NO335602B1 - A pharmaceutical composition for down-regulation of amyloid containing an immunogen comprising a polyamino acid which induces production of antibody to the animal's autologous amyloid precursor protein (APP) or amyloid-beta (A-beta), nucleic acid fragment encoding the polyamino acid, vector and transformed cell. - Google Patents

Abstract

SUMMARY New methods for combating diseases characterized by amyloid deposition are described. The methods generally relate to immunization against amyloid precursor protein (APP) or beta amyloid (AP). Immunization is preferably performed by administering analogues of autologous APP or AP, said analogs being able to induce antibody production against the autologous amyloidogenic polypeptides. Particularly preferred as an immunogen is autologous AP which has been modified by introducing a single or a few foreign, immunodominant and promiscuous T-cell epitopes. Also disclosed is nucleic acid vaccination against APP or AP and in addition vaccination using live vaccines as well as methods and agents useful for the vaccination. Such methods and agents include methods for the preparation of analogs and pharmaceutical preparations as well as nucleic acid fragments, vectors, transformed cells, polypeptides and pharmaceutical preparations.

Description

FIELD OF THE INVENTION

The present invention relates to improvements in the therapy and prevention of Alzheimer's disease (AD) and other diseases characterized by amyloid deposits, for example characterized by amyloid deposits in the central nervous system (CNS). More specifically, the present invention provides a pharmaceutical composition suitable for the down-regulation of (unwanted) amyloid deposits by enabling the production of antibodies against a relevant protein (APP or Ap) or components thereof in individuals suffering from or at risk of suffering from diseases with a pathology involving amyloid deposits. The invention also relates to a polyamino acid or conjugate derived from APP or Ap, as well as an immunogenic composition comprising said polyamino acid or conjugate. The invention further relates to nucleic acid fragments encoding the modified polypeptides as well as vectors for incorporation of these nucleic acid fragments and the host cells and cell lines transformed thereby.

BACKGROUND OF THE INVENTION

Amyloidosis is the extracellular deposits of insoluble protein fibrils that cause tissue damage and disease (Pepys, 1996; Tan et al., 1995; Kelly, 1996). The fibrils are formed when normally soluble proteins and peptides associate with themselves in an abnormal way (Kelly, 1997).

Amyloid is associated with serious diseases including systemic amyloidosis, AD, onset of adult-onset diabetes, Parkinson's disease, Huntington's disease, frontotemporal dementia and the prion-related transmissible spongiform encephalopathies (kuru and Creutzfeldt-Jacob disease in humans and scrapie and BSE in sheep and cattle, respectively) and the amyloid plaque formation in, for example, Alzheimer's appears to be closely associated with the development of disease in humans. In animal models, overexpression or expression of modified forms of proteins found in deposits, such as the β-amyloid protein, has been shown to induce a variety of disease symptoms, such as Alzheimer's-like symptoms. There is no specific treatment for amyloid deposition, and these diseases are usually fatal.

The subunits of amyloid fibrils can be wild-type, variant 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 the polypeptide component of the fibrils defines the nature of the amyloidosis. Despite wide differences in the size, native structure, and function of amyloid proteins, all amyloid fibrils are of indeterminate length, unbranched, 70 to 120 Å in diameter, and show characteristic Congo red staining

(Pepys, 1996). They are characteristic of a cross-β structure (Pauling & Corey, 1951), in which the polypeptide chain is organized into β-sheets. Although the amyloid proteins have very different precursor structures, they can also undergo a structural transformation, perhaps along a similar reaction pathway, to a misfolded form that is the building block of the β-sheet helical protofilament.

The characteristic fiber patterns led the amyloidosis to be called β-fibrillosis (Glenner, 1980a, b) and the fibril protein of AD was called β-protein before its secondary structure was known (Glenner & Wong, 1984). The characteristic cross-β diffraction pattern, together with fibril appearance and color characteristics, are now the accepted diagnostic hallmarks of amyloid, suggesting that the fibrils, although formed from quite different protein precursors, share a degree of structural similarity and comprise a structural superfamily, independent of the property of their precursor proteins (Sunde M, Serpell LC, Bartlam M, Fraser PE, Pepys MB, Blake CCFJ Mol Biol 1997 Oet 31; 273(3): 729-739).

One of the most widespread and well-known diseases where amyloid deposits in the central nervous system are proposed to have a central role in the development of the disease is

A.D.

A.D

Alzheimer's disease (AD) is an irreversible, progressive brain condition that occurs gradually and results in memory loss, behavioral and personality changes, and a decline in mental abilities. These losses are related to brain cell death and the breakdown of the connections between them. The course of the disease varies from person to person and so does the rate of decline. On average, AD patients live for 8-10 years after diagnosis, although the disease can last up to 20 years.

AD develops in stages, from early, mild forgetfulness to severe loss of mental function. This loss is known as dementia. In most people with AD, symptoms first appear after the age of 60, but earlier onset is not rare. The earliest symptoms often include short-term memory loss, impaired judgment, and personality changes. People with early stages of AD often think less clearly and forget names of family members and familiar objects. Later in the illness, they may forget how to perform even the simplest tasks. Eventually, people with AD lose all reasoning ability and become dependent on other people for daily care. Eventually, the disease becomes so debilitating that patients are bedridden and likely to develop other ailments and infections. People with AD usually die from pneumonia.

Although the risk of developing AD increases with age, AD and dementia symptoms are not part of normal aging. AD and other dementia conditions are caused by diseases that affect the brain. In normal ageing, nerve cells in the brain are not lost in greater numbers. In contrast, AD disrupts three key processes: Neuron cell communication, metabolism and repair. This destruction eventually causes many nerve cells to stop working, lose connection with other nerve cells, and die.

First, AD destroys neurons in parts of the brain that control memory, particularly in the hippocampus and related structures. As nerve cells in the hippocampus cease to function adequately, short-term memory fails and a person's ability to perform simple and familiar tasks is often reduced. AD also attacks the cerebral cortex, especially the area responsible for language and reasoning. Eventually, many other areas of the brain become involved, all these brain areas wither (shrink), and the AD patient becomes bedridden, incontinent, totally helpless and passive in relation to the outside world (source: National Institute on Aging Progress Report on Alzheimer's Disease, 1999).

The impact of AD

AD is the most common cause of dementia among people aged 65 or older. It is a major health problem due to its enormous impact on individuals, families, the health and care system and society as a whole. Scientists estimate that up to 4 million people currently suffer from the disease, and the general prevalence doubles every 5 years after the age of 65. It is also estimated that approximately 360,000 new cases (incidences) will occur each year, although this number will increase as the population ages (Brookmeyer et al, 1998).

AD imposes a heavy financial burden on society. A recent study in the United States calculated the annual cost of caring for an AD patient at $18,400 for a patient with mild AD, $30,096 for a patient with moderate AD, and $36,132 for a patient with severe AD. The annual national cost of AD patient care in the United States is estimated to be slightly over $50 billion (Leon et al, 1998).

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

The most important characteristics of AD

Two abnormal structures in the brain are characteristic of AD: Amyloid plaques and neurofibrillary tangles (NFT). Plaques are dense, large insoluble deposits of protein and cellular material on the outside of and around the brain's neurons. Tangles are insoluble tangled fibers that build up inside neurons.

There are two types of AD: Familial AD (FAD) which follows a certain inheritance pattern, and sporadic AD where no obvious inheritance pattern can be seen. Because of differences in age at onset, AD is further described as early-onset (occurring in people younger than 65 years old) or late-onset (occurring in those 65 years or older). Early-onset AD is rare (about 10% of cases) and usually affects people in their 30s to 60s. Some forms of early-onset AD are hereditary and run in families. Early-onset AD often develops more quickly than the more common late-onset form.

All FADs known so far have an early onset, and as many as 50% of FAD cases are now known to be caused by defects in three genes located on three different chromosomes. These are mutations in the APP gene on chromosome 21; mutations in a gene on chromosome 14 called presenilin 1; and mutations in a gene on chromosome 1 called presenilin 2. However, there is no evidence yet that any of these mutations play an important role in the more common, sporadic or non-familial form of late-onset AD. (National Institute on Aging Progress Report on Alzheimer's Disease, 1999).

Amyloid plaques

In AD, amyloid plaques first develop in areas of the brain used for memory and other cognitive functions. They consist of large insoluble deposits of beta-amyloid (hereafter called Aβ) - a protein fragment of a larger protein called amyloid precursor protein (APP, whose amino acid sequence is described in SEQ ID NO: 2) - mixed with parts of neurons and with non - nerve cells such as microglia and astrocytes. It is not known whether amyloid plaques themselves constitute the main cause of AD or whether they are a by-product of the AD process. Changes in the APP protein can indeed cause AD as shown in the hereditary form of AD caused by mutations in the APP gene and Aβ plaque formation appears to be closely associated with the development of the human disease (Lippa CF. et al, 1998).

APP

APP is one of several proteins that are associated with cell membranes. After it has been produced, APP is anchored in the nerve cell's membrane, partly on the inside and partly on the outside of the cell. Recent studies using transgenic mice show that APP appears to play an important role in the growth and survival of neurons. Certain forms and amounts of APP can, for example, protect neurons against both short- and long-term damage and can make damaged neurons better able to repair themselves and help parts of the neurons' growth after brain damage.

While APP is anchored in the cell membrane, proteases act on specific APP sites and cleave these into protein fragments. One protease cleaves APP and forms Aβ, and another protease cleaves APP in the middle of the amyloid fragment so that Aβ is not formed. The formed Aβ is of two different lengths, a shorter 40 (or 41) amino acid Aβ which is relatively soluble and aggregates slowly, and a somewhat longer, 42 amino acid "sticky" Aβ, which rapidly forms insoluble clumps. While Ap is formed, it is not yet known exactly how it moves through and around nerve cells. The final stage of this process aggregates the "sticky" Aβ into long filaments on the outside of the cell and, together with fragments of dead and dying neurons and microglia and astrocytes, plaques characteristic of AD are formed in brain tissue.

There is some evidence that the mutations in APP make it more likely that Aβ will be excised from the APP precursor, thereby causing more of either total Aβ or relatively more of the "sticky" form to be formed. It also seems that mutations in the presenile genes can contribute to the breakdown of neurons in at least two ways: by modifying Aβ production or by triggering cell death more directly. Other researchers suggest that mutated presenilin 1 and 2 may be involved in increasing the rate of apoptosis.

It is also expected that more and more plaques form and fill more and more of the brain as the disease progresses. Studies suggest that it may be that Ap aggregates and disaggregates at the same time, in a kind of dynamic equilibrium. This gives hope that it may be possible to break down plaques even after they are different. (National Institute on Aging Progress Report on Alzheimer's Disease, 1999).

It is believed that Ap is toxic to neurons. In tissue culture studies, researchers have observed an increase in death of hippocampal neuronal cells engineered to overexpress mutated forms of human APP compared to neurons overexpressing normal human APP (Luo et al, 1999).

Furthermore, overexpression or the expression of modified forms of the Aβ protein in animal models has been shown to induce Alzheimer-like symptoms (Hsiao K. et al, 1998).

Given that increased Aβ formation, its aggregation into plaques and resulting neurotoxicity can lead to AD, it is of therapeutic interest to investigate conditions under which Aβ aggregation into plaques can be slowed and even blocked.

Presenilins

Mutations in presenilin-1 (S-180) account for almost 50% of all cases of early-onset familial AD (FAD). Around 30 mutations have been identified that give rise to AD. The onset of AD varies with the mutations. Mutations in presenilin-2 account for a much smaller proportion of FAD cases, but are still a significant factor. It is not known whether presenilin is involved in sporadic non-familial AD. The function of presenilin is not known, but they appear to be involved in the processing of APP, yielding Ap-42 (the longer and stickier form of the peptide, SEQ ID NO: 2, residues 673-714), as AD -patients with presenilin mutations have increased levels of this peptide. It is unclear whether the presenilins also play a role in causing the formation of NFTs. Some suggest that presenilins may also have a more direct role in the breakdown of neurons and nerve death. Presenilin-1 is located on chromosome 14, while presenilin-2 is linked to chromosome 1. If a person carries a mutated version of only one of these genes, he or she will almost certainly develop early-onset AD.

It is somewhat uncertain whether presenilin-1 is identical to the hypothetical gamma-secretase involved in the processing of APP (Naruse et al., 1998).

Apolipoprotein E

Apolipoprotein E is usually associated with cholesterol, but is also found in plaques and tangles in AD brains. While alleles 1-3 do not appear to be involved in AD, there is a significant correlation between the presence of the APOE-e4 allele and the development of late-onset AD (Strittmatter et al., 1993). However, it is a risk factor and not a direct cause, which is the case for the presenilin and APP mutations and it is not limited to familial AD.

The ways in which the APOE e4 protein increases the likelihood of developing AD is not known with certainty, but one possible theory is that it facilitates Aβ build-up and that this helps to lower the age of onset of AD, or the presence or absence of specific APOE alleles can affect the way neurons respond to damage (Buttini et al., 1999).

Apo Al has also been known to be amyloidogenic. Intact apo Al can itself form amyloid-like fibrils in vitro which are Congo red-positive (Am J Pathol 147 (2): 238-244 (Aug 1995), Wisniewski T, Golabek AA, Kida E, Wisniewski KE, Frangione B).

There seem to be some conflicting results indicating that there is a positive effect of the APOE-e4 allele in the reduction of symptoms of mental loss compared to other alleles (Stern, Brandt, 1997, Annals of Neurology 41).

Neurofibrillary tangles

The other hallmark of AD consists of abnormal collections of twisted filaments found on the inside of nerve cells. The main component is tangles in the form of a protein called tau (x). In the central nervous system, tau proteins are best known for their ability to bind and help stabilize microtubules as a component of the cell's internal support structure or skeleton. However, tau in AD is chemically altered and this altered tau can no longer stabilize microtubules and causes them to break down. This collapse of the transport system can initially result in poorly functioning communication between nerve cells and can later lead to nerve death.

In AD, chemically altered rope twists into paired helical filaments - two strands of rope twisted around each other. These filaments are the main substance found in neurofibrillary tangles. In a recent study, researchers found neurofibrillary changes in fewer than 6% of the nerves in a specific part of the hippocampus in healthy brains, in more than 43% of those nerves in people who died with severe AD. When the loss of nerves was studied, a similar progression was found. Evidence of this kind supports the idea that the formation of tangles and the loss of neurons develop together during the course of AD. (National Institute on Aging Progress Report on Alzheimer's Disease, 1999).

Tauopathies and tangles

Several neurodegenerative diseases, besides AD, are characterized by the aggregation of tau into insoluble filaments in neurons and glia, leading to dysfunction and death. Very recently, several research groups studying families with multiple hereditary dementias other than AD found the first mutation in the tau gene 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 caused nerve cell death and dementia. These disorders, which share some of the characteristics with AD but differ in several important aspects, are collectively called "frontotemporal dementia and parkinsonism linked to chromosome 17" (FTDP-17). These are diseases such as Parkinson's disease, some forms of amyotrophic lateral sclerosis (ALS), corticobasal degeneration, progressive supranuclear palsy ("palsy") and Pick's disease and all are characterized by abnormal aggregation of tau protein.

Other AD-like neurological diseases.

There are important parallels between AD and other neurological diseases including prion diseases (such as kuru, Creutzfeld-Jacob disease and bovine spongiform encephalitis), Parkinson's disease, Huntington's disease and frontotemporal dementia. All involve the deposition of abnormal proteins in the brain. AD and prion diseases cause dementia and death and both are associated with the formation of insoluble amyloid fibrils, but from membrane proteins that are different from each other.

Researchers studying Parkinson's disease, the second best-known neurodegenerative disease after AD, discovered the first gene associated with the disease. This gene codes for a protein called synuclein which, interestingly enough, has been found in the amyloid plaques in the brains of AD patients (Lavedan C, 1998, Genome Res. 8(9): 871-80). Researchers have also discovered that genetic defects in Huntington's disease, another progressive neurodegenerative disease that causes dementia, cause the Huntington protein to form into insoluble fibrils that closely resemble the Aβ fibrils in AD and the protein fibrils in prion disease (Scherzinger E, et al, 1999, PNAS U.S.A. 96 (8): 4604-9).

Researchers have also discovered a new gene that, when mutated, is responsible for familial British dementia (FBD), a rare inherited disease that causes severe movement disorders and progressive dementia similar to that seen in AD. In a biochemical analysis of the amyloid fibrils found in FBD plaques, a unique peptide called AB ri was found (Vidal et al, 1999). A mutation at a specific point along this gene resulted in the production of a longer than normal Bri protein. The ABri peptide that is cleaved from the mutated end of the Bri protein is deposited as amyloid fibrils. These plaques are thought to lead to the nerve dysfunction and dementia that characterize FBD.

Immunization with Ap.

The immune system will normally take part in the removal of foreign protein and proteinaceous particles in the organism, but the deposits associated with the above-mentioned diseases consist mainly of self-proteins, and therefore make the role of the immune system in the control of these diseases less obvious. Furthermore, the deposits are located in an area (CNS) that is normally separate from the immune system, and both facts suggest that any vaccine or immunotherapeutic approach will not be successful.

Nevertheless, researchers have recently attempted to immunize mice with a vaccine composed of heterologous human Aβ and a compound known to stimulate the immune system (Schenk et al, 1999 and WO 99/27944). The vaccine was tested in a specific transgenic mouse model of AD with a human mutated gene of APP inserted into the mouse's DNA. The mice produced the modified APP protein and developed amyloid plaques as they aged. This mouse model was used to test whether vaccination against the modified transgene human APP had an effect on plaque build-up. In a first experiment, a group of transgenic mice were given monthly injections of the vaccine, starting at 6 weeks of age and ending at 11 months of age. A second group of transgenic mice received no injections and served as a control group. At 13 months of age, the mice in the control group had plaques covering 2-6% of their brains. In contrast, the immunized mice had virtually no plaque.

In a second experiment, the researchers began the injections at 11 months of age when some plaque had already developed. Over a 7-month period, the transgenic control mice had a 17-fold increase in the amount of plaque in their brains, while those receiving the vaccine had a 99% reduction compared to 18-month-old transgenic control mice. In some mice, some of the pre-existing plaque deposits appear to have been removed by the treatment. It was also found that other plaque-associated damage such as inflammation and abnormal nerve cell processes decreased as a result of the immunization.

The results stated above are thus a preliminary study in mice and researchers need to find out, for example, whether vaccinated mice remain healthy in relation to other aspects and whether the memory of the vaccinated mice remains normal. Because the mouse model is not a complete representation of AD (the animals do not develop neurofibrillary tangles, nor do many of their nerves die), further studies are needed to further determine whether humans have similar or different responses compared to mice. Another aspect that is considered is that the procedure may be able to "cure" amyloid deposition, but not stop the development of dementia.

Technical aspects can also provide major challenges. For example, it is unlikely that it is even possible to apply this technology and create a vaccine that enables humans to produce antibodies against their own proteins. Several safety and effectiveness aspects must therefore be resolved before any tests on humans can be considered.

The work of Schenk et al. consequently shows that if it were possible to form a strong immune response against one's own proteins in proteinaceous deposits in the central nervous system such as the plaques formed in AD, it is possible to both prevent the formation of the deposits and also possible to remove already formed plaques.

Clinical studies using the above-mentioned Ap vaccines have recently been terminated as a result of side effects. A number of vaccinated individuals developed chronic encephalitis which may be due to an uncontrolled autoimmunity against Ap in the central nervous system.

In the international patent application WO93/15760, a two-part immunogenic carrier construct is described which is indicated to enhance the effect of active immunization in animals and humans. More specifically, a two-part immunogenic construct is described comprising at least one primary carrier comprising a molecule having a molecular weight greater than 70 KD, and at least one secondary carrier comprising a T-dependent antigen conjugated to the primary carrier. Examples of the primary carriers include dextran, ficoll, polyacrylamide, microbial polysaccharides, and combinations thereof. Examples of secondary carriers include viral, bacterial, parasitic, animal and fungal proteins.

In the international patent application WO93/23076, the use of conjugates applicable to the induction of the immune system for the production of immunoglobulins which can function as alternative receptors for a specific ligand is described, where the conjugate has a molecular weight of at least 100,000 D and comprises at least 20 antigenic epitopes. It is stated that the ligand can, for example, be an opiate, a benzodiazepine, a phencyclidine, cocaine or nicotine, a hormone, or a growth factor.

OBJECTIVES OF THE INVENTION

The purpose of the present invention is to provide new therapies against conditions characterized by the deposition of amyloid, such as AD. A further aim is to develop an autovaccine against amyloid to achieve a new treatment for AD and for other pathological conditions involving amyloid deposition.

SUMMARY OF THE INVENTION

Described herein is the application of an autovaccination technology to generate strong immune responses against otherwise non-immunogenic APP and Ap. It also describes the preparation of such vaccines for the prevention, possible cure or alleviation of the symptoms of such diseases which are associated with amyloid deposits.

Accordingly, in its broadest and most general sense, the present invention relates to a pharmaceutical composition containing an immunogen which induces the production of antibody against the animal's autologous APP or Aβ, in which the immunogen incorporates

a) a polyamino acid consisting of a polyamino acid selected from the group consisting of: - amino acid residues 1-12, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, followed by the amino acid residues of the tetanus toxoid P30 epitope, followed by the amino acid residues of the tetanus toxoid P2 epitope, followed by amino acid residues 13-28, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2 Ap, - amino acid residues 1-12, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, followed by the amino acid residues of the tetanus toxoid P30 epitope, followed by amino acid residues 1-12, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, followed by the amino acid residues of the tetanus toxoid P2 epitope, followed by amino acid residues 1-12, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, - amino acid residues 13-28, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, followed by the amino acid residues of the tetanus toxoid P30 epitope, followed by amino acid residues 13-28, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, followed by the amino acid residues of the tetanus toxoid P2 epitope, followed by amino acid residues 13-28, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap,

in which all the amino acid sequences are indicated in the direction from the N- to the C-terminal end, or b) is a conjugate comprising a polyhydroxy polymer backbone to which a polyamino acid as defined in a) is separately attached; or

c) is a nucleic acid encoding the polyamino acid as defined in a); or

d) is a non-pathogenic microorganism or virus carrying a nucleic acid fragment that encodes and expresses the polyamino acid as defined

in a),

for use in the treatment, prevention or alleviation of Alzheimer's disease or other diseases characterized by amyloid deposition.

The present applicants have previously submitted an international patent application aimed at safe vaccination strategies against amyloidogenic polypeptides such as APP and Ap, cf. WO 01/62284. This application does not contain details regarding the above-mentioned applicable analogues of APP and Ap.

The invention also relates to analogues of APP and Aβ as well as nucleic acid fragments encoding a subset of these. Immunogenic compositions comprising the analogues or nucleic acid fragments are also part of the invention.

FIGURES

Figure 1: Schematic image of Autovac variants derived from the amyloid precursor protein for the purpose of generating antibody responses against the Ap protein Ap-43 (or C-100). APP is shown schematically at the top of the figure and the remaining schematic constructs show that the model epitopes P2 and P30 are substituted or inserted into different truncations of APP. In the figure, the black pattern indicates the APP signal sequence, bidirectional cross pattern is the extracellular part of APP, dark vertical pattern is the transmembrane domain of APP, light vertical pattern is the intracellular domain of APP, coarse cross pattern indicates the P30 epitope and fine cross pattern indicates P2 -epitope. The full-length box indicates Ap-42/43 and the full-line box and dashed-line box together indicate C-100. "Abeta" denotes Ap. Figure 2: Schematic image of an embodiment of the synthesis of generally applicable immunogenic conjugates. Peptide A (any antigenic sequence, such as an Aβ sequence described herein) and peptide B (an amino acid sequence including a foreign T-helper epitope) are synthesized and mixed. After they have been brought into contact with a suitable activated polyhydroxy polymer, the peptides A and B are bound via the activation group in a ratio corresponding to the initial ratio between these two compounds in the peptide mixture. See example 4 for details.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the following, a number of expressions used in the present description and claims will be defined and explained in detail to clarify the limits of the invention.

The terms "amyloid" and "amyloid protein" used interchangeably herein denote a class of proteinaceous unbranched fibrils of indeterminate length. Amyloid fibrils show characteristic staining with Congo red and share a cross-β structure in which the polypeptide chain is organized into β-sheets. Amyloid is generally derived from amyloidogenic proteins that have very different precursor structures, but all of which can undergo a structural transformation into a misfolded form that is the building block of the β-sheet helical protofilament. Normally, the diameter of the amyloid fibrils ranges from about 70 to about 120 Å.

The term "amyloidogenic protein" is intended to denote a polypeptide involved in the formation of amyloid deposits, either as part of the deposits as such or by being part of the biosynthetic pathway leading to the formation of the deposits. Examples of amyloidogenic proteins are therefore APP and Ap, but also proteins that are involved in the metabolism of these can be amyloidogenic proteins.

An "amyloid polypeptide" is intended herein to denote polypeptides comprising the amino acid sequence of the above-discussed amyloidogenic proteins derived from humans or from other mammals (or truncated variants thereof that share a substantial amount of B-cell epitopes with an intact amyloidogenic protein) - an amyloidogenic polypeptide may therefore, for example, comprise substantial parts of a precursor for the amyloidogenic polypeptide (in the case of Aβ, a possible amyloid polypeptide may be APP-derived). Unglycosylated forms of amyloidogenic polypeptides produced in prokaryotic systems are also included within the scope of expression, and there are also forms with different glycosylation patterns as a result of, for example, the use of yeast or other eukaryotic expression systems that do not originate from mammals. However, it should be noted that when the term "an amyloidogenic polypeptide" is used, it is intended that the polypeptide in question is normally non-immunogenic when presented to the animal being treated. In other words, the amyloidogenic polypeptide is a separate protein or an analogue of such a separate protein which normally does not give rise to an immune response against the relevant amyloidogenic polypeptide in the animal.

An "analog" is an APP- or Aβ-derived molecule that incorporates one or more changes in its molecular structure. Such a change can, for example, be in the form of a fusion of APP or Aβ polyamino acids to a suitable fusion partner (that is, a change in the primary structure that exclusively involves C- and/or N-terminal additions of amino acid residues) and/or it can be in the form of insertions and/or deletions and/or substitutions in the polypeptide's amino acid sequence. Also covered by the term are derivatized APP- and Ap-derived molecules, cf. the discussion below regarding modifications of APP or Ap. In some cases, the analog can be engineered so that it is less able, or even unable, to elicit antibodies against the normal precursor protein(s) of the amyloid, thereby avoiding unwanted interference with the (physiologically normal) non-aggregated form of the polypeptide that is a precursor to the amyloid protein.

It should be noted that the use as a vaccine in a human of a xeno-analogue (eg a rabbit or pig analogue) of a human APP or Aβ is conceivable to produce the desired immunity against APP or Aβ. Such use of a xeno-analog for immunization is also considered part of the invention.

The term "polypeptide" in the present context is intended to mean both short peptides with from 2 to 10 amino acid residues, oligopeptides with from 11 to 100 amino acid residues and polypeptides with more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, for example functional biomolecules comprising at least one polypeptide; when it comprises at least two polypeptides these may form complexes, be covalently bound or may be non-covalently bound. The polypeptide or polypeptides in a protein may be glycosylated and/or lipidated and/or may comprise prosthetic groups. The term "polyamino acid" is also an equivalent of the term "polypeptide".

The terms "T lymphocyte" and "T cell" are used interchangeably for lymphocytes of thymic origin that are responsible for a variety of cell-mediated immune responses as well as for helper activity in the humoral immune response. In the same way, the terms "B-lymphocyte" and "B-cell" will be used interchangeably for antibody-producing lymphocytes.

The term "subsequence" means any contiguous stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a naturally occurring amyloid amino acid sequence or nucleic acid sequence, respectively.

The term "animal" in the present context is generally intended to denote an animal species (preferably mammal) such as Homo sapiens, Canis domesticus etc. and not just a single animal. However, the term also means a population of such an animal species, as it is important that all the individuals immunized according to the method according to the invention all carry essentially the same APP or Ap which allows immunization of the animals with the same immunogen(s). It will be clear to those skilled in the art that an animal in the present context is a living being that has an immune system. It is preferred that the animal is a vertebrate such as a mammal.

With the expression "in vivo down-regulation of APP or Ap" here is meant the reduction in the living organism of the total amount of deposited amyloid protein (or amyloid as such) of the relevant type. The downregulation can be achieved by several mechanisms: of these, the simple interaction with amyloid of antibody binding to prevent misaggregation is the simplest. However, it is also within the scope of the present invention that the antibody binding results in the removal of amyloid by means of "renovation cells" (such as macrophages or other phagocytic cells) and that the antibodies work together with other amyloidogenic polypeptides leading to amyloid formation. A further possibility is that antibodies bind Ap on the outside of the CNS and thus effectively remove Ap from the CNS via a simple mass action principle.

The phrase "affecting the presentation ... to the immune system" is intended to mean that the animal's immune system is exposed to an immunogenic challenge in a controlled manner. As will be apparent from the description below, such challenge of the immune system can be effected in a number of different ways, the most important of which is vaccination with polypeptide containing "pharmaceuticals" (ie, a vaccine administered to treat or alleviate ongoing disease). or nucleic acid "pharmaceutical" vaccination. The important result to be achieved is that immunocompetent cells in the animal are confronted with the antigen in an immunologically effective manner, while the exact way of achieving this result is of less importance to the inventive idea behind the invention.

The term "immunogenic effective amount" has its usual meaning in the art, that is, an amount of an immunogen capable of inducing an immune response that significantly primes pathogens that share immunological features with the immunogen.

When the term APP or Aβ has been "modified" as used herein, it is meant that a chemical modification of the polypeptide has been performed on APP or Aβ. Such a modification can for example be derivatization (for example alkylation) of certain amino acid residues in the sequence, but as will be clear from the description below, the preferred modifications include changes to the primary structure of the amino acid sequence.

When "autotolerance to APP or Aβ" is discussed, it should be understood that since the polypeptide is a self-protein in the population to be vaccinated, normal individuals in the population will not mount an immune response to the polypeptide; it cannot be excluded although random individuals in an animal population may be able to produce antibodies against the natural polypeptide, for example as part of an autoimmune disease. At any rate, an animal will normally only be autotolerant to its own APP or Ap, but it cannot be ruled out that analogues derived from other animal species or from a population with a different phenotype will also be tolerated by said animal.

A "foreign T cell epitope" (or: "foreign T lymphocyte epitope") is a peptide capable of binding to an MHC molecule and stimulating T cells in a mammalian species. Preferred foreign T-cell epitopes according to the invention are promiscuous epitopes, i.e. epitopes which bind to a significant fraction of a specific class of MHC molecules in an animal species or population. Only a very limited number of such promiscuous T-cell epitopes are known, and they will be discussed in more detail below. Promiscuous T-cell epitopes are also termed "universal" T-cell epitopes. It should be noted that in order for the immunogens used according to the present invention to be effective in as large a part of the animal population as possible, it may be necessary to 1) insert several foreign T-cell epitopes into the same analog or 2) prepare several analogs in which each analog has a different promiscuous epitope inserted. It should also be noted that the concept of foreign T-cell epitopes also encompasses the use of cryptic T-cell epitopes, i.e. epitopes which are derived from a self-protein and which only elicit immunogenic behavior when they exist in isolated form without being part of the specific eigenprotein.

A "foreign T helper epitope" (a foreign Tn epitope) is a foreign T cell epitope that binds an MHC class II molecule that can be presented on the surface of an antigen presenting cell (APC) bound to the MHC class II molecule.

A "functional part" of a (bio)molecule is meant in the present context to mean the part of the molecule which is responsible for at least one of the biochemical or physiological effects exerted by the molecule. It is well known to those skilled in the art that many enzymes and other effector molecules have an active site that is responsible for the effects exerted by the molecule in question. Other parts of the molecule can serve as a stabilizing or solubility-enhancing purpose and can therefore be set aside if these purposes are not relevant in the context of a specific embodiment of the present invention. For example, it is possible to use certain cytokines as a modifying unit in APP or Aβ (cf. the detailed description below), and in such a case the stability aspect may be irrelevant as the link to APP or Aβ may provide the required stability.

The term "adjuvant" has its usual meaning in the field of vaccine technology, i.e. a compound or a composition which is 1) not in itself capable of eliciting a specific immune response against the immunogen in the vaccine, but which is 2) nevertheless in able to enhance the immune response against the immunogen. Or in other words, vaccination with the adjuvant alone does not elicit an immune response against the immunogen, vaccination with the immunogen may or may not elicit an immune response against the immunogen, but the combined vaccination with immunogen and adjuvant induces an immune response against the immunogen that is stronger than that induced of the immunogen alone.

"Targeting" of a molecule is, in the present context, intended to denote the situation where a molecule, upon introduction into the animal, will preferentially appear in certain tissues or will preferentially be associated with certain cells or cell types. The effect can be achieved in several ways, including the formation of the molecule in a composition that facilitates targeting or by introducing groups into the molecule that facilitate targeting. This topic will be discussed in more detail below.

"Stimulation of the immune system" means that a compound or composition exerts a general non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. The result of using an immunostimulant is an increased "alertness" of the immune system, meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated application of the immunogen.

"Productive binding" means binding of a peptide to an MHC molecule (class I or II) such that it is capable of stimulating T cells that engage a cell presenting the peptide bound to the MHC molecule. For example, a peptide that is bound to an MHC class II molecule on the surface of an APC is said to be productively bound if this APC will stimulate a Tn cell that binds to the presented peptide-MHC class II complex.

Preferred embodiments of amyloid downregulation

The analog used as an immunogen in the application of the invention is a modified APP or Ap molecule, in which at least one change is present in the amino acid sequence of APP or Ap, as the changes to achieve the important breaking of autotolerance are greatly facilitated this way - this is for example evident from the results presented in comparative example 2 herein, where immunization with wild-type Aβ is compared to immunization with an Aβ variant molecule. It has been shown (in Dalum I et al, 1996, J. Immunol. 157: 4796-4804) that potentially self-reactive B lymphocytes that recognize self proteins are physiologically present in normal individuals. However, for these B lymphocytes to be induced to actually produce antibodies reactive with the relevant self-proteins, assistance is needed from cytokine-producing T helper lymphocytes (Th cells or Tn lymphocytes). Normally, this help is not provided because T-lymphocytes generally do not recognize T-cell epitopes derived from self-proteins when presented on antigen-presenting cells (APC). However, by providing an element of "foreignness" in a self-protein (that is, by introducing an immunologically significant modification), T cells that recognize the foreign element will be activated by recognizing the foreign epitope on an APC (such as initially a mononuclear cell). Polyclonal B-lymphocytes (which are also APCs) capable of recognizing self-epitopes on the modified self-protein also internalize the antigen and then present the foreign T-cell epitope(s) thereof, and the activated T-lymphocytes then provide cytokine help to these self-reactive polyclonal B lymphocytes. Since the antibodies produced by these polyclonal B lymphocytes are reactive with different epitopes on the modified polypeptide, including those also present in the native polypeptide, an antibody is induced that is cross-reactive with the unmodified self-protein. To summarize, the T-lymphocytes can be tricked into acting as if the population of polyclonal B-lymphocytes has recognized a totally foreign antigen, when in fact only the inserted epitope(s) are foreign to the host. In this way, antibodies are induced which are able to cross-react with unmodified self-antigens.

Several ways of modifying a peptide self-antigen to achieve and break autotolerance are known in the art. It is nevertheless preferred that the analogue according to the present invention includes

- at least a first unit is introduced which provides targeting of the modified molecule to an antigen presenting cell (APC), and/or - at least a second unit is introduced which stimulates the immune system, and/or - at least a third unit is introduced which optimizes the presentation of the analogue to the immune system.

However, all these modifications should be carried out while maintaining a substantial part of the original B-lymphocyte epitopes in APP or Aβ, as the B-lymphocyte recognition of the native molecule is thereby enhanced.

In a preferred embodiment, side groups (in the form of foreign T-cell epitopes or the above-mentioned first, second and third units) are covalently or non-covalently introduced. This means that stretches of amino acid residues derived from APP or Ap are derivatized without changing the primary amino acid sequence, or at least without introducing changes in the peptide bonds between the individual amino acids in the chain.

An alternative and preferred embodiment utilizes amino acid substitution and/or deletion and/or insertion and/or addition (which can be accomplished by recombinant means or by peptide synthesis; modifications involving longer stretches of amino acids can give rise to fusion polypeptides). A particularly preferred version of this embodiment is the technique described in WO 95/05849, which describes a method for down-regulation of self-proteins by immunization with analogues of the self-proteins, in which a number of amino acid sequences have been substituted with a corresponding number of amino acid sequences, each comprising a foreign immunodominant T-cell epitope while simultaneously maintaining the total tertiary structure of the self-protein in the analogue. For the purposes of the present invention, however, it is sufficient if the modification (be it an insertion, addition, deletion or substitution) gives rise to a foreign T-cell epitope and at the same time preserves a significant number of the B-cell epitopes in APP or Ap. However, in order to achieve the maximum effect of the immune response induced, it is preferred that the overall tertiary structure of APP or Aβ is maintained in the modified molecule.

In some cases, it is preferred that APP or Ap or fragments thereof are mutated. Particularly preferred are substitution variants where methionine in position 35 of Ap-43 has been substituted, preferably with leucine or isoleucine or simply deleted. Particularly preferred analogs contain a single methionine located at the C-terminus, either because it is naturally occurring in the amyloidogenic polypeptide or the foreign TH epitope, or because it has been inserted or added. Accordingly, it is also preferred that the portion of the analog that includes the foreign Tn epitope be free of methionine, except for the possible C-terminal location of a methionine.

The main reason for removing all but one methionine is that it becomes possible to recombinantly produce multimeric analogues which can then be cleaved using cyanogen bromide, leaving the single analogue. The advantage is that recombinant production is facilitated in this way.

In fact, it is usually preferred that all analogs of APP or Aβ used share the characteristic of including only a single methionine positioned as the C-terminal amino acid of the analog and that other methionines in either the amyloidogenic polypeptide or in the foreign Tn epitope are deleted or substituted with another amino acid.

A further interesting mutation is a deletion or substitution of the phenylalanine in position 19 of Ap-43, and it is particularly preferred that the mutation is a substitution of this phenylalanine residue with a proline.

Other interesting polyamino acids that can be used in the analogue are shortened parts of Ap-43 proteins. These can also be used in immunogenic analogues according to the present invention. Particularly preferred are the abbreviations Ap(l-42), Ap(l-40), Ap(l-39), Apl-35), Ap(l-34), Ap(l-34), Ap(l-28) , Ap(l-12), Ap(l-5),

Ap(13-28), Ap(13-35), Ap(17-28), Ap(25-35), Ap(35-40), Ap(36-42) and Ap(35-42)

(where the number in the brackets indicates the amino acid stretches of Ap-43 that make up the relevant fragment - Ap(35-40) is, for example, identical to amino acids 706-711 in SEQ ID NO: 2). All these variants with shortened parts of Ap-43 can be prepared with the Ap fragments described herein, especially with variants 9, 10, 11, 12 and 13 as mentioned in example 1.

The following formula describes the molecular constructs generally covered by the invention: - where amyloidei-amyloidexer x B-cell epitopes containing subsequences of APP or Aβ which are independently identical or non-identical and which may or may not contain foreign side groups , x is an integer > 3, nl-nx is x integer > 0 (at least one is >), MODi-MODxer x modifications introduced between the conserved B-cell epitopes, and Si-sxer x integer > 0 (at least one is > 1 if no side groups are introduced in the amyloidex sequences). Accordingly, given the general functional relatedness of the immunogenicity of the constructs, the invention allows all kinds of permutations in the original sequence of APP or Aβ, and all kinds of modifications therein. Included in the invention is consequently modified APP or Ap obtained by omitting parts of the sequence which, for example, exhibit side effects in vivo or omit parts which are normally intracellular and which can consequently give rise to unwanted immunological reactions.

A preferred version of the constructs indicated above are, when applicable, those in which the B-cell epitope containing subsequences of an amyloid protein is not extracellularly exposed in the precursor polypeptide from which the amyloid is derived. By making such a choice of epitopes, it is ensured that no antibodies are formed that could be reactive with the cells that produce the precursor and thereby limit the formed immune response to an immune response against the unwanted amyloid deposits. In this case, for example, it would be useful to induce immunity against epitopes in APP or Aβ which are only exposed on the extracellular phase when they are free from any link to the cells from which they are produced.

Maintenance of a substantial portion of the B-cell epitopes or even the overall tertiary structure of a protein subjected to modification as described herein can be achieved in several ways. One way is simply to prepare a polyclonal antiserum directed against the polypeptide of interest (for example an antiserum produced in a rabbit) and then use this antiserum as a test reagent (for example in a competitive ELISA) against the modified proteins produced. Modified versions (analogs) that react to the same extent with the antiserum as APP or Ap must be considered to have the same overall tertiary structure as APP or Ap, while analogs that have a limited (but still significant and specific) reactivity with such an antiserum, considered to have retained a substantial fraction of the original B-cell epitopes.

Alternatively, a selection of monoclonal antibodies reactive with distinct epitopes on APP or Aβ can be prepared and used in a test panel. This approach has the advantage of allowing 1) an epitope mapping of APP or Aβ and 2) a mapping of the epitopes maintained in the produced analogs.

Of course, a third approach would be to resolve the three-dimensional structure of APP or Ap or of a biologically active truncate thereof (see above) and compare this with the resolved three-dimensional structure of the prepared analogs. Three-dimensional structure can be resolved using X-ray diffraction studies and NMR spectroscopy. Additional information related to the tertiary structure can be obtained to some extent from circular dichroism studies which have the advantage that only the polypeptide in pure form is required (whereas X-ray diffraction requires the acquisition of crystallized polypeptide and NMR requires the acquisition of isotopic variants of the polypeptide) to provide useful information about the tertiary structure of a given molecule. Finally, however, X-ray diffraction and/or NMR is necessary to obtain definitive data, as circular dichroism can only provide indirect evidence of correct three-dimensional structure via information on secondary structure elements.

A preferred embodiment according to the invention utilizes multiple presentations of B-lymphocyte epitopes in APP or Ap (that is, formula I in which at least one B-cell epitope is present in two positions). This effect can be achieved in a number of different ways, for example by simply preparing fusion polypeptides comprising the structure (APP or Aβ derived polypeptide) where m is an integer 2 and then introducing the modifications described herein into at least one of the APP or Aβ the sequences. It is preferred that the introduced modifications include at least one duplication of a B-lymphocyte epitope and/or introduction of a hapten. These embodiments, including multiple presentations of selected epitopes, are particularly preferred in situations where only minor portions of APP or Ap are usable as components of a vaccine.

As mentioned above, the introduction of a foreign T-cell epitope can be achieved by introducing at least one amino acid insertion, addition, deletion or substitution. Of course, the normal situation would be the introduction of more than one change in the amino acid sequence (eg insertion or substitution of a complete T-cell epitope), but the important goal to achieve is that the analogue, when produced by an antigen-presenting cell (APC ) will give rise to such a foreign immunodominant T-cell epitope that is presented in the sense of an MHC class II molecule on the surface of APC. Consequently, if the amino acid sequence of APP or Ap in appropriate positions comprises a number of amino acid residues that can also be found in a foreign Tn epitope, the introduction of a foreign TV epitope can then be achieved by providing the remaining amino acids of the foreign epitope by means of amino acid insertion, addition , deletion and substitution. In other words, it is not necessary to introduce a complete TH epitope by insertion or substitution to satisfy the purpose of the present invention.

It is preferred that 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. It is further preferred that the number of amino acid insertions, substitutions, additions or deletions does not exceed 150, such as no more than 100, no more than 90, no more than 80 and no more than 70. It is particularly preferred that the number of substitutions, insertions, deletions and additions do not exceed 60 and in particular the number should not exceed 50 or even 40. Most preferred is a number of no more than 30. With regard to amino acid additions, it should be noted that when the resulting construct is in the form of a fusion polypeptide , is often significantly higher than 150.

Preferred embodiments of the invention include modifications by introducing at least one foreign immunodominant T-cell epitope. It should be understood that the question of immunodominance of a T-cell epitope depends on the animal species involved. As used herein, the term "immunodominance" simply refers to epitopes which in the vaccinated individual/population give rise to a significant immune response, but it is a well-known fact that a T-cell epitope which is immunodominant in an individual/population is not necessarily immunodominant in another individual of the same species, even if it is able to bind MHC-II molecules in the latter individual. Accordingly, for the purposes of the present invention, an immunodominant T-cell epitope is a T-cell epitope that will effectively provide T-cell help when presented in an antigen. Typically, immunodominant T-cell epitopes will have as an inherent feature that they will essentially always be presented bound to an MHC class II molecule, regardless of the polypeptide in which it appears.

Another important point is the aspect of MHC restriction of T-cell epitopes. In general, naturally occurring T-cell epitopes are MHC-restricted, that is, certain peptides that constitute a T-cell epitope will only effectively bind to a subset of MHC class II molecules. This in turn has the effect that in most cases the use of a specific T-cell epitope will result in a vaccine component that is only effective in a fraction of the population, and depending on the size of the fraction, it may be necessary to include multiple T-cell epitopes in the same molecule, or alternatively prepare a multicomponent vaccine in which the components are variants of APP or Aβ that differ from each other through the property of the introduced T-cell epitope.

If MHC restriction of the T cells used is completely unknown (for example in a situation where the vaccinated animal has an ill-defined MHC composition), the part of the population covered by a specific vaccine composition can be approximated using the following formula

where pt is the frequency in the population of responders where the/foreign T-cell epitopes are present in the vaccine composition, and n is the total number of foreign T-cell epitopes in the vaccine composition. Accordingly, a vaccine composition containing 3 foreign T-cell epitopes having population response rates of 0.8, 0.7 and 0.6, respectively, will

that is, 97.6 of the population will statistically have an MHC-II-mediated response to the vaccine.

The formula stated above does not apply in situations where one or more less accurate MHC restriction patterns of the peptides used are known. If, for example, a certain polypeptide only binds the human MHC-II molecules coded for by the HLA-DR alleles DR1, DR3, DR5 and DR7, then the use of this peptide together with another peptide that binds the remaining MHC-II the molecules coded for by the HLA-DR alleles provide 100% coverage in the population of interest. Likewise, if the second peptide only binds DR3 and DR5, the addition of this peptide will not increase coverage at all. If one bases the population response calculations only on MHC restriction of T-cell epitopes in the vaccine, the minimum fraction of the population covered by a specific vaccine formulation can be determined using the following formula:

where ty is the sum of frequencies in the population of allelic haplotypes encoding MHC molecules that bind any of the T-cell epitopes in the vaccine and that belong to j of the 3 known HLA loci (DP, DR, and DQ); in practice, it is first determined which MHC molecules will recognize each T-cell epitope in the vaccine and then these are indicated by type (DP, DR and DQ) - then the individual

the frequencies of the various indicated allelic haplotypes summed up for each type, thereby giving ( pu q>2and q>3.

It may happen that the value /?, in the formula II exceeds the corresponding theoretical value jz;-: where V is the sum of the frequencies in the population of the allelic haplotype that codes for MHC molecules that bind the /T-cell epitope in the vaccine and that belongs to j of the 3 known HLA loci (DP, DR and DQ). This means that 1 -jt; of the population is a frequency of responders of frest_i= ( prXi) I Therefore formula III can be adjusted to give formula V:

where the expression l-/rest_is set to zero if it is negative. It should be noted that Formula V requires all epitopes to be haplotype mapped against identical sets of haplotypes.

When choosing T-cell epitopes to be introduced into the analogue, it is therefore important to include all knowledge about the epitopes that is available: 1) The frequency of responders in the population to each epitope, 2) MHC restriction data and 3) frequency in the population against the the relevant haplotypes.

There are a number of naturally occurring "promiscuous" T-cell epitopes which are active in a larger part of the individuals in an animal species or an animal population and these are preferably introduced in the vaccine in order to thereby reduce the need for a very large number of different analogues in the same vaccine.

According to the invention, the promiscuous epitope can be a naturally occurring human T-cell epitope such as epitopes from tetanus toxoid (for example the P2 and P30 epitopes), diphtheria toxoid, influenza virus hemagglutinin (HA) and P. falciparum CS antigen.

Over the years, a number of other promiscuous T-cell epitopes have been identified. In particular, peptides capable of binding a greater proportion of HLA-DR molecules encoded by the various HLA-DR alleles have been identified, and these are all possible T-cell epitopes that can be introduced into the analogues used according to to the present invention. See also the epitopes discussed in the following references: WO 98/23635 (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 reference also relates to HLA-DQ and -DP ligands. All epitopes indicated in these 5 references are relevant as natural candidate epitopes that can be used in the present invention, so are epitopes that share common motifs with them.

Alternatively, the epitope can be any artificial T-cell epitope capable of binding a greater proportion of MHC class II molecules. In this context, the pan DR epitope peptides ("PADRE") described in WO 95/07707 and in the corresponding article Alexander J et al, 1994, Immunity 1: 751-761 are interesting candidates as epitopes for use according to the present invention. It should be noted that the most effective PADRE peptides described in these publications carry D-amino acids at the C- and N-termini to improve stability upon administration. However, the present invention primarily seeks the incorporation of the relevant epitopes as part of the analogue which should then be degraded enzymatically inside the lysosomal parts of the APC to allow subsequent presentation in the sense of an MHC-II molecule and is therefore not expected to incorporate D-amino acids in the epitopes used in the present invention.

A particularly preferred PADRE peptide is that with the amino acid sequence AKFVAAWTLKAAA (SEQ ID NO: 17) or an immunologically effective subsequence thereof. This and other epitopes with the same lack of MHC restriction are preferred T-cell epitopes that should be present in the analog used in the inventive method. Such superpromiscuous epitopes will allow the simplest embodiments of the invention in which only a single analog is present in the immune system of the vaccinated animal.

As mentioned above, the modification of APP and Aβ may also include the introduction of a first unit that targets the modified amyloidogenic polypeptide to an APC or a B-lymphocyte. For example, the first entity can be a specific binding partner for a B-lymphocyte-specific surface antigen or for an APC-specific surface antigen. Many such specific surface antigens are known in the art. For example, the entity may be a carbohydrate for which there is a receptor on the B-lymphocyte or APC (eg mannan or mannose). Alternatively, the second entity may be a hapten. An antibody fragment that specifically recognizes a surface molecule on APC or lymphocytes can also be used as a first entity (the surface molecule can for example be an FCy receptor for macrophages and monocytes, such as FCyRI or alternatively any other specific surface marker such as CD40 or CTLA-4) . It should be noted that all of these exemplary targeting molecules can also be used as part of an adjuvant, see below.

As an alternative or supplement to targeting the analog to a certain cell type to achieve an enhanced immune response, it is possible to increase the level of responsiveness of the immune system by including the above-mentioned second entity that stimulates the immune system. Typical examples of such other entities are cytokines and heat shock proteins or molecular chaperones, as well as effective parts thereof.

Suitable cytokines that can be used according to the invention are those that normally also function as adjuvants in a vaccine composition, i.e. for example interferon-y (IFN-y), 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, the functional part of the cytokine molecule may be sufficient as the second unit. With respect to the use of such cytokines as adjuvants, see the discussion below.

According to the invention, suitable heat shock proteins or molecular chaperones used as the second entity can be HSP70, HSP90, HSC70, GRP94 (also known as gp96, cf. Wearsch PA et al, 1998, Biochemistry 37: 5709-19) and CRT (calreticulin) .

Alternatively, the second entity can be a toxin such as listeriolysin (LLO), lipid A and heat labile enterotoxin. A number of mycobacterial derivatives such as MDP (muramyl dipeptide), CFA (Freund's complete adjuvant) and the trehalose diesters TDM and TDE may also be interesting possibilities.

The possibility of introducing a third unit which enhances the presentation of the analogue to the immune system is also an important embodiment of the invention. The prior art has shown several examples of this principle. For example, it is known that the palmitoyl lipidation anchor in the Borrelia burgdorferi protein OspA can be utilized to provide self-adjuvant polypeptides (cf. for example WO 96/40718) - it appears that the lipidated proteins form micelle-like structures with a core consisting of the lipidation anchor parts of the polypeptides and the the remaining parts of the molecule that stand out from these, and result in multiple presentations of the antigenic determinants. The use of these and related approaches using different lipidation anchors (for example, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI anchor, and an N-acyldiglyceride group) are also preferred embodiments of the invention, especially since the acquisition of such a lipidation anchor in a recombinantly produced protein is quite simple and only requires the use of a naturally occurring signal sequence as a fusion partner for the analog. Another possibility is the application of the C3d fragment to complement factor C3 or C3 itself (cf. Dempsey et al, 1996, Science 271, 348-350 and Lou & Kohler, 1998, Nature Biotechnology 16, 458-462).

An alternative embodiment of the present invention which also results in the preferential presentation of multiple (eg at least 2) copies of the major epitope regions of APP or Aβ for the immune system is the covalent linkage of the analogue to certain molecules, i.e. the variants d and e mentioned above. For example, polymers can be used, for example carbohydrates such as dextran, see for example Lees A et al., 1994, Vaccine 12: 1160-1166; Lees A et al., 1990 J Immunol. 145: 3594-3600, but also mannose and mannan are useful alternatives. Integral membrane proteins from, for example, E. coli and other bacteria are also useful conjugation partners. The traditional carrier molecules such as keyhole limpet hemocyanin (KLH), tetanus toxoid, diphtheria toxoid and bovine serum albumin (BSA) are also preferred and useful conjugation partners.

Preferred embodiments of covalently linking APP- or Aβ-derived material to polyhydroxy polymers such as carbohydrates involve the use of at least one APP- or Aβ-derived peptide and at least one foreign T-helper epitope that is separately linked to the polyhydroxy polymer (that is, say that the foreign T-helper epitope and APP- and Aβ-derived amino acid sequence are not condensed with each other, but rather are bound to the polyhydroxy polymer which as such serves as a carrier backbone). Again, such an embodiment is most preferred when the appropriate B cell epitope bearing regions of the APP or Aβ derived peptides are composed of short peptide stretches - this is because this approach is a very suitable way to achieve multiple presentations of selected epitopes in the resulting immunogenic agent. However, it is also possible to easily link analogs already described herein to the polyhydroxy polymer backbone, that is, the APP- or Aβ-derived material is not bound to the backbone separately from the foreign Tn epitopes.

It is particularly preferred that the connection of the foreign T-helper epitope and the APP- or Aβ-derived (poly)peptide is achieved by means of an amide bond which can be cleaved with a peptidase. This strategy has the effect that the APC will be able to take up the conjugate and at the same time be able to process the conjugate and then present the foreign T-cell epitope in an MHC class II context.

One way to achieve coupling of peptides (both the APP- or Aβ-derived peptide of interest as well as the foreign epitope) is to activate a suitable polyhydroxy polymer with tresyl (trifluoroethylsulfonyl) groups or other suitable activating groups such as maleimide, p- nitrophenylchloroformate (for activation of OH groups and formation of a peptide bond between peptide and polyhydroxy polymer), and tosyl (p-toluenesulfonyl). For example, it is possible to prepare activated polysaccharides as described in WO 00/05316 and US 5,874,469 and link these to APP- or Aβ-derived peptides or polyamino acids as well as to the T-cell epitopes prepared by conventional solid- or liquid-phase peptide synthesis techniques . The resulting product consists of a polyhydroxy polymer backbone (for example, a dextran backbone) which has, attached thereto by its N-terminus or by means of other available nitrogen moieties, polyamino acids derived from APP or Aβ and from foreign T-cell epitopes. If desired, it is possible to synthesize the APP or Aβ peptides so that all available amino groups except that unit at the N-terminus are protected, then link the resulting protected peptides to the trisylated dextran unit and finally deprotect the resulting conjugate. A specific example of this approach is described in the examples below.

Instead of using the water-soluble polysaccharide molecules as described in WO 00/05316 and in US 5,874,469, it is correspondingly possible to utilize cross-linked polysaccharide molecules and thereby obtain a specific conjugate between polypeptides and polysaccharide - this is believed to lead to an improved presentation to the immune system of the polypeptides, as two goals are achieved, namely to achieve a local deposition effect when the conjugate is injected and to achieve particles that are attractive targets for APC. The approach for using such specific systems is also described in detail in the examples.

Assessments underlying selected areas to introduce modifications in APP or Ap are a) conservation of known and putative B-cell epitopes,

b) preservation of tertiary structure, c) avoidance of B-cell epitopes present on "producer cells", etc. At any rate as discussed above, it is quite

easily assay a set of analogs that have all been subjected to introduction of a T-cell epitope at different sites.

Accordingly, since the most preferred embodiments of the present invention involve the down-regulation of human Aβ, it is preferred that the APP or Aβ polypeptide discussed above be a human Aβ polypeptide. In this embodiment, it is particularly preferred that the APP or Aβ polypeptide has been modified by substitution of at least one amino acid sequence in SEQ ID NO. NO. 2 with at least one amino acid sequence of equal or different length and containing a foreign Tn epitope. Preferred examples of modified amyloidogenic APP and Aβ are shown schematically in Figure 1 using the P2 and P30 epitopes as examples. The logical explanation behind such constructs is described in detail in the examples.

More specifically, a Tn-containing (or complete) amino acid sequence introduced in SEQ ID NO. NO. 2 is introduced at any amino acid in SEQ ID NO. NO. 2. That is, the introduction is possible after any of the amino acids 1-770, but preferably after any of the amino acids 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, 708, 709 , 710, 711, 712, 713 and 714 in SEQ ID NO. NO. 2. This can be combined with deletion of any or all of amino acids 1-671, or any of all of amino acids 715-770. Utilizing the substitution technique, each of the amino acids 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, 707, 708, 709, 710, 711, 712, 713 and 714 in SEQ.ID . NO. 2 is deleted in combination with the introduction.

According to the present invention, the analogue does not include any subsequence of SEQ ID NO. NO. 2 that binds productively to MHC class II molecules and initiates a T-cell response.

The rationale behind such a strategy to engineer the immunogen that primes the immune system and induces, for example, an anti-Aβ immune response, is the following: It has been discovered that when amounts of autologous proteins are immunized formulated in an adjuvant sufficiently strong to to break the body's tolerance to the autologous protein, there is a danger that the immune response in simply vaccinated individuals does not cease simply by breaking the immunization. This is because the induced immune response in such individuals is likely driven by a natural TV epitope in the autologous protein, and this has the side effect that the vaccinated individuals own protein will be able to act as an immunizing agent against its own targets: An autoimmune condition has consequently been established.

The use of foreign Tn epitopes, to the inventors' knowledge, has never been observed to produce this effect because the anti-self immune response is driven by a foreign Tn epitope, and it has been repeatedly demonstrated by the inventors that the induced immune response elicited by the preferred technology actually declines after discontinuation of the immunization. Theoretically, however, it should happen in a small number of individuals that the immune response will also be driven by an autologous Tn epitope (of the relevant self-protein against which one is immunized) - this is particularly relevant when considering self-proteins that are abundant, such as such as Ap, while other therapeutically relevant self-proteins are only present locally or in such low amounts in the body that a "self-immunization effect" is not possible. Consequently, a very simple way to avoid this is to completely avoid including peptide sequences in the immunogen that could serve as Tn epitopes (and since peptides shorter than about 9 amino acids cannot serve as Tn epitopes, the use of shorter fragments a simple and feasible approach). This embodiment of the invention therefore also serves to ensure that the immunogen does not include peptide sequences of the target APP or Aβ that could serve as "self-stimulatory Tn epitopes" including sequences that only contain conservative substitutions in a sequence of the target protein that could otherwise function as a Tn- epitope.

Preferred embodiments of the immune system presentation of the analogs of APP or Aβ involve the use of a chimeric peptide comprising at least one APP- or Aβ-derived peptide that does not productively bind to MHC class II molecules, and at least one foreign T-helper epitope . It is particularly advantageous if the immunogenic analogue is one in which the amino acid sequences comprising one or more B-cell epitopes are represented either as a continuous sequence or as a sequence including inserts, wherein the insert comprises foreign T-helper epitopes.

Again, such an embodiment is most preferred when the appropriate B cell epitope bearing regions of APP or Aβ consist of short peptide stretches that would not be capable of binding productively to an MHC class II molecule in any way. The selected B-cell epitope or epitopes of the amyloidogenic polypeptide should therefore comprise at least 9 contiguous amino acids of SEQ ID NO. NO. 2. Shorter peptides are preferably such as those that have at least 8, 7, 6, 5, 4 or 3 contiguous amino acids from the amyloidogenic polypeptide's amino acid sequence.

It is preferred that the analogue comprises at least one subsequence of SEQ ID NO. NO. 2 so that each such at least one subsequence consists of amino acid stretches from APP or Ap which are selected from the group consisting of 9 contiguous amino acids, 8 contiguous amino acids, 7 contiguous amino acids, 6 contiguous amino acids, 5 contiguous amino acids, 4 contiguous amino acids and 3 contiguous amino acids.

It is particularly preferred that the contiguous amino acids begin with an amino acid residue selected from the group consisting of the 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, 713 and 714 of SEQ ID NO. NO. 2.

Protein/peptide vaccination; formulation and administration of the analogues

In eliciting presentation of the analog to an animal's immune system by administering it to the animal, the formulation of the polypeptide follows the general principles of the prior art.

The preparation of vaccines containing peptide sequences as active ingredients is generally well understood in the art as exemplified by U.S. Pat. patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770,. Such vaccines are usually prepared as injectable vaccines, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared. The preparation can also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. If desired, the vaccine can also contain smaller amounts of auxiliary compounds such as wetting agents or emulsifiers, pH buffers or adjuvants that increase the effectiveness of the vaccines; see the detailed description of adjuvants below.

The vaccines are usually administered parenterally by injection, for example either subcutaneously, intracutaneously or intramuscularly. Additional formulations suitable for other routes of administration include suppositories and in some cases oral, buccal, sublingual, intraperitoneal, intravaginal, anal, epidural, spinal and intracranial formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in a range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally used excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, delayed release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%. For oral formulations, cholera toxin is an interesting formulation partner (and also a possible conjugation partner).

The polypeptides can be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups in the peptide) and which are formed with inorganic acids such as hydrochloric or phosphoric acid, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

The vaccines are administered in a manner compatible with the dosage formulation and in such an amount as to be therapeutically effective and immunogenic. The amount administered depends on the individual being treated, including, for example, the capacity of the individual's immune system to elicit an immune response and the degree of protection desired. Suitable dosage ranges are in the order of several hundred micrograms of active ingredient per vaccination with a preferred range from about 0.1 fig to 2000 fig (although even higher amounts in the range of 1-10 mg are encompassed), such as in the range from about 0.5 µg to 1000 µg, preferably in the range from 1 µg to 500 µg and especially in the range from 10 µg to 100 µg. Suitable regimens for initial administration and subsequent administration also vary, but a typical example is an initial administration followed by subsequent inoculations and other administrations.

The method of application can vary greatly. Any of the conventional methods of administering a vaccine are acceptable. These include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and the formulation of the antigen.

Some of the polypeptides in the vaccine are sufficiently immunogenic in a vaccine, but for some of the others the immune response will be enhanced if the vaccine further comprises an adjuvant compound.

Various methods for achieving an adjuvant effect for the vaccine are known. General principles and methods are detailed in "The Theory and Practical Application of Adjuvants", 1995, Duncan E.S. Stewart-Tull (ed.), John Wiley & Sons Ltd, ISBN 0-471-95170-6, and also in "Vaccines: New Generation Immunological Adjuvants", 1995, Gregoriadis G et al. (ed.), Plenum Press, New York, ISBN 0-306-45283-9.

It is particularly preferred to use an adjuvant which can be shown to facilitate the breaking of the autotolerance against autoantigens; in fact, this is essential in cases where unmodified amyloidogenic polypeptide is used as the active ingredient in the autovaccine. Non-limiting examples of suitable adjuvants are selected from the group consisting of an immune targeting adjuvant; an immunomodulatory adjuvant such as a toxin, a cytokine and a mycobacterial derivative; an oil formulation; a polymer; a micelle-forming adjuvant; a saponin; an immunostimulatory complex matrix (ISCOM matrix); a particle; DDA; aluminum adjuvants; DNA adjuvants; γ-insulin; and an encapsulation adjuvant. In general, it should be noted that the above descriptions relating to compounds and agents useful as first, second and third units in the analogues also refer mutatis mutandis to their use in the adjuvant of a vaccine according to the invention.

The application of adjuvants includes the use of agents such as aluminum hydroxide or phosphate (alum), usually used as a 0.05 to 0.1% solution in buffered saline, mixed with synthetic polymers of sugar (eg Carbopol®) used as 0.25 % solution, aggregation of the protein in the vaccine by heat treatment with temperatures in the range between 70°C and 110°C for periods of 30 seconds to 2 minutes respectively and also aggregation using cross-linking agents is possible. Aggregation by reactivation with pepsin-treated antibodies (Fab fragments) to albumin, mixing with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of Gram-negative bacteria, emulsion in a physiologically acceptable oil carrier such as mannide monooleate (Aracel A) or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA) used as a blocking substitute can also be used. Mixing with oils such as squalene and IFA is also preferred.

According to the invention, DDA (dimethyldioctadecylammonium bromide) is an interesting candidate as an adjuvant such as DNA and γ-inulin, but also Freund's complete and incomplete adjuvant as well as ^«/7/a/a-saponins such as QuilA and QS21 as well like RIBI, are interesting. Additional possibilities are monophosphoryl lipid A (MPL), the above mentioned C3 and C3d and muramyl dipeptide

(MDP).

Liposome formulations are also known to include adjuvant effects, and liposome adjuvants are therefore preferred according to the invention.

Also immunostimulatory complex matrix type (ISCOM® matrix) adjuvants are preferred choices according to the invention, especially as it has been shown that this type of adjuvant is able to upregulate an MHC class II expression through APC. An ISCOM® matrix consists of (possibly fractionated) saponins (triterpenoids) from Quillaja saponaria, cholesterol and phospholipid. When mixed with the immunogenic protein, the resulting particle formulation becomes what is known as an ISCOM particle where the saponin makes up 60-70% w/w, the cholesterol and phospholipid 10-15% w/w and the protein 10-15% w/w . Details dealing with the composition and use of immunostimulating complexes can be found, for example, in the above-mentioned textbooks dealing with adjuvants, but also in Morein B et al, 1995, Clin. Immunother. 3: 461-475 as well as in Barr IG and Mitchell GF, 1996, Immunol. and Cell Biol. 74: 8-25 provides useful instructions for the preparation of complete immunostimulatory complexes.

Another very interesting (and therefore preferred) possibility to achieve adjuvant effect is to use the technique described in Gosselin et al., 1992. Briefly, the presentation of a relevant antigen such as an antigen according to the present invention can be enhanced by conjugation of the antigen to antibodies (or antigen-binding antibody fragments) against the Fcy receptors on monocytes/macrophages. In particular, conjugates between antigen and anti-FcγRI have been shown to enhance immunogenicity for vaccination purposes.

Other possibilities involve the use of targeting and immunomodulatory compounds (including cytokines) mentioned above as candidates for the first and second units of the modified versions of amyloidogenic polypeptides. In this context, synthetic inducing compounds of cytokines such as poly I:C are also possible.

Suitable mycobacterial derivatives are selected from the group consisting of muramyl dipeptide, Freund's complete adjuvant, RIBI and a diester of trehalose such as TDM and TDE.

Suitable immunotargeting adjuvants are selected from the group consisting of CD40 ligand and CD40 antibodies or specific binding fragments thereof (see discussion above), mannose, a Fab fragment and CTLA-4.

Suitable polymer adjuvants are selected from the group consisting of carbohydrate such as dextran, PEG, starch, mannan and mannose; a plastic polymer such as<*>; and latex such as latex balls.

Yet another interesting way to modulate an immune response is to include the immunogen (optionally together with adjuvants and pharmaceutically acceptable carriers and vehicles) in an "artificial lymph node" (VLN) (a proprietary device developed by ImmunoTherapy, Inc., 360 Lexington Avenue, New York, NY 10017-6501). The VLN (a thin tube device) mimics the structure and function of a lymph node. Insertion of a VLN under the skin forms a site of sterile inflammation with an increase in cytokines and chemokines. T and B cells as well as APC respond quickly to the danger signals, arrive at the site of inflammation and accumulate inside the porous matrix of the VLN. It has been shown that the necessary antigen dose required to elicit an immune response against an antigen is reduced by the use of VLN and that the immune protection achieved by vaccination using VLN exceeds conventional immunization using Ribi as an adjuvant. The technology is, among other things, briefly described 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 Lymph Node", in: "From the Laboratory to the Clinic, Book of Abstracts, October 12-15, 1998, Seascape Resort, Aptos, California".

Microparticle formulations of vaccines have in many cases been shown to increase the immunogenicity of protein antigens and are therefore another preferred embodiment of the invention. Microparticles are formed either as co-formulations of antigen with a polymer, a lipid, a carbohydrate or other molecules suitable for forming the particles, or the microparticles can be homogeneous particles consisting only of the antigen itself.

Examples of polymer-based microparticles are PLGA- and PVP-based particles (Gupta, R.K. et al., 1998) where the polymer and antigen are condensed into a solid particle. Lipid-based particles can be formed as micelles of the lipid (so-called liposomes) that trap the antigen inside the micelle (Pietrobon, PJ. 1995). Carbohydrate-based particles are typically made from a suitable degradable carbohydrate such as starch or chitosan. The carbohydrate and antigen are mixed and condensed into particles in a process similar to that used for polymer particles (Kas, H.S. et al. 1997).

Particles consisting only of the antigen can be produced using various spray and freeze-drying techniques. Particularly suitable for the purpose of the present invention is the supercritical fluid technology used to produce highly uniform particles of controlled size (York, P. 1999 & Shekunov, B. et al. 1999).

It is expected that the vaccine should be administered 1-6 times per year, such as 1, 2, 3, 4, 5 or 6 times per year to an individual in need of it. It has previously been shown that the memory immunity induced by the use of the preferred autovaccines according to the present invention is not permanent, and the immune system therefore needs periodic challenge with the amyloidogenic polypeptide or the modified amyloidogenic polypeptides.

As a result of genetic variation, different individuals may respond with immune responses of varying strength to the same polypeptide. The vaccine according to the present invention may therefore comprise several different polypeptides to increase the immune response, cf. also the discussion above on the choice of foreign T-cell epitope introductions. The vaccine may comprise two or more polypeptides where all the polypeptides are as defined above.

Accordingly, the vaccine may comprise 3-20 different modified or unmodified polypeptides, such as 3-10 different polypeptides.

Nucleic acid vaccination

As an alternative to classical administration of a peptide-based vaccine, the nucleic acid vaccination technology (also known as "nucleic acid immunization", "genetic immunization" and "gene immunization") offers a number of attractive features.

First, unlike the traditional vaccine approach, nucleic acid vaccination does not require resource-intensive large-scale production of the immunogenic agent (eg, in the form of industrial-scale fermentation of microorganisms that produce modified amyloidogenic polypeptides). Furthermore, there is no need for device cleaning and refolding forms for the immunogen. Finally, since nucleic acid vaccination depends on the biochemical apparatus of the vaccinated individual to produce the expressed product of the nucleic acid introduced, it is expected that optimal post-translational processing of the expression product occurs; this is particularly important in the case of autovaccination, since, as mentioned above, a significant part of the original B-cell epitopes should be preserved in the modified molecule, and since the B-cell epitopes can in principle be made up of parts of any (bio)molecule (for e.g. carbohydrate, lipid, protein, etc.). Natural glycosylation and lipidation patterns in the immunogen may therefore just as well be important for the overall immunogenicity and this is best ensured by the host producing the immunogen.

A preferred embodiment of the invention's variants a-c consequently comprises effecting presentation of the analogue to the immune system by introducing nucleic acid(s) that code for the analogue in the animal's cells and thereby achieving in vivo expression with the help of the cells in which the nucleic acid or nucleic acids have been introduced.

In this embodiment, the introduced nucleic acid is preferably DNA which may be in the form of naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA included in a viral vector, DNA formulated with a transfection-deleting protein or polypeptide, DNA formulated with a target protein or polypeptide, DNA formulated with calcium precipitating agents, DNA linked to an inert carrier molecule, DNA encapsulated in a polymer, for example in PLGA (cf. the microencapsulation technology described in WO 98/31398) or in chitin or chitosan and DNA formulated with an adjuvant. In this sense, it is noted that virtually all considerations regarding the use of adjuvants in traditional vaccine formulations apply to the formulation of DNA vaccines. Accordingly, the descriptions herein relating to the use of adjuvants in the sense of polypeptide-based vaccines apply mutatis mutandis to their use in nucleic acid vaccination technology.

As for administration routes and administration schemes for polypeptide-based vaccines which have been described in detail above, these are also applicable to nucleic acid vaccines according to the invention and all discussions above regarding administration routes and administration schemes for polypeptides apply mutatis mutandis to nucleic acids. To this it should be added that nucleic acid vaccines can conveniently be administered intravenously and intra-arterially. Furthermore, it is well known to experts in the field that nucleic acid vaccines can be administered using so-called "gene guns", and consequently this and similar administration methods are also considered part of the present invention. Finally, the use of a VLN in the administration of nucleic acids has also been reported to give good results and is therefore a particularly preferred mode of administration.

Furthermore, the nucleic acid or nucleic acids used as an immunizing agent may contain regions coding for the first, second and/or third unit, for example in the form of the immunomodulatory compound described above such as the cytokines discussed as useful adjuvants. A preferred version of this embodiment comprises having the coding region of the analog and the coding region of the immunomodulator in different reading frames or at least under the control of different promoters. Thereby, it is avoided that the analogue or epitope is produced as a fusion partner of the immunomodulator. Alternatively, two separate nucleotide fragments can be used, but this is less preferred due to the advantages that ensure co-expression when both coding regions are included in the same molecule.

The invention therefore also relates to a composition for inducing the production of antibodies against APP or Ap, where the composition comprises - a nucleic acid fragment or a vector according to the invention (cf. the discussion of vectors below), and - a pharmaceutically and immunologically acceptable vehicle and/or carrier and /or adjuvant as described above.

Under normal conditions, the variant encoding nucleic acid is introduced in the form of a vector in which expression is under the control of a viral promoter. For more detailed description of vectors according to the invention, see the discussion below. Detailed descriptions regarding the formulation and use of nucleic acid vaccines are also available, cf. Donnelly JJ et al, 1997, Annu. Fox. Immunol. 15: 617-648 and Donnelly JJ et al, 1997, Life Sciences 60: 163-172. Both of these references are incorporated by reference herein.

Live vaccines

A third option to effect presentation of the analogues as defined in variants a-c for the immune system is the use of live vaccine technology. In live vaccination, presentation to the immune system is effected by administering to the animal a non-pathogenic microorganism that has been transformed with a nucleic acid fragment encoding an analog or with a vector incorporating such a nucleic acid fragment. The non-pathogenic microorganism can be any suitable attenuated bacterial strain (attenuated by repeated passages or by removing pathogenic expression products by recombinant DNA technology), for example Mycobacterium bovis BCG., non-pathogenic Streptococcus spp., E. coli, Salmonella spp., Vibrio cholerae, Shigella, etc. Overviews concerning the production of live vaccines in the state of the art today can be found, for example, in Saliou P, 1995, Rev. Talk. 45: 1492-1496 and Walker PD, 1992, Vaccine 10: 977-990, both incorporated by reference herein.

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

As an alternative to bacterial live vaccines, the nucleic acid fragment of the invention as discussed below can be incorporated into a non-viral viral vaccine vector such as a vaccinia strain or any other suitable smallpox virus.

Normally, the non-pathogenic microorganism or virus is administered only once to the animal, but in certain cases it may be necessary to administer the microorganism more than once during its lifetime to maintain protective immunity. It is even expected that immunization schedules such as those described above for polypeptide vaccination will be applicable when using live or viral vaccines.

Alternatively, live or virus vaccination is combined with previous or subsequent polypeptide and/or nucleic acid vaccination. For example, it is possible to effect primary immunization with a live or viral vaccine followed by subsequent reimmunization using the polypeptide or nucleic acid approach.

The microorganism or virus can be transformed with the nucleic acid or nucleic acids containing regions encoding the first, second and/or third unit, for example in the form of the immunomodulatory compounds described above such as the cytokines described as useful adjuvants. A preferred version of this embodiment comprises having the coding region of the analog and the coding region of the immunomodulator in different reading frames or at least under the control of different promoters. Thereby, it is avoided that the analogue or epitopes are produced as fusion partners for the immunomodulator. Alternatively, two separate nucleotide fragments can be used as transformation agents. Of course, by having the first and/or second and/or third unit in the same reading frame, an analog according to the invention can be provided as an expression product, and such an embodiment is particularly preferred according to the present invention.

Application of the invention in disease treatment

As will be apparent from the above description, the provision of the invention allows the control of diseases characterized by amyloid deposits. In this context, AD is the main target of the inventive method, but other diseases characterized by Aβ-containing amyloid deposits are also possible targets. An important aim of the invention is consequently to treat and/or prevent and/or alleviate AD or other diseases characterized by amyloid deposition, including down-regulation of APP or Ap to such an extent that the amount of amyloid is significantly reduced.

It is particularly preferred that the reduction in amyloid results in an inversion in the balance between amyloid formation and amyloid degradation/removal, i.e. that the rate of amyloid degradation/removal is brought so that it exceeds the rate of amyloid formation. By carefully controlling the number and immunological impact of the immunization in the individual in need of such treatment, it will be possible to achieve a balance over time that results in a net reduction of amyloid deposits without excessive side effects.

Alternatively, if it is not possible to remove or reduce existing amyloid deposits in an individual, the invention enables a clinically significant reduction in the formation of new amyloid and thereby significantly extends the period of time during which the disease state is not debilitating. It should be possible to monitor the deposition rate of amyloid by either measuring the serum concentration of amyloid (which is assumed to be in equilibrium with deposited material) or by using positron emission tomography (PET) scanning, cf. Small GW, et al, 1996, Ann N Y Acad Sci 802: 70-78.

Other diseases and conditions in which the present agents and applications can be used in treatment or relief in a similar manner have been mentioned above in "background of the invention" or are indicated below in the section "other amyloidogenic diseases and proteins associated therewith".

Peptides, polypeptides and compositions according to the invention

As will be apparent from the above description, the present invention is based on the concept of immunizing individuals against APP or Aβ antigen to achieve a reduced amount of pathology-related amyloid deposits. The preferred way of achieving such immunization is to use the analogues described herein and thereby provide molecules that have not previously been described in the state of the art.

It is believed that the analogs discussed herein are inventive in themselves, and an important part of the invention therefore relates to an analog as described above. Any description presented herein therefore relates to modified APP or Ap which is relevant for the purpose of describing the amyloidogenic analogues according to the invention and any such description applies mutatis mutandis to the description of these analogues.

It should be noted that preferred modified APP or Aβ molecules include modifications that result in a polypeptide with a sequence identity of at least 70% to APP or Aβ, or with a subsequence thereof that is at least 10 amino acids in length. Higher sequence identities are preferred, for example at least 75% or even at least 80, 85, 90 or 95%. The sequence identity for the proteins and nucleic acids can be calculated as (Nref- Ndif)- 100/ Nref, where Ndifer is the total number of non-identical residues in the two sequences when these are combined and where Nrefers the number of residues in one of the sequences. The DNA sequence AGTCAGTC will therefore have a sequence identity of 75% with the sequence AATCAATC (Ndif= 2 and Nref= 8).

The invention also includes compositions which are useful for carrying out the method according to the invention. Accordingly, the invention also relates to an immunogenic composition comprising an immunogenically effective amount of an analogue as described above, said composition further comprising a pharmaceutical and immunogenically acceptable diluent and/or vehicle and/or carrier and/or excipient and optionally an adjuvant. In other words, this part of the invention concerns formulations of analogues, mainly as described above. The choice of adjuvants, carriers and vehicles is according to what is described above with reference to the formulation of modified and unmodified amyloidogenic polypeptide for use in the inventive method for down-regulation of APP or Ap.

The polypeptides are prepared according to methods well known to those skilled in the art. Longer polypeptides are normally produced by recombinant genetic engineering, including introducing a nucleic acid sequence encoding the analog into a suitable vector, transforming a suitable host cell with the vector, expressing the nucleic acid sequence by the host cell, recovering the expressed product from the host cells and their culture supernatant and subsequent purification and possibly further modification, for example refolding or derivatization.

Other peptides are preferably prepared using well-known techniques for solid or liquid phase peptide synthesis. However, recent advances in this technology have made it possible to produce full-length polypeptides and proteins using these agents, and these are therefore also within the scope of the present invention for the production of longer constructs using synthetic agents.

Nucleic acid fragments and vectors according to the invention

It should be understood from the above discussion that polyamino acid analogs can be produced by recombinant genetic engineering, but also by chemical synthesis or semisynthesis; the latter two options are particularly relevant when the modification consists of linking two protein carriers (such as KLH, diphtheria toxoid, tetanus toxoid and BSA) and non-proteinaceous molecules such as carbohydrate polymers and of course also when the modification includes the addition of side chains or side groups on an APP or Ap -derived peptide chain.

For the purpose of recombinant genetic engineering and of course also for the purpose of nucleic acid immunization, nucleic acid fragments encoding analogs are important chemical products. An important part of the invention therefore relates to a nucleic acid fragment that codes for an analogue according to the invention, that is, an APP- or Ap-derived polypeptide that either comprises the natural sequence to which a fusion partner has been added or inserted, or preferably an APP or Ap -derived polypeptide in which a foreign T-cell epitope has been inserted by means of insertion and/or addition, preferably by means of substitution and/or deletion. The nucleic acid fragments according to the invention are either DNA or RNA fragments.

The nucleic acid fragments according to the invention will normally be inserted into suitable vectors to form cloning or expression vectors that carry the nucleic acid fragments according to the invention; such new vectors are also part of the invention. Details regarding the construction of these vectors of the invention will be discussed in the context of transformed cells and microorganisms below. The vector can, depending on the purpose and type of application, be in the form of plasmids, phages, cosmids, minichromosomes or viruses, but also naked DNA which is only transiently expressed in cells as an important vector. Preferred cloning and expression vectors of the invention are capable of autonomous replication thereby enabling high copy numbers for high level expression or high level replication purposes for subsequent cloning.

The general outline of a vector according to the invention comprises the following features in the 5'->3' direction and is operatively linked to: A promoter to drive expression of the nucleic acid fragment according to the invention, optionally a nucleic acid sequence which codes for a leader peptide which enables secretion ( to the extracellular phase or, when applicable, into the periplasm) or integration of the polypeptide fragment into the membrane, the nucleic acid fragment according to the invention and optionally a nucleic acid sequence encoding a termination signal. When operating with expression vectors in production strains or cell lines, it is preferred for the purpose of genetic stability in the transformed cells that the vector, when introduced into a host cell, integrates into the host cell's genome. In contrast, it is preferred for safety reasons that the vector is capable of being integrated into the host cell's genome when working with vectors that are to effect in vivo expression in an animal (that is, when the vector is used in DNA vaccination); naked DNA or non-integrating virus vectors are typically used, and the choices are well known to those skilled in the art.

The vectors according to the invention are used to transform host cells to produce the analogue according to the invention. Such transformed cells, which are also part of the invention, can be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors according to the invention, or can be used for recombinant production of the analogues according to the invention. Alternatively, the transformed cells can be suitable live vaccine strains in which the nucleic acid fragment (a single or several copies) has been inserted so that secretion or integration of the analogue in the bacterial membrane or cell wall is effected.

Preferred transformed cells according to the invention are microorganisms such as bacteria (such as the species Escherichia (for example E. coli), Bacillus (for example Bacillus subtilis), Salmonella or Mycobacterium (preferably non-pathogenic, for example M. bovis BCG), yeast ( such as Saccharomyces cerevisiaé) and protozoans. Alternatively, the transformed cells are derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. More preferably, the cells are derived from a human, cf. the discussion of cell lines and vectors below. Newer results give hope for the use of a commercially available Drosophilia melanogaster cell line (Schneider 2 (S2) cell line and vector system available from Invitrogen) for the recombinant production of the polypeptides in the inventors' laboratory and this expression system is therefore particularly preferred.

For cloning and/or optimized expression purposes, it is preferred that the transformed cell is capable of replicating the nucleic acid fragment according to the invention. Cells expressing the nucleic acid fragment are preferred useful embodiments of the invention; they can be used for small-scale and large-scale production of the analogue according to the invention or, in the case of non-pathogenic bacteria, as a vaccine component in a live vaccine.

When the analog according to the invention is produced by means of transformed cells, it is appropriate, although far from essential, that the expression product is either exported into the culture medium or carried on the surface of the transformed cell.

When an efficient production cell has been identified, it is preferred on this basis to establish a stable cell line which carries the vector according to the invention and which expresses the nucleic acid fragment which codes for the modified amyloidogenic polypeptide. Preferably, the cell line secretes or carries the analogue according to the invention, thereby facilitating its purification.

In general, plasmid vectors containing replication origins and control sequences derived from species compatible with the host cells are used in conjunction with the hosts. The vector usually carries a site of replication as well as marker sequences capable of providing phenotypic selection in transformed cells. For example, E. coli is usually transformed using pBR322, a plasmid derived from an E. coli species (see, for example, Bolivar et al, 1977). The pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides a simple means of identifying transformed cells. The pBR plasmid or other microbial plasmids or phage must also contain or be modified so that they contain promoters that can be used by the prokaryotic microorganism for expression.

These promoters commonly used in recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al, 1978; Itakura et al, 1977; Goeddel et al, 1979) and a tryptophan (trp) promoter system (Goeddel et al, 1979; EP-A-0 036 776). While these are the most widely used, other microbial promoters have been discovered and exploited and details regarding their nucleotide sequences have been published, enabling those skilled in the art to functionally ligate them with plasmid vectors (Siebwenlist et al, 1980). Certain genes from prokaryotes can be efficiently expressed in E. coli from their own promoter sequences, which precludes the need for the addition of other promoters by artificial means.

In addition to prokaryotes, eukaryotic microbes such as yeast cultures can also be used, and here the promoter should be able to drive expression. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are widely available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al, 1979; Kingsman et al, 1979; Tschemper et al, 1980). This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan such as ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpl lesion as a feature of the yeast host cell genome thereby provides an efficient environment for detecting transformation upon growth in the absence of tryptophan.

Suitable promoter sequences in yeast vectors include the promoter for 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 and glucokinase. In constructing suitable expression plasmids, the termination sequence associated with these genes is also ligated into the expression vector's 3' end of the sequence desired for expression to provide polyadenylation of the mRNA and termination.

Other promoters that have the additional advantage of being transcriptionally controlled by growth conditions are the promoter region of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms can also be used as hosts. In principle, any such cell culture is applicable, regardless of whether it is from a vertebrate or non-vertebrate culture. However, interest has been greatest for vertebrate cells and the propagation of vertebrates in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973). Examples of such applicable host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines and W138, BHK, COS-7, 293, Spodoptera frugiperda (SF) cells (commercially available as complete expression systems from, among others, Protein Sciences, 1000 Research Parkway, Meriden, CT 06450, U.S.A. and from Invitrogen), and MDCK cell lines. In the present invention, a particularly preferred cell line is S2 which is available from Invitrogen, PO Box 2312, 9704 CH Groningen, The Netherlands. Expression vectors for such cells usually include (if necessary) an origin of replication, a promoter located upstream of the gene to be expressed together with all necessary ribosome binding sites, RNA splice sites, polyadenylation sites and transcription termination sequences.

For use in mammalian cells, the control functions on the expression vectors are often provided through viral material. For example, commonly used promoters are derived from polyoma, adenovirus 2 and most often simian virus 40 (SV40). SV40 virus early and late promoters are particularly useful because both are readily obtained from the virus as a fragment that also contains the SV40 virus origin of replication (Fiers et al, 1978). Smaller and larger SV40 fragments can also be used, provided they include the approximately 250 bp sequence that runs from the i/wcflll site toward the Bgll site located in the viral origin of replication. Furthermore, it is also possible and often desirable to utilize promoter or control sequences which are normally associated with the desired gene sequence, provided that such control sequences are compatible with the host cell systems.

An origin of replication may be provided either by engineering the vector to include an exogenous origin such as may be derived from SV40 or other viruses (eg polyoma, adeno, VS V, BP V) or may be provided through the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is usually sufficient.

Identification of applicable analogues

It will be clear to those skilled in the art that not all possible variants or modifications of naturally occurring APP or Ap will have the ability to elicit antibodies in an animal that are cross-reactive with the natural form. However, it is not difficult to set up an effective standard assay for modified amyloidogenic molecules that meet the minimum requirements for immunological reactivity as described herein. Accordingly, it is possible to utilize a method for identifying a modified amyloidogenic polypeptide which is capable of inducing antibodies against unmodified amyloidogenic polypeptide in an animal species where the unmodified amyloidogenic polypeptide is a (non-immunogenic) own protein, characterized in that the method comprises

- production by means of peptide synthesis or genetic engineering techniques of a set of mutually different analogues according to the invention, in which amino acids have been added, inserted into, deleted from or substituted into the amino acid sequence of an APP or Ap of an animal species and which thereby give rise to amino acid sequences in the set comprising T-cell epitopes foreign to the animal species, or prepare a set of nucleic acid fragments encoding the set of mutually different analogues, - test members of the set of analogues or nucleic acid fragments for their ability to induce the production of antibodies by the animal species against it unmodified APP or Ap, and - identify and optionally isolate the members of the set of analogues that significantly induce antibody production against unmodified APP or Ap in the species or to identify and optionally isolate the polypeptide expression product encoded by the members of the set of nucleic acid fragments that significantly induce antibody production oduction against unmodified APP or Ap in the animal species.

In this context, "set of mutually different modified amyloidogenic polypeptides" is a collection of non-identical analogs that have been selected, for example, due to the criteria discussed above (for example, in combination with studies of circular dichroism, NMR spectra and/or X-ray diffraction patterns ). The set may consist of only a few members, but it should be understood that sets may contain several hundred members.

The test of the members of the set can finally be performed in vivo, but a number of in vitro tests can be used to narrow down the number of modified molecules that will serve the purpose of the invention.

As the aim of introducing the foreign T-cell epitopes is to support the B-cell response by T-cell help, there is a caveat that T-cell proliferation is induced by the analogue. T-cell proliferation can be tested using standardized in vitro proliferation assays. Briefly, a sample enriched with T cells is obtained from an individual and then kept in culture. The cultured T cells are contacted with APCs from the individual that have previously taken up the modified molecule and processed it to present its T cell epitopes. The proliferation of the T cells is monitored and compared to a suitable control (eg T cells in culture contacted with APC that has processed intact native amyloidogenic polypeptide). Alternatively, proliferation can be measured by determining the concentration of relevant cytokines released by the T cells in response to their recognition of foreign T cells.

Since it is very likely that at least one analog of each type of the set is capable of inducing antibody production against APP or Aβ, it is possible to prepare an immunogenic composition comprising at least one analog capable of inducing antibodies against unmodified APP or Ap in an animal species where the unmodified APP or Ap is a separate protein, where the method comprises mixing the members of the kit that significantly induces the production of antibodies in the animal species that are reactive with APP or Ap with a pharmaceutically and immunogenically acceptable carrier and /or vehicle and/or diluent and/or excipient, possibly in combination with at least one pharmaceutically and immunogenically acceptable adjuvant.

The above-mentioned tests of the polypeptide sets are suitably performed by initially producing a number of mutually distinct nucleic acid sequences or vectors according to the invention, inserting these into suitable expression vectors, transforming suitable host cells (or host animals) with the vectors and effecting expression of the nucleic acid sequences according to the invention. These steps can be followed by isolating the expression products. It is preferred that the nucleic acid sequences and/or vectors are prepared using methods that include the use of a molecular amplification technique such as PCR or by means of nucleic acid synthesis.

Specific amyloidogenic targets

In addition to the proteins most commonly associated with Alzheimer's, APP, ApoE4 and Tau, there is a long list of other proteins that have somehow been linked to AD, either through their direct presence in plaques or tangles in AD brains or by their clear genetic association with an increased risk of developing AD. Most, if not all, of these antigens, along with the above discussed Aβ, APP, presenilin and ApoE4, are possible target proteins in certain embodiments of the present invention. These possible targets have already been thoroughly discussed in WO 01/62284. These possible targets will therefore only be briefly mentioned here, while a more careful background discussion can be found in WO 01/62282 which is hereby incorporated by reference herein: Alpha-antichymotrypsin (ACT); alpha2-macroglobulin; AB AD (Aβ peptide-binding alcohol dehydrogenase); APLP1 and -2 (amyloid precursor like protein 1 and -2); AMY117; Bax; Bcl-1; bleomycin hydrolase; BRI/ABRI; chromogranin A, clusterin/apoJ; CRF (corticotropin-releasing factor) binding protein; EDTF (endothelial derived toxin factor); heparan sulfate proteoglycans; human collapsin response mediator protein-2; huntingtin (Huntington's disease protein); ICAM-I; IL-6; lysosome-associated antigen CD68; P21 crash; PLC-delta 1 (phospholipase C isoenzyme delta 1); serum amyloid P component (SAP); synaptophysin; synuclein (alpha-synuclein or NACP); and TGF-bl (transforming growth factor bl).

The newly described agents and methods for down-regulating APP or Aβ can be combined with therapies, for example, active specific immunotherapy against any of these other amyloidogenic polypeptides.

Apart from Alzheimer's disease, cerebral amyloid angiopathy is also a disease that would be a suitable target for the technology described herein.

It is believed that most methods of immunization against APP or Aβ should be limited to immunization that gives rise to antibodies cross-reactive with the natural APP or Aβ. Nevertheless, in some cases it will be interesting to induce cellular immunity in the form of CTL responses against cells presenting MHC class I epitopes from amyloidogenic polypeptides - this may be useful in those cases in which reduction in the number of cells producing APP or Aβ does not constitute a serious side effect. In such cases where the CTL responses are desirable, it is preferred to utilize the teachings in the applicant's WO 00/20027. The descriptions of these two documents are hereby incorporated by reference herein.

IMMUNOGENIC CARRIERS

Molecules comprising a T-helper epitope and APP or Aβ peptides representing or including B-cell epitopes covalently bound to a non-immunogenic polymer molecule acting as a carrier, for example a multivalent activated polyhydroxy polymer, will, as mentioned above, act as a vaccine molecule which contain only the immunologically relevant parts and can be obtained and are interesting embodiments of the variants d and e described above. Promiscuous or so-called universal T-helper epitopes can be used if, for example, the target of the vaccine is a self-antigen such as APP or Ap. Furthermore, elements that enhance the immunological response can also be connected to the vehicle at the same time and thus act as an adjuvant. Such elements may be mannose, tuftsin, muramyl dipeptide, CpG motifs, etc. In that case, subsequent adjuvant formulation of the vaccine product may be unnecessary and the product may be administered in pure water or saline.

By linking cytotoxic T-cell (CTL) epitopes together with the T-helper epitopes, it will also be possible to generate CTL specific for the antigen from which the CTL epitope was derived. Elements that promote uptake of the product into the cytosol, such as mannose, into the APC, such as a macrophage, should also be linked to the vehicle along with the CTL and T helper epitope and enhance the CTL response.

The ratio of B-cell epitopes to T-helper epitopes (P2 and P30) in the final product can be varied by varying the concentration of these peptides in the synthesis step. As mentioned above, the immunogenic molecule can be labeled with, for example, mannose, tuftsin, CpG motifs or other immunostimulating substances (described herein) by adding these, if necessary by using, for example, aminated derivatives of the substances, to the carbonate buffer in the synthesis step.

If an insoluble activated polyhydroxy polymer is used to combine the peptides containing the APP or Ap B-cell epitope and the T-helper epitope, as mentioned above, it can be performed as a solid phase synthesis and the final product can be harvested and purified by washing and filtration. The elements to be linked to a tresyl-activated polyhydroxy polymer (peptides, labels, etc.) can be added to the polyhydroxy polymer at low pH, for example pH 4-5, and allowed to be evenly distributed in the "gel" by passive diffusion. The pH can then be increased to pH 9-10 to initiate reaction of the primary amino groups on the peptides and the tags of the triacyl groups on the polyhydroxy polymer. After linking the peptides and, for example, immunostimulating elements, the gel is ground to form particles of suitable size for immunization.

Such an immunogen therefore includes

a) at least one first amino acid sequence derived from APP or Ap, as indicated above, and b) at least one second amino acid sequence that includes a foreign T helper cell epitope,

wherein each of the at least first and at least second amino acid sequences is linked to a pharmaceutically acceptable activated polyhydroxy polymer carrier.

In order for the amino acid sequence to be linked to the polyhydroxy polymer, it is normally necessary to "activate" the polyhydroxy polymer with a suitable reactive group which can form the necessary bond to the amino acid sequence.

The term "polyhydroxy polymer" is intended to have the same meaning as in WO 00/05316, that is, the polyhydroxy polymer may have exactly the same properties as specifically taught in that application. The polyhydroxy polymer may therefore be water-soluble or insoluble (and thereby require different synthesis steps during the preparation of the immunogen). The polyhydroxy polymer can be selected from naturally occurring polyhydroxy compounds and synthetic polyhydroxy compounds.

Specific and preferred polyhydroxy polymers are polysaccharides selected from acetate, amylopectin, agar-agar gum, agarose, alginates, gum arabic, carrageenan, cellulose, cyclodextrins, dextran, furcellaran, galactomannan, gelatin, ghatti, glucan, glycogen, guar, karaya, konjac /A, locust bean gum, mannan, pectin, psyllium, pullulan, starch, tamarind, tragacanth, xanthan, xylan and xyloglucan. Dextran is particularly preferred.

However, the polyhydroxy polymer can also be selected from highly branched poly(ethyleneimine) (PEI), tetrathienylenevinylene, Kevlar (long chains of polyparaphenylterephthalamide), poly(urethanes), poly(siloxanes), polydimethylsiloxane, silicone, poly(methyl methacrylate) (PMMA), poly( vinyl alcohol), poly(N-vinyl pyrrolidone), poly(2-hydroxyethyl methacrylate), poly(N-vinyl pyrrolidone), poly(vinyl alcohol), poly(acrylic acid), polytetrafluoroethylene (PTFE), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol) and derivatives, poly(methacrylic acid), polylactic acids (PLA), polyglycolides (PGA), poly(lactic co-glycolides) (PLGA), polyanhydrides and polyorthoesters.

The average molecular weight of the polyhydroxy polymer of interest (ie before activation) is usually at least 1000, such as at least 2000, preferably in the range of 2500-200000, more preferably in the range of 3000-1000000, especially in the range of 5000-500000. It has been shown in the examples that polyhydroxy polymers with an average molecular weight in the range of 10,000-200,000 are particularly advantageous.

The polyhydroxy polymer is preferably water-soluble to an extent of 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. It is known that dextran, even when activated as described herein, satisfies the requirements with regard to water solubility.

For some of the most interesting polyhydroxy polymers, the ratio of C-(carbon atoms) to OH groups (hydroxy groups) of the unactivated polyhydroxy polymers (that is, the natural polyhydroxy polymers before activation) is in the range of 1.3 to 2.5, such as 1, 4-2.3, preferably 1.6-2.1, especially 1.85-2.05. Without being bound by any specific theory, it is believed that such a C/OH ratio of the unactivated polyhydroxy polymers represents a highly advantageous level of hydrophilicity. Polyvinyl alcohol and polysaccharides are examples of polyhydroxy polymers that satisfy this requirement. It is believed that the above ratio should be about the same for the activated polyhydroxy polymer, as the activation ratio should be rather low.

The term "polyhydroxypolymer carrier" is intended to denote the portion of the immunogen that carries the amino acid sequences. As a general rule has

the polyhydroxy polymer carrier an outer boundary where the amino acid sequences can be cleaved by a peptidase, for example in an antigen-presenting cell that processes the immunogen. The polyhydroxy polymer carrier can therefore be the polyhydroxy polymer with an activation group where the bond between

the activation group and the amino acid sequence can be cleaved using a peptidase in an APC, or the polyhydroxy polymer carrier can be a polyhydroxy polymer with an activation group and, for example, a linker such as a single L-amino acid or a number of D-amino acids, where the last part of the linker can be attached to the amino acid sequence and is cleaved by a peptidase in an APC.

As mentioned above, the polyhydroxy polymers carry functional groups (activating groups) that facilitate the anchoring of the peptides to the support. Several possible functional groups are known in the art, for example tresyl (trifluoroethylsulfonyl), maleimide, p-nitrophenyl chloroformate, cyanogen bromide, tosyl (p-toluenesulfonyl), triflyl (trifluoromethanesulfonyl), pentafluorobenzenesulfonyl and vinylsulfone groups. Preferred examples of functional groups within the present invention are tresyl, maleimide, tosyl, triflyl, pentafluorobenzenesulfonyl, p-nitrophenylchloroformate and vinyl sulfone groups, among which tresyl, maleimide and tosyl groups are particularly relevant.

Tresyl-activated polyhydroxy polymers can be prepared using tresyl chloride as described for activation of dextran in Example 1 in WO 00/05316 or as described in Gregorius et al., J. Immunol. Meth. 181 (1995) 65-73.

Maleimide-activated polyhydroxy polymers can be prepared using p-maleimide phenyl isocyanate as described for the activation of dextran in example 3 of WO 00/05316. Alternatively, maleimide groups can be introduced into a polyhydroxy polymer such as dextran by derivatization of a tresyl-activated polyhydroxy polymer (such as tresyl-activated dextran (TAD)) with a diamine compound (generally H2N-CnH2n-NH2, where n is 1-20, preferably 1-8) , for example 1,3-diaminopropane, in excess and then react the amino groups introduced in TAD with reagents such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB), N-y-maleimidobutyryloxysuccinimide ester (GMBS) or N-y- maleimidobutyryloxysulfosuccinimide esters. Although the different reagents and routes of activation formally result in somewhat different maleimide-activated products with respect to the bond between the maleimide functionality and the remainder of the parent hydroxy group upon which the activation is performed, all are considered as "maleimide-activated polyhydroxy polymers".

Tosyl-activated polyhydroxy polymers can be prepared using tosyl chloride as described for the activation of dextran in example 2 in WO 00/05316. Triflyl- and pentafluorobenzenesulfonyl-activated polyhydroxy polymers are prepared as tosyl- or tresyl-activated analogues, for example by using the corresponding acid chlorides.

Cyanogen bromide activated polyhydroxy polymers can be prepared by reacting the polyhydroxy polymer with cyanogen bromide using conventional methods. The resulting functional groups are normally cyanate esters with two hydroxy groups on the polyhydroxy polymer.

The degree of activation can be expressed as the ratio between the free hydroxy groups and the activation groups (that is, functionalized hydroxy groups). It is believed that a ratio between the free hydroxy groups of the polyhydroxy polymer and the activated groups should be between 250:1 and 4:1 to achieve a favorable balance between the hydrophilicity and reactivity of the polyhydroxy polymer. Preferably the ratio is between 11:1 and 6:1, more preferably between 60:1 and 8:1, especially between 40:1 and 10:1.

Particularly interesting activated polyhydroxy polymers for use in the method for producing the generally applicable immunogen according to the invention are tresyl-, tosyl- and maleimide-activated polysaccharides, especially tresyl-activated dextran (TAD), tosyl-activated dextran (TosAD) and maleimide-activated dextran (MAD).

It is preferred that the bond between the polyhydroxy polymer carrier and the amino acid sequences bound thereto be cleaved by means of a peptidase, for example a peptidase which is active in the processing of antigens in an APC. It is therefore preferred that at least the first and at least the second amino acid sequence is connected to the activated polyhydroxy polymer carrier via an amide bond or a peptide bond. It is particularly preferred that the at least first and the at least second amino acid sequence each provide the nitrogen unit to their respective amide bond.

The polyhydroxy polymer carrier can be substantially free of amino acid residues, which necessitate that the activation group provides part of a peptidase-cleaving bond, but as mentioned above, the carrier can also easily include a spacer that includes at least one L-amino acid. Nevertheless, at least the first and at least the second amino acid sequence are normally attached to the activated version of the polyhydroxy polymer via the nitrogen at the N-terminus of the amino acid sequence.

The above-mentioned generally used immunogen according to the present invention can be used in immunization methods essentially as described herein for polypeptide vaccines. That is to say, all the descriptions regarding dosages, methods of administration and formulation of polypeptide vaccines for down-regulation of amyloidogenic polypeptides described herein apply mutatis mutandis to the generally applicable immunogens.

GENERALLY APPLICABLE SAFE VACCINATION TECHNOLOGY

As discussed above, a preferred embodiment of the present invention involves the use of variants of amyloidogenic polypeptides that are unable to provide self-derived Tn epitopes capable of driving an immune response against the amyloidogenic polypeptide.

However, the present inventors believe that this strategy for designing anti-self vaccines and for effecting anti-self immunity is a generally applicable technology that is inventive in itself. It should be particularly suitable in cases where the self-antigen that is sought to be down-regulated is present in sufficient quantities in the body so that it is possible for the self-stimulation of an immune response to occur. Accordingly, all descriptions above of this embodiment regarding the acquisition of an anti-self immune response against APP or Aβ apply mutatis mutandis to immunization against other self-polypeptides, especially those present in sufficient quantities to maintain the immune response in the form of an uncontrolled autoimmune condition due to autologous Tn epitopes of the relevant self-polypeptide that derives the immune response.<*>

COMPARISON EXAMPLE 1

Autovaccination approach for immunization against AD

The fact that Aβ protein "knock out" mice show no abnormalities or side effects suggests that removing or lowering Aβ levels is safe, Zheng H.

(1996).

Published experiments in which transgenic animals are immunized against the transgenic human Aβ protein suggest that if it were possible to break self-tolerance, the downregulation of Aβ could be achieved by means of autoreactive antibodies. These experiments further suggest that such a downregulation of Ap would potentially both prevent the formation of plaques and even remove formed Ap plaques from the brain, cf. Schenk et al. (1999). Traditionally, however, it is not possible to elicit antibodies against self-proteins.

Accordingly, the published data do not provide the means for breaking down true self-tolerance against true self-proteins. Nor does the data provide information on how to ensure that the immune reaction is exclusively or mainly directed against Aβ deposits and not against the cell membrane-bound Aβ precursor protein APP) if this appears to be necessary. An immune response that is formed using existing technology would presumably form an immune response against self-proteins in an unregulated manner so that unwanted and exaggerated autoreactivity against parts of the Aβ protein can be formed. Consequently, the application of existing immunization strategies will probably not be able to generate strong immune responses against self-proteins and will further be uncertain due to possible strong cross-reactivity against membrane-bound APP that is present on a greater number of cells in the CNS.

The present invention provides the means for the efficient formation of a

highly regulated immune response against true self-proteins that could possibly form plaques and cause severe disease in the CNS or in other parts of the body. A safe and effective human Aβ protein therapeutic vaccine will be developed using this technology for the treatment of AD.

In light of this, it is possible to count that AD, a disease that is expected to destroy the healthcare system in the next century can be cured, or that such described vaccines at least constitute an effective therapeutic approach for treating the symptoms and the development of this the disease. This technique represents a completely new immunological approach to block amyloid deposition in AD, as well as other neurological diseases.

In the following table, 35 considered constructs are indicated. All the positions given in

the table is relative to the methionine start in APP (first amino acid in SEQ ID NO: 2) and includes both start and end amino acids, for example the 672-714 fragment includes both amino acids 672 and 714. The start and end positions for P2 and P30 indicates that the epitope substitutes part of the APP fragment at the indicated positions (both positions are included in the substitution) - in most constructs the introduced epitope substitutes a fragment of the length of the epitope. The stars in the table have the following meaning:

<*>) Only one position for P2 and P30 indicates that the epitope has been inserted into the APP derivative at the indicated position (the epitope begins at the C-terminal amino acid immediately adjacent to the given position).<**>) Construction 34 contains three identical APP fragments separated by P30 and P2, respectively.<***>) Construct 35 contains nine identical APP fragments separated by changing the P30 and P2 epitopes.

The part of APP against which it is most interesting to form a response is the 43 amino acid long Aβ core peptide (Aβ-43 corresponding to SEQ ID NO: 2, residues 672-714) which is the main component of amyloid plaques in AD - brains. This APP fragment is part of all the constructs indicated above.

Variants 1 and 2 comprise a part of APP upstream of Ap-43 where the model epitopes P2 and P30 have been placed. Variants 1 and 3-8 all include the C-100 fragment that has been shown to be neurotoxic - the C-100 fragment corresponds to amino acid residues 714-770 of SEQ ID NO: NO. 2.1 variants 3-5 the epitopes substitute part of the C-100 fragment, while the epitope in variants 6-8 has been inserted into C-100.

Variants 9-35 contain only the Ap-43 core protein. In variants 9-13, P2 and P30 are fused to either the Ap-43 end; in 14-21, P2 and P30 substitute parts of Ap-43; in 22-33, P2 and P30 are inserted into Ap-43; 34 contains three identical Ap-43 fragments separated by P30 and P2, respectively; 35 contains 9 Ap-43 repeats at alternating P2 and P30 epitopes.

Truncated parts of the above-mentioned Ap-43 protein can also be used in immunogenic analogues according to the present invention. Particularly preferred are the truncations Ap(l-42), Ap(l-40), Ap(l-39), Ap(l-35), Ap(l-34), Ap(l-28), Ap(l- 12), Ap(l-5), Ap(13-28), Ap(13-35), Ap(17-28), Ap(25-35), Ap(35-40), Ap(36-42 ), and Ap(35-42) (where the number in brackets indicates the amino acid stretches of Ap-43 that make up the relevant fragment - Ap(35-40) is, for example, identical to amino acids 706-711 in SEQ ID NO. 2). All of these truncated variants of Ap-43 can be prepared with the Ap fragments described herein, particularly with variants 9, 10, 11, 12 and 13.

In some cases, it is preferred that Ap-43 or fragments thereof are mutated. Particularly preferred are substitution variants where methionine position 35 in Ap-43 is substituted, preferably with leucine or isoleucine, or simply deleted. Particularly preferred analogs contain a single methionine located at the C-terminus, either because it is naturally occurring in the amyloidogenic polypeptide or the foreign Tn epitope, or because it has been inserted or added. Accordingly, it is also preferred that the portion of the analog that includes the foreign Th epitope be free of methionine, except for the possible C-terminal localization of a methionