antichymotrypsin (ACT); α2-macroglobulin; ABAD (Aβ peptide-binding alcohol dehydrogenase); APLP1 and -2 (amyloid precursor-like proteins 1 and -2); AMY117; Bax Bcl-2; Bleomycin hydrolase; BRI/ABRI; Chromogranin A; Clusterin/ApoJ; CRF (corticotropin releasing factor) binding protein; EDTF (endothelium-derived toxin factor); heparan sulfate Proteoglycan; human brain failure protein response mediator protein-2; Huntingtin (Huntington's disease protein); ICAM-I; IL-6; lysosome-associated antigen CD68; P21 ras; PLC-δ1 (phospholipase C isoform serum amyloid P component (SAP); synaptophysin; Synuclein (alpha-synuclein or NACP); and TGF-b1 (transforming growth factor b1).

The previously described means and methods for down-regulating APP or A[beta] can be combined with therapy against any of these other amyloidogenic polypeptides, eg, activity-specific immunotherapy.

In addition to Alzheimer's disease, cerebral amyloid angiopathy is also a disease that is a suitable target for the presently disclosed technology.

It should be considered that most methods of immunization against APP or Aβ are limited to immunizations that generate antibodies that cross-react with native APP or Aβ. In certain circumstances, however, it is of interest to induce cellular immunity in the form of a CTL response against cells presenting MHC class I epitopes from amyloidogenic polypeptides - this is advantageous if , that is, the decrease in the number of cells producing APP or Aβ does not cause serious side effects. Where a CTL response is desired, the teaching in Applicant's WO 00/20027 is preferably utilized. The contents of both documents are hereby incorporated by reference.

Immunogen carrier

As described above, APP or Aβ peptides comprising T helper epitopes and representing or comprising B cell epitopes can be obtained, covalently linked to non-immunogenic polymers as vehicles, e.g. multivalent activated multivalent Molecules on the hydroxyl polymer that will function as vaccine molecules comprising only the immune-relevant moiety are also embodiments of interest in variants d and e disclosed above. For example, if the target of the vaccine is an autoantigen, such as APP or A[beta], promiscuous or so-called universal T helper epitopes can be used. In addition, components that enhance the immune response can also be co-coupled to the vehicle, thereby acting as adjuvants. These components may be mannose, phagocytosis hormones, muramyl dipeptides, CpG motifs, and the like. In this case, subsequent adjuvant formulation of the vaccine product is not necessary and the product can be administered in the form of pure water or saline.

It is also possible to generate CTLs specific for antigens that generate CTL epitopes by coupling cytotoxic T cell (CTL) epitopes together with T helper epitopes. Substances of APCs such as macrophages that promote product uptake into the cytosol, such as mannose, can also be co-coupled to the vehicle along with CTL and T helper epitopes and enhance the CTL response.

The ratio of B cell epitopes and T helper epitopes (P2 and P30) in the final product was varied according to the concentration of these peptides during the synthesis steps. As mentioned above, immunogenic molecules can be labeled during the synthesis step by adding substances such as mannose, phagocytosis hormones, CpG motifs or other immunostimulatory substances (described here) to the carbonate buffer , if necessary, using aminated derivatives of these substances for labeling.

As described above, if insoluble activated polyols are used to link peptides containing APP or AβB cell epitopes and T helper cell epitopes, this can be done by solid phase synthesis, the final product harvested and filtered by washing and filtration. purification. Components (peptides, labels, etc.) coupled to trifluoroethylsulfonyl-activated polyols can be added to the polyols at low pH, e.g. Glue" for uniform distribution. Subsequently, the pH is raised to pH 9-10 to initiate the reaction of the primary amino groups on the peptide and label with the trifluoroethylsulfonyl groups on the polyol. After the peptide is coupled to, for example, an immunostimulatory substance, the gel is ground to form particles of a size suitable for immunization.

Therefore this immunogen contains

a) at least one first amino acid sequence derived from APP or Aβ, wherein at least one first amino acid sequence comprises at least one B cell and/or at least one CTL epitope, and

b) at least one second amino acid sequence comprising an exogenous T helper epitope, wherein each of the at least first and at least second amino acid sequences is coupled to a pharmaceutically active polyol carrier.

In order to couple an amino acid sequence to a polyol, it is generally necessary to "activate" the polyol with a suitable reactive group capable of forming the necessary linkage with the amino acid sequence.

The term "polyol" is intended to have the same meaning as in WO 00/05316, ie the polyol does have the same characteristics as taught in detail in that application. Thus, the polyol can be water soluble or water insoluble (and thus require different synthetic steps in preparing the immunogen). The polyol may be selected from naturally occurring polyols and synthetic polyols.

Particular and preferred polyols are polysaccharides selected from the group consisting of acetan, pullulan, gum agar-agar, agarose, alginate, gum arabic, carregeenan, cellulose, cyclodextrin , dextran, Danish agar, galactomannan, gelatin, ghatti, dextran, glycogen, guar gum, karaya, konjac/A, locust bean gum, mannan, pectin, plantain , pullulan, starch, tamarine, tragacanth, xanthan, xylan, and xyloglucan, with dextran being particularly preferred.

However, the polyol polymer can also be selected from highly branched polyethyleneimine (PEI), tetrathienylene vinylene, fiber B (long chain of poly-paraphenyl terephtalamide), polyurethane, polysiloxane, polydimethyl Polysiloxane, Silicone, Polymethylmethacrylate (PMMA), Polyvinyl Alcohol, Polyvinylpyrrolidone, Poly(2-Hydroxyethylmethacrylate), Poly(N-Vinylpyrrolidone), Polyvinyl alcohol, polyacrylic acid, polytetrafluoroethylene (PTFE), polyacrylamide, polyethylene-co-vinyl acetate, polyethylene glycol and derivatives, polymethacrylic acid, polylactide (PLA), Polyglycolide (PGA), poly(glycolide-co-lactide) (PLGA), polyanhydrides and polyorthoesters.

The (weight) average molecular weight (i.e. before activation) of the polyol polymer is typically at least 1,000, such as at least 2,000, preferably in the range of 2,500-2,000,000, more preferably in the range of 3,000-1,000,000, especially in the range of 5,000 -500,000 range. It has been shown in the examples that polyol polymers having an average molecular weight in the range from 10,000 to 200,000 are particularly advantageous.

Preferably, the degree of water solubility of the polyol is at least 10 mg/ml, preferably at least 25 mg/ml, such as at least 50 mg/ml, especially at least 100 mg/ml, such as at least 150 mg/ml at room temperature. Dextran is known to meet water solubility requirements even after activation as here.

For some polyols of most interest, the ratio between C (carbon atoms) and OH groups (hydroxyl groups) in unactivated polyols (i.e., natural polyols before activation) is between 1.3- The range of 2.5, for example 1.5-2.3, preferably 1.6-2.1, especially in the range of 1.85-2.05. Without being bound by any particular theory, it is believed that this C/OH ratio of the unactivated polyol represents a highly favorable level of hydrophilicity. Polyvinyl alcohol and polysaccharides are examples of polyols that meet this requirement. The above ratios are believed to be about the same for activated polyols since the activated ratio should be relatively low.

The term "polyhydroxypolymer carrier" is intended to mean the part of the immunogen that carries the amino acid sequence. As a general rule, polyol carriers have external constraints in which the amino acid sequence is cleavable by peptidases, eg, in antigen-presenting cells that process the immunogen. Thus, the polyol carrier can be a polyol with an activating group where the bond between the activating group and the amino acid sequence can be cleaved by a peptidase in APC, or the polyol carrier can be a polyol with an activating group Groups and eg linkers, such as polyhydroxypolymers of a single L-amino acid or multiple D-amino acids, wherein the last part of the linker can be attached to the amino acid sequence and be cleaved by peptidases in APC.

As mentioned above, the polyhydroxy polymer has functional groups (activating groups) which facilitate the anchoring of the peptide to the support. Many applicable functional groups are known in the art, for example, tresyl (trifluoroethylsulfonyl), maleimide, p-nitrophenyl cloroformate, cyanogen bromide, tosyl (p-toluenesulfonyl), triflyl (trifluoromethanesulfonyl), pentafluorobenzenesulfonyl and vinylsulfone groups. Preferred examples of functional groups in the present invention are trifluoroethylsulfonyl, maleimide, tosyl, trifluoromethanesulfonyl, pentafluorobenzenesulfonyl, p-nitrophenyl cloroformate and vinylsulfone groups, Among them trifluoroethylsulfonyl, maleimide and tosyl are particularly relevant.

Trifluoroethylsulfonyl-activated polyols can be used with three Fluoroethylsulfonyl chloride to prepare.

Maleimide-activated polyols can be prepared by activating dextran with p-maleimidophenylisocyanate as described in Example 3 of WO 00/05316. Alternatively, by derivatizing the tris in excess with a diamine compound (typically H ₂ NC _n H _2n -NH ₂ , where n is 1-20, preferably 1-8), such as 1,3-diaminopropane Fluoroethylsulfonyl-activated polyols such as trifluoroethylsulfonyl-activated dextran (TAD), maleimide groups can be introduced into polyols such as dextran, Subsequent reagents such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfo-succinimidyl 4-(N- Maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC), sulfo-succinimidyl 4-(p-maleimidophenyl)butanoic acid ester (SMPB), sulfo-succinimidyl 4-(p-maleimidophenyl) butyrate (sulfo-SMPB), N-gamma-maleimido butyryl Oxy-succinimidyl ester (GMBS) or N-γ-maleimidobutyryloxy-sulfosuccinimidyl ester reacts with the amino group introduced into TAD. Although different reagents and routes of activation result in slightly different linkages between the maleimide functionality of the maleimide-activated product and the remainder of the parent hydroxyl group from which activation proceeds, all are viewed as " Maleimide-activated polyols".

Tosyl-activated polyols can be prepared using tosyl chloride as described for activation of dextran in Example 2 of WO 00/05316. Trifluoromethanesulfonyl- and pentafluorobenzenesulfonyl-activated polyols such as tosyl- or trifluoroethylsulfonyl-activated analogs are prepared, for example, using the corresponding acid chlorides.

Cyanogen bromide activated polyols can be prepared by reacting polyols with cyanogen bromide using conventional methods. The resulting functional group is usually a cyanate ester with two hydroxyl groups of the polyol.

The degree of activation can be expressed as the ratio between free hydroxyl groups and activated groups (ie, functionalized hydroxyl groups). It is believed that the ratio between free hydroxyl groups and activated groups of the polyol should be between 250:1 and 4:1 to achieve a favorable balance between hydrophilicity and reactivity of the polyol. Preferably the ratio is between 100:1 and 6:1, more preferably between 60:1 and 8:1, especially between 40:1 and 10:1.

Activated polyhydroxypolymers of particular interest for use in methods of producing commonly used immunogens according to the invention are trifluoroethylsulfonyl, toluenesulfonyl and maleimide activated polysaccharides, especially trifluoroethylsulfonyl, toluenesulfonyl and maleimide Fluoroethylsulfonyl-activated dextran (TAD), tosyl-activated dextran (TosAD) and maleimide-activated dextran (MAD).

Preferably, the bond between the polyhydroxypolymer carrier and the attached amino acid sequence is cleavable by a peptidase, eg, a peptidase active in APC during antigen processing. Thus preferably at least a first and at least a second amino acid sequence are coupled to the activated polyhydroxy polymer via an amide bond or a peptide bond. It is particularly preferred that the at least first and at least second amino acid sequences each donate the nitrogen moiety of their respective amide bond.

The polyol carrier can be sufficiently free of amino acid residues to force the activating group to provide a peptidase cleavage bond moiety, but as noted above, the carrier can also include only a spacer comprising at least one L-amino acid. Typically, however, at least the first and at least the second amino acid sequence are linked to the active form of the polyol polymer through the N-terminal nitrogen of the amino acid sequence.

The immunogens described above for general use in the invention may be used in a polypeptide vaccine immunization method substantially as described herein. That is, all descriptions of dosages, administration regimens, and polypeptide vaccine formulations for down-regulating amyloidogenic polypeptides discussed herein are adapted to commonly used immunogens with modifications.

Routinely Applied Safe Vaccination Techniques

As stated above, a preferred embodiment of the present invention is the use of variants of the amyloidogenic polypeptide which do not present _TH epitopes from the source which elicit an immune response against the amyloidogenic polypeptide.

However, the inventors consider the design of anti-self vaccines and strategies for effective anti-autoimmunity to be generally available techniques, inventive in their own right. It should prove particularly suitable in cases where the self-antigen it is trying to down-regulate is sufficiently plentiful in the body that self-stimulation of the immune response is possible. Thus, all of the above references within the scope of this embodiment to provide an anti-autoimmune response against APP or Aβ can be adapted for immunity against other self-polypeptides, particularly those present in sufficient numbers to maintain an uncontrolled form of autoimmune disease. Immune response, due to the _TH epitope of the relevant self-polypeptide eliciting an immune response.

Example 1

Automated Vaccination Method for Anti-AD Immunization

The fact that Aβ protein knockout mice did not show any abnormal or adverse side effects indicates that the clearance or reduction in the amount of Aβ is safe, Zheng H. (1996).

Published experiments on transgenic animals immunized against transgenic human Aβ protein suggest that downregulation of Aβ may be achieved by autoreactive antibodies, if self-tolerance may be broken. These experiments further suggested that this down-regulation of A[beta] would potentially simultaneously block plaque formation and even clear formed A[beta] lesions from the brain, see Schenk et al. (1999). But traditionally, it has not been possible to increase antibodies against self-proteins.

Published data therefore do not provide a way to block self-tolerance against bona fide self-proteins. These data also do not provide information on how to ensure that the immune response is directed or primarily directed only at A[beta] deposits and not at membrane-bound A[beta] precursor protein (APP), if this appears to be necessary. The immune response generated with the prior art assumes that it is possible to generate an immune response against self proteins in an uncontrolled manner, so that unnecessary and additional auto-responses against the Aβ protein moiety can be generated. Therefore, the use of existing immunization strategies is most likely to fail to generate a strong immune response against self-proteins. In addition, it is also unsafe due to the potential strong cross-reactivity to membrane-bound APP present on a large number of cells in the CNS.

The present invention provides methods to efficiently generate a strong regulated immune response against bona fide self proteins that could potentially form lesions and cause serious disease in the CNS or other areas of the body. A safe and effective human Aβ protein therapeutic vaccine was developed by this technology for the treatment of AD.

Accordingly, it may be expected that AD, a disease that is expected to cripple the health care system over the next century, can be cured, or that such a vaccine as described can at least establish an effective treatment for the symptoms and progression of the disease. This technology describes a completely novel immune approach to blocking amyloid deposition in AD and other neurological diseases.

In the table below, the 35 expected constructs are shown. The positions given in all tables are relative to the starting methionine of APP (the first amino acid in SEQ ID NO: 2) and include the starting and ending amino acids, for example the 672-714 fragment includes amino acids 672 and 714. The start and end positions of P2 and P30 indicate that the epitope replaces a portion of the APP fragment at the indicated position (both positions are included in the substitution) - in most constructs the introduced epitope replaces a fragment the length of the epitope. The asterisks in the table have the following meanings:

^* ) Only one P2 and P30 position indicates that the epitope has been inserted into the APP derivative at the position indicated (the epitope starts C-terminal to the amino acid adjacent to the given position).

^** ) Construct 34 contains three identical APP fragments separated by P30 and P2, respectively.

^*** ) Construct 35 contains nine identical APP fragments separated by alternating P30 and P2 epitopes.

The portion of APP to which it is most interesting to respond is the 43 amino acid Aβ core peptide (Aβ-43, corresponding to residues 672-714 in SEQ ID NO: 2), which is the major group of amyloid plaques in AD brains. point. This APP fragment was part of all the constructs listed above.

Variants 1 and 2 comprise the upstream portion of APP of A[beta]-43, where the model epitopes P2 and P30 have been placed. Variants 1 and 3-8 both contain the C-100 fragment, which has been shown to be neurotoxic - the C-100 fragment corresponds to amino acid residues 714-770 in SEQ ID NO:2. In variants 3-5, the epitope replaced part of the C-100 fragment, while in variants 6-8 C-100 had been inserted.

Variant 9-35 contains only the core A[beta]-43 protein. In variants 9-13, P2 and P30 are fused to either end of Aβ-43; in 14-21, P2 and P30 replace part of Aβ-43; in 22-33, P2 and P30 are inserted into Aβ-43 Middle; 34 contains three identical Aβ-43 fragments separated by P30 and P2, respectively; 35 contains 9 repeats of Aβ-43 separated by alternating P2 and P30 epitopes.

According to the present invention, truncated portions of the above-mentioned Aβ-43 protein are also used in immunogenic analogues. Particularly preferred are truncated forms Aβ(1-42), Aβ(1-40), Aβ(1-39), Aβ(1-35), Aβ(1-34), Aβ(1-34), Aβ (1-28), Aβ(1-12), Aβ(1-5), Aβ(13-28), Aβ(13-35), Aβ(17-28), Aβ(25-35), Aβ( 35-40), Aβ(36-42) and Aβ(35-42) (wherein the numbers in brackets indicate the amino acid sequence of Aβ-43 constituting the relevant fragment, one such as Aβ(35-42) and SEQ ID NO: 2 Amino acids 706-711 are the same). All of these variants, with truncated portions of A[beta]-43, can be prepared using the A[beta] fragments described herein, particularly variants 9, 10, 11, 12 and 13.

In certain instances, it is preferred that A[beta]-43 or a fragment thereof is mutated. Particularly preferred are substitution variants in which the methionine at position 35 of A[beta]-43 has been substituted, preferably by leucine or isoleucine, or simply deleted. Particularly preferred are analogues comprising only a single methionine at the C-terminus due to its natural occurrence in the amyloidogenic polypeptide or to the exogenous _TH epitope, or because it has been inserted or added . Thus, analogs comprising exogenous _TH epitopes are preferably partially free of methionine, apart from possibly a methionine at the C-terminus.

Indeed, it is generally preferred that all analogues of APP or Aβ for use according to the invention are characterized in that they comprise only a single methionine as the C-terminal amino acid in the analogue, the other being present in the amyloidogenic polypeptide or The methionine in the exogenous _TH epitope is deleted or replaced by another amino acid.

A more interesting mutation is the deletion or substitution of the phenylalanine at position 19 in Aβ-43, a particularly preferred mutation is the substitution of the phenylalanine with a proline.

A particularly preferred group of constructs made with truncations or mutants of Aβ-43 is listed in the table below:

variant number Aβ fragments used in molecules relative to Aβ(1-42/43)aa1 Aβ fragment position relative to aa1 of the molecule Position of the P2 epitope relative to aa1 of the molecule Position of the P30 epitope relative to aa1 of the molecule The total length of the molecule (aa) 36 1-28 22-49 50-64 1-21 64 37 1-12(a)+13-28(b) 1-12(a)+49-64(b) 34-48 13-33 64 38 1-12(x3) 1-12, 34-45, 61-72 46-60 13-33 72 39 13-28(x3) 1-16, 38-53, 69-84 54-68 17-37 84 40 1-12(a)+13-35(b)+36-42(c) 1-12(a)+34-56(b)+72-78(c) 57-71 13-33 78 41 1-28(x3) 1-28, 50-77, 93-120 78-92 29-49 120 42 1-43(F19P/M35K) 1-43 65-79 44-64 79

In this table, the Aβ fragment used in the molecule is indicated by the number of amino acids relative to aa1 of the Aβ(1-42/43) molecule, i.e., 1-28 indicates the fragment of Aβ(1-42/43) used in the molecule 1-28. If 2 or more different fragments are used, both are indicated in the table, i.e., 1-12(a) + 13-28(b) indicates the use of fragment 1- of Aβ(1-42/43) in the molecule 12 and fragments 13-28.

Also, if more than 1 copy of the same fragment is present in the construct, it is indicated in the table, i.e., 1-12(x3) indicates that fragments 1-12 of Aβ(1-42/43) are present in the construct 3 copies.

In addition, the position of the Aβ fragment in the molecule is indicated by the amino acid position relative to the first amino acid of the molecule, ie, 22-49 indicates that the Aβ fragment is located in the molecule at positions from amino acid 22 to amino acid 49, both inclusive. The positions of the P2 and P30 epitopes are also shown. If 2 or more different Aβ fragments are used in the molecule, their positions are indicated, i.e., 1-12(a)+49-64(b) indicates the position of fragment (a) aa 1-12 in the molecule , and fragment (b) is at the position of aa 49-64.

Also, if more than 1 copy of the same fragment is present in the molecule, the positions of all these copies are indicated, i.e., 1-12, 34-45, 61-72 indicate the presence of 3 copies of the Aβ fragment in the molecule, At positions 1-12, 34-45 and 61-72 respectively.

Finally, the total length shown per molecule includes A[beta] fragments and P2 and P30 epitopes.

Variant 42 contained 2 amino acid substitutions, at position 19 (phenylalanine replaced by proline), at position 35 (methionine replaced by lysine), as indicated in the columns showing Aβ fragments.

See Figure 1 and the diagram above for details on introducing exogenous T cell epitopes at specific sites.

An additional type of construct is particularly preferred. Since one goal of the present invention is to avoid destruction of APP-producing cells, whereas clearance of Aβ is required, it seems feasible to make autovaccine constructs comprising only the portion of Aβ that is not exposed to the extracellular phase when present in APP. Thus, such constructs need to contain at least one B cell epitope derived from the amino acid fragment identified by amino acids 700-714 of SEQ ID NO:2. Because such short polypeptide fragments are expected to be only weakly immunogenic, it is preferred that such self-vaccine constructs include several copies of B-cell epitopes, e.g., to have the Constructs of structures, see above. In the form of formula I, the term amyloid _el -amyloid _ex refers to x B cell epitopes comprising the amino acid sequence derived from amino acids 700-714 in SEQ ID NO:2. A preferred alternative is the coupling of the above-mentioned amyloidogenic (poly)peptides and selected exogenous T helper cell epitopes via amide bonds to a polysaccharide carrier molecule - in this method it is possible to carry out the sequence represented by SEQ ID NO: Multiple presentation of "weak" epitopes composed of amino acids 700-714 in 2, and it is also possible to select the optimal ratio of B-cell and T-cell epitopes.

Example 2

Immunization of transgenic mice with Aβ and modified protein of the present invention

Construction of DNA encoding hAβ43+-34

The hAβ43+-34 gene was constructed in several steps. First, use primers ME#801 (SEQ ID NO: 10) and ME#802 (SEQ ID NO: 11), and use primer ME#800 (SEQ ID NO: 9) as a template to generate a PCR fragment. ME#800 encodes a human Aβ-43 fragment with E. coli-optimized codons. ME#801 and 802 added appropriate restriction sites to the fragment.

The PCR fragment was purified, digested with Nco I and Hind III, purified again, and cloned into Nco I-Hind III digested and purified pET28b+ E. coli expression vector. The resulting plasmid encoding wild-type human Aβ-43 was named pAB1.

In a next step, the T helper epitope P2 was added to the C-terminus of the molecule. Primer ME#806 (SEQ ID NO: 12) contains the sequence encoding the P2 epitope, thereby obtaining the fusion of P2 and Aβ-43 by PCR reaction.

Cloning was performed by preparing a PCR fragment using primers ME#178 (SEQ ID NO: 8) and ME#806, and using pAB1 as a template. This fragment was purified, digested with Nco I and Hind III, purified again, and cloned into Nco I-Hind III digested and purified pET28b+ vector. The resulting plasmid was named pAB2.

In a similar manner, another plasmid was prepared that had the Aβ-43 coding sequence and added another T helper epitope, P30, to the N-terminus. This was done by preparing a PCR fragment using primers ME#105 (SEQ ID NO: 7) and ME#807 (SEQ ID NO: 13) using pAB1 as a template.

This fragment was purified, digested with Nco I and Hind III, purified again, and cloned into Nco I-Hind III digested and purified pET28b+ vector. The resulting plasmid was named pAB3.

In the third step, a second Aβ-43 repeat was added to the C-terminus of the P2 epitope of plasmid pAB2 by primer ME#809 (SEQ ID NO: 14). At the same time ME#809 follows the Aβ-43 repeat, creating a BamHI site. The PCR fragment was prepared using primers ME#178 and ME#809 using pAB2 as a template. This fragment was digested with Nco I and Hind III, purified and cloned into Nco I-Hind III digested and purified pET28b+ vector. The resulting plasmid was named pAB4.

Finally, the P30 epitope-Aβ-43 repeat sequence from pAB3 was cloned into the pAB4 plasmid. This was done by preparing a PCR fragment using primers ME#811 (SEQ ID NO: 16) and ME#105 using pAB3 as a template. This fragment was purified and used as a primer together with ME#810 (SEQ ID NO: 15) in subsequent PCR, and PCR was performed using pAB3 as a template. The resulting fragment was purified, digested with BamHI and Hind III, and cloned into BamHI-Hind III digested and purified pAB4 plasmid. The resulting plasmid pAB5 encodes the hAB43+-34 molecule.

All PCR and cloning procedures were performed essentially as described by Sambrook, J., Fritsch, E.F. & Maniatis, T. 1989 "Molecular cloning: a laboratory manual". 2nd Ed. Cold Spring Harbor Laboratory, N.Y.

For all cloning procedures E. coli K-12 cells, strain Top-10F' (Stratagene, USA) were used. The pET28b+ vector was purchased from Novagen, USA. All primers were synthesized at DNA Technology, Denmark.

Expression and purification of hAB43+-34

The hAB43+-34 protein encoded by pAB5 was expressed in E. coli BL21-Gold (Novagen) cells as described by the supplier of the pET28b+ system (Novagen).

The expressed hAB43+-34 protein was purified to more than 85% purity by washing the inclusion bodies followed by cation exchange chromatography with a BioCad purification workstation (PerSeptive Biosystems, USA) in the presence of 6M urea. Urea is then removed by sequential dialysis with solutions containing decreasing amounts of urea. The final buffer is 10mM Tris, pH 8.5.

immune research

Human APP (Alzheimer's Disease Precursor Protein) transgenic mice were used for the research. These mice, termed TgRND8+, express a mutant form of APP that results in the production of high concentrations of Aβ-40 and Aβ-42 in the mouse brain (Janus, C et al.).

Mice (8-10 mice per group) were synthesized with Aβ-42 (SEQ ID NO: 2, residues 673-714 by standard Fmoc strategy) or hAB43+-34 variant (construct 34 in the table of Example 1). , recombinantly produced) for immunization, a total of 4 times at 2-week intervals. The dose was 100mg Aβ or 50mg AB43+-34. Mice were bled on day 43 (after three injections) and day 52 (after four injections) and serum was used to determine the level of anti-Aβ-42 specific titers by direct Aβ-42 ELISA.

Shown in the table below are the mean relative anti-Aβ-42 titers.

Clearly, antibody titers obtained after three and four immunizations when immunized with the hAB43+-34 Aβ variant were approximately 4-fold and 7.5-fold higher than those obtained with unaltered wild-type Aβ-42 as antigen, respectively. This fact is properly taken into account when considering the fact that the amount of variant used for immunization was only 50% of the amount of wild-type sequence used for immunization.

Example 3

Synthesis of Aβ Peptide Copolymer Vaccine Using Activated Polyol as Cross-linking Agent

Introduction

Traditional conjugate vaccines comprise a (poly)peptide covalently coupled to a carrier protein. The peptide contains B cell epitopes and the carrier protein provides T helper cell epitopes. However, the bulk of the carrier protein is usually irrelevant as a source of T helper epitopes, since only a small fraction of the entire sequence contains relevant T helper epitopes. Such epitopes are defined and synthesized as peptides, eg, 12-15 amino acid peptides. If these peptides are covalently linked to peptides comprising B-cell epitopes, for example via polyvalently activated polyols, vaccine molecules comprising only the relevant parts can be obtained. It is further possible to provide a vaccine conjugate comprising an optimized ratio between B cell and T cell epitopes.

Synthesis of activated polyols

Polyhydroxyl polymers, such as dextran, starch, agarose, etc., can be used with 2,2,2-trifluoroethylsulfonyl chloride (trifluoroethylsulfonyl chloride), or by dissolving in N-methylpyrrolidone ( Activation by homogeneous synthesis (dextran) in NMP) or by heterogeneous synthesis (starch, agarose, cross-linked dextran) in eg acetone.

Under dry conditions, 225 ml of dry N-formazan was added to lyophilized water-soluble dextran (4.5 g, 83 mmol, clinical grade, average molecular weight 78,000) in a 500 ml round bottom flask supplemented with a magnet for stirring. Nylpyrrolidone (NMP). Place the flask in a 60° oil bath and stir magnetically. The temperature increased to 92°C within 20 minutes. When the dextran dissolved, the flask was immediately removed from the oil bath and the temperature of the oil bath was lowered to 40°C. The flask was again placed in the oil bath, still under magnetic stirring, and trifluoroethylsulfonyl chloride (2.764ml, 25mmol) was added dropwise. After 15 minutes, dry pyridine (anhydrous, 2.020ml, 25mmol) was added dropwise. The flask was removed from the oil bath and stirred at room temperature for 1 hour. The product (trifluoroethylsulfonyl-activated dextran, TAD) was precipitated in 1200 ml of cold ethanol (99.9%). Centrifuge at 2000 rpm in a centrifuge, decant the supernatant, and collect the precipitate in a 50 ml polypropylene tube. The precipitate was dissolved in 50ml 0.5% acetic acid, dialyzed twice in 5000ml 0.5% acetic acid, and freeze-dried. TAD was stored at -20°C as a freeze-dried powder.

Insoluble polyols, such as agarose or cross-linked dextran, can be activated with trifluoroethylsulfonyl groups by preparing a suspension of the polyol in, for example, acetone and synthesized by solid-phase synthesis techniques . The activated polyol can be collected by filtration. Suitable methods are reported, for example, in Nilsson K and Mosbach K (1987), Methods in Enzymology 135, p. 67 and in Hermansson GT et al. (1992), in "Immobilized Affinity Ligand Techniques", Academic Press, Inc., p. 87.

Synthesis of Aβ Peptide Copolymer Vaccine

TAD (10 mg) was dissolved in 100 μl H ₂ O and added to 1000 μl carbonate buffer, pH 9.6, which contained 5 mg Aβ-42 (SEQ ID NO: 2, residues 673-714), 2.5 mg P2 (SEQ ID NO: 4) and 2.5 mg of P30 (SEQ ID NO: 6). Both Aβ-42 and P2 and P30 peptides contain protected lysine groups: they are 1-(4,4-dimethyl-2,6-dioxan-1-ylidene)ethyl (Dde ) protected form of the lysine group. Peptides were prepared by the standard Fmoc strategy, in which the traditional Fmoc-Lys(Boc)-OH has been replaced by Fmoc-Lys(Dde)-OH (obtained from Novabiochem, catalog number 04-12-1121), i.e., The ε-amino group is protected by replacing Boc with Dde.

The pH was measured and adjusted to 9.6 with 1M HCl. After 2.5 hours at room temperature, 80% hydrazine solution was added to a final concentration of 8% hydrazine, the solution was incubated at room temperature for another 30 minutes, and then immediately subjected to lyophilization. The lyophilized product was dissolved in water and extensively dialyzed against water before final lyophilization.

The ratio of B cell epitopes (Aβ) and T helper epitopes (P2 and P30) in the final product can be varied during the synthesis steps by using different concentrations of these peptides. Alternatively, the final product can be labeled, for example, with mannose (to localize the conjugate to the APCs), by adding aminated mannose to the carbonate buffer during the synthesis step.

If insoluble activated polyols are used to link peptides comprising B cell epitopes and T helper epitopes, coupling to the polymers can be accomplished in solid phase synthesis and the final product harvested and purified by washing and filtration.

As mentioned in the general description, the presently described methods for preparing peptide-based vaccines can be applied in any other polypeptide antigen where it is convenient to prepare a purely synthetic peptide vaccine, and where the polypeptide antigen is expressed as a A single peptide provides sufficient immunogenicity.

Example 4

Synthesis of Peptide Copolymer Vaccines

Dissolve TAD (10 mg) in 100 μl _H2O and add 1000 μl carbonate buffer, pH 9.6, containing 1-5 mg peptide A (any immunogenic peptide of interest!), 1-5 mg P2 (diphtheria toxoid P2 epitope) and 1-5 mg P30 (diphtheria toxoid P30 epitope). The pH was measured and adjusted to 9.6 with 0.1M HCl. After 2.5 hours at room temperature, the solution was then immediately freeze-dried. The freeze-dried product was dissolved in water and dialyzed extensively against water or desalted on a gel filtration column before final freeze-drying. In the case of lysine in the peptide sequence, the ε-amino group of the lysine side chain was protected by Dde during synthesis using the Fmoc-Lys(Dde)-OH derivative (Gregorius and Theisen 2001, in contribution). After coupling, add 80% hydrazine solution to a final hydrazine concentration of 1-20%, and incubate the solution for an additional 30 minutes at room temperature, followed by immediate lyophilization and extensive dialysis with water before final lyophilization, or Desalting was performed on a gel filtration column. This principle is illustrated in the schematic diagram in FIG. 2 .

Such immunogens have been used by the present inventors, with a short C-terminal fragment of the Borrelia bruceli protein OspC as "peptide A" and a diptheria toxoid epitope (P2 or P30) as peptide B. The results of the immunization studies with this antigen showed that only the immunogen of the present invention comprising an OspC fragment and an exogenous diphtheria toxoid epitope matching the MHC haplotype of the vaccinated mice could induce OspC-reactive OspC in these mice. Antibody production. In contrast, a molecule containing only the OspC peptide failed to induce antibody production, as did a mixture of 2 immunogens, one containing OspC and the other containing the epitope. It was therefore concluded that inclusion bodies in the same polyol carrier are preferred, if not essential, to induce antibody production against short peptide haptens, such as OspC.

references

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Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu

180 185 190

Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val

195 200 205

Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys

210 215 220

Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu Glu

225 230 235 240

Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu

245 250 255

Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile

260 265 270

Ala Thr Thr Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg

275 280 285

Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile

290 295 300

Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe

305 310 315 320

Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr

325 330 335

Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr

340 345 350

Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala

355 360 365

Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp

370 375 380

Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala

385 390 395 400

Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala

405 410 415

Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile

420 425 430

Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn

435 440 445

Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met

450 455 460

Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu

465 470 475 480

Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys

485 490 495

Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe

500 505 510

Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser

515 520 525

Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser

530 535 540

Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp

545 550 555 560

Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val

565 570 575

Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala

580 585 590

Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro

595 600 605

Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe

610 615 620

Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val

625 630 635 640

Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser

645 650 655

Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp

660 665 670

Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu

675 680 685

Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly

690 695 700

Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu

705 710 715 720

Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val

725 730 735

Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met

740 745 750

Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met

755 760 765

Gln Asn

770

<210>3

<211>45

<212>DNA

<213> Clostridium tetani

<220>

<221> CDS

<222>(1)..(45)

<223> DNA encoding P2 epitope

<400>3

cag tac atc aaa gct aac tcc aaa ttc atc ggt atc acc gag ctg 45

Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

1 5 10 15

<210>4

<211>15

<212>PRT

<213> Clostridium tetani

<400>4

Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

1 5 10 15

<210>5

<211>63

<212>DNA

<213> Clostridium tetani

<220>

<221> CDS

<222>(1)..(63)

<223> DNA encoding P30 epitope

<400>5

ttc aac aac ttc acc gta agc ttc tgg ctg cgt gtt ccg aaa gtt agc 48

Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser

1 5 10 15

gct agc cac ctg gaa 63

Ala Ser His Leu Glu

20

<210>6

<211>21

<212>PRT

<213> Clostridium tetani

<400>6

Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser

1 5 10 15

Ala Ser His Leu Glu

20

<210>7

<211>21

<212>DNA

<213> Synthetic

<223> Synthetic PCR primers

<400>7

caactcagct tcctttcggg c 21

<210>8

<211>21

<212>DNA

<213> Synthetic

<223> Synthetic PCR primers

<400>8

agatctcgat cccgcgaaat t 21

<210>9

<211>135

<212>DNA

<213> Synthetic

<223> Synthetic PCR primers

<400>9

atggatgcag aattccgtca cgactccggt tacgaagttc accaccagaa actggttttc 60

ttcgcagaag atgttggttc caacaaaggt gcaatcatcg gtctgatggt tggcggtgtt 120

gttatcgcga cctag 135

<210>10

<211>31

<212>DNA

<213> Synthetic

<223> Synthetic PCR primers

<400>10

gccggccatg gatgcagaat tccgtcacga c 31

<210>11

<211>39

<212>DNA

<213> Synthetic

<223> Synthetic PCR primers

<400>11

gccggaagct tctaggtcgc gataacaaca ccgccaacc 39

<210>12

<211>84

<212>DNA

<213> Synthetic

<223> Synthetic PCR primers

<400>12

ccggcaagct tctacagctc ggtgataccg atgaatttgg agttagcttt gatgtactgg 60

gtcgcgataa caacaccgcc aacc 84

<210>13

<211>101

<212>DNA

<213> Synthetic

<223> Synthetic PCR primers

<400>13

gccggccatg ggtttcaaca acttcaccgt tagcttctgg ctgcgtgttc cgaaagttag 60

cgcgagccac ctggaagatg cagaattccg tcacgactcc g 101

<210>14

<211>172

<212>DNA

<213> Synthetic

<223> Synthetic PCR primers

<400>14

gggccaagct tggatccggt cgcgataaca acaccgccaa ccatcagacc gatgattgca 60

cctttgttgg aaccaacatc ttctgcgaag aaaaccagtt tctggtggtg aacttcgtaa 120

ccggagtcgt gacggaactc tgcatccagc tcggtgatac cgatgaattt gg 172

<210>15

<211>30

<212>DNA

<213> Synthetic

<223> Synthetic PCR primers

<400>15

ctggaagatg cagagttccg tcacgactcc 30

<210>16

<211>35

<212>DNA

<213> Synthetic

<223> Synthetic PCR primers

<400>16

gcgccggatc cttcaacaac ttcaccgtta gcttc 35

<210>17

<211>13

<212>PRT

<213> Synthetic

<223> Synthetic HLA DR binding sequence

<400>17

Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala

1 5 5 10

Claims (57)

1. The application of an immunogen in the preparation of a pharmaceutical preparation for treating, preventing or improving Alzheimer's disease or other diseases characterized by amyloid deposition in animals, said pharmaceutical preparation comprising said immune original, the immunogen a) It is a polyamino acid, which integrates a substantial part of the B cell antigen epitope of APP and/or Aβ in the same molecule, so that the polyamino acid has the same degree of anti-APP or Aβ as APP or Aβ. A polyclonal serum response to Aβ, and at least one exogenous T-helper epitope ( _TH epitope), and which comprises at least one exogenous _TH epitope and an interrupted APP or Aβ sequence, such that the The polyamino acid does not include any subsequence of SEQ ID NO: 2 that operatively binds to an MHC class II molecule that elicits a T-cell response; or b) is a conjugate comprising a polyhydroxy polymer backbone independently coupled to a polyamino acid as defined in a); or C) is a nucleic acid encoding the polyamino acid as defined in a); or d) is a non-pathogenic microorganism or virus that carries a nucleic acid encoding and expressing a polyamino acid as defined in a). 2. The application according to claim 1, wherein said immunogen is a conjugate comprising a polyhydroxy polymer backbone independently coupled to 1) said at least one exogenous _TH epitope, and 2) a disruptor sequence of APP or A[beta] as described in a). 3. The application according to claim 1, wherein said polyamino acid or combination comprises - at least one first moiety which localizes said polyamino acid to antigen presenting cells (APC) or B-lymphocytes, and/or - at least one second moiety, which stimulates said immune system, and/or - at least one third moiety which optimizes presentation of said polyamino acid to said immune system. 4. The use according to claim 3, wherein said first and/or second and/or third moieties are flanked by covalent or non-covalent linkages to suitable chemical groups in said APP or Aβ sequence Chain groups are attached. 5. The use according to claim 1, wherein said polyamino acid or combination comprises a fusion polypeptide. 6. Use according to claim 1, wherein the polyamino acid or combination comprises at least one replica of a B-cell epitope of APP or A[beta] and/or introduces a hapten. 7. The use according to claim 1, wherein said exogenous T-cell epitope is immunodominant in said animal. 8. The use according to claim 1, wherein said exogenous T-cell epitopes are promiscuous. 9. The use according to claim 8, wherein said exogenous T-cell epitope is an exogenous T-cell epitope selected from natural promiscuous T-cell epitopes and artificial MHC-II binding peptide sequences. 10. according to the application of claim 9, wherein said natural T-cell epitope is selected from tetanus toxoid epitope, diphtheria toxoid epitope, influenza virus hemagglutinin epitope and Plasmodium falciparum (P.falciparum) CS gauge. 11. The use according to claim 10, wherein the tetanus toxoid epitope is P2 or P30. 12. The use according to claim 1, wherein said polyamino acid or combination comprises a B-cell epitope which is not exposed to the extracellular phase when present in the cell-bound form of the precursor polypeptide of A[beta]. 13. The use according to claim 1, wherein said polyamino acid or combination lacks at least one B-cell epitope exposed to the extracellular phase when present in the cell-bound form of the precursor polypeptide of A[beta]. 14. The use according to claim 1, wherein said polyamino acid or combination comprises at most 9 consecutive amino acids of SEQ ID NO:2. 15. The application according to claim 14, wherein said polyamino acid or combination comprises at most 8 consecutive amino acids of SEQ ID NO:2. 16. The use according to claim 14, wherein said polyamino acid or combination comprises at most 7 consecutive amino acids of SEQ ID NO:2. 17. The use according to claim 14, wherein said polyamino acid or combination comprises at most 6 consecutive amino acids of SEQ ID NO:2. 18. The use according to claim 14, wherein said polyamino acid or combination comprises at most 5 consecutive amino acids of SEQ ID NO:2. 19. The use according to claim 14, wherein said polyamino acid or combination comprises at most 4 consecutive amino acids of SEQ ID NO:2. 20. The use according to claim 14, wherein said polyamino acid or combination comprises at most 3 consecutive amino acids of SEQ ID NO:2. 21. The application according to claim 14, wherein said polyamino acid or combination comprises at least one subsequence of SEQ ID NO: 2, such that each such at least one subsequence of SEQ ID NO: 2 is independently composed of an amino acid sequence The amino acid sequence is selected from 9 consecutive amino acids of SEQ ID NO: 2, 8 consecutive amino acids of SEQ ID NO: 2, 7 consecutive amino acids of SEQ ID NO: 2, and 6 consecutive amino acids of SEQ ID NO: 2 Amino acids, 5 contiguous amino acids of SEQ ID NO:2, 4 contiguous amino acids of SEQ ID NO:2, and 3 contiguous amino acids of SEQ ID NO:2. 22. Use according to claim 14 or 21, wherein said consecutive amino acids start with an amino acid residue selected from residues 672, 673, 674, 675, 676, 677, 678, 679 and 680. 23. Use according to claim 14 or 21, wherein said consecutive amino acids start with an amino acid residue selected from residues 681, 682, 683, 684, 685, 686, 687, 688 and 689. 24. Use according to claim 14 or 21, wherein said consecutive amino acids start with an amino acid residue selected from residues 690, 691, 692, 693, 694, 695, 696, 697 and 698. 25. Use according to claim 14 or 21, wherein said consecutive amino acids start from an amino acid residue selected from residues 699, 700, 701, 702, 703, 704, 705, 706 and 707. 26. Use according to claim 14 or 21, wherein said consecutive amino acids start from an amino acid residue selected from residues 708, 709, 710, 711, 712, 713 and 714. 27. The use according to claim 1, wherein at least 2 copies of said polyamino acid or conjugate are covalently or non-covalently linked to a carrier molecule capable of multi-copy presentation of an antigenic determinant. 28. The use according to claim 1, wherein said polyamino acid is linked to said polyhydroxy polymer through an amide bond. 29. The use according to claim 2, wherein said _TH epitope is attached to said polyhydroxy polymer through an amide bond. 30. Use according to claim 1, wherein said polyhydroxy polymer is a polysaccharide. 31. Use according to claim 1, wherein said polyamino acid or conjugate has been formulated with an adjuvant which promotes the breaking of autotolerance to self-antigens. 32. The use according to claim 1, wherein the effective amount of the polyamino acid or the conjugate is 0.5 μg-2,000 μg. 33. The use according to claim 1, wherein the pharmaceutical preparation comprises a nucleic acid encoding the polyamino acid, and the nucleic acid is introduced into cells of the animal, thereby obtaining in vivo expression of the introduced nucleic acid. 34. Use according to claim 33, wherein the nucleic acid introduced is selected from the group consisting of naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA included in viral vectors , DNA formulated with transfection-promoting proteins or peptides, DNA formulated with targeting proteins or peptides, DNA formulated with reagents for calcium precipitation, DNA coupled to inert carrier molecules, encapsulated in chitin or chitosan DNA in and DNA formulated with adjuvant. 35. The use according to claim 1, wherein the treatment, prevention or improvement requires down-regulation of APP or Aβ to the extent that the total amount of amyloid is reduced or the rate of amyloid formation has a clinically significant reduction. 36. according to the application of claim 1, wherein said pharmaceutical preparation comprises a kind of non-pathogenic microorganism or virus, described non-pathogenic microorganism or virus carries the nucleic acid of encoding and expressing the polyamino acid as defined in a) fragment. 37. A polyamino acid or a conjugate derived from animal APP or Aβ, wherein a modification is introduced, the modification leading to immunization of the animal with the polyamino acid or the conjugate to induce the production of anti-APP or Aβ of the animal itself An antibody, and wherein said polyamino acid or combination is as defined in any one of claims 1-30. 38. An immunological composition comprising an immunologically effective amount of the polyamino acid or the conjugate according to claim 37, said composition further comprising a pharmaceutically and immunologically acceptable carrier and/or vehicle. 39. The immunological composition according to claim 38, further comprising an adjuvant. 40. A nucleic acid fragment encoding the polyamino acid of claim 37. 41. A vector carrying the nucleic acid fragment of claim 40. 42. The vector according to claim 41, which is an autonomously replicating vector. 43. The vector according to claim 41, which is selected from the group consisting of plasmids, bacteriophages, cosmids, minichromosomes and viruses. 44. The vector of claim 41 or 43, comprising a promoter for promoting expression of the nucleic acid fragment of claim 40, and the nucleic acid fragment of claim 40 in the 5'→3' direction and operably linked. 45. The vector according to claim 44, which additionally comprises a nucleic acid sequence encoding a leader peptide enabling secretion or integration into membranes of the polypeptide fragment encoded by said nucleic acid fragment. 46. The vector according to claim 44, which additionally comprises a terminator. 47. The vector of claim 41 or 43 which, when introduced into a host cell, is capable or incapable of being integrated into the genome of said host cell. 48. The vector of claim 44, wherein said promoter promotes expression in eukaryotic cells and/or prokaryotic cells. 49. A transformed cell carrying the vector of any one of claims 41-48, capable of replicating the nucleic acid fragment of claim 40. 50. The transformed cell according to claim 49, which is a transformed cell. 51. The transformed cell of claim 49, which is a microorganism selected from the group consisting of bacteria, yeast, protozoa, or a cell derived from a multicellular organism selected from the group consisting of fungi, insect cells, Plant cells and mammalian cells. 52. The transformed cell according to claim 51, wherein said insect cell is an _S2 or SF cell. 53. The transformed cell of claim 49 or 51 expressing the nucleic acid fragment of claim 40. 54. The transformed cell according to claim 53, which is a transformed cell which secretes or carries the polyamino acid or the conjugate of claim 37 on its surface. 55. A composition that induces the production of anti-amyloid antibodies, said composition comprising - the nucleic acid fragment of claim 40 or the vector of any one of claims 41-48, and - Pharmaceutically and immunologically acceptable carriers and/or vehicles and/or adjuvants. 56. A stable cell line carrying the vector of any one of claims 41-48 and expressing the nucleic acid fragment of claim 40. 57. The cell line according to claim 56, which secretes or carries on its surface the polyamino acid or conjugate of claim 37.

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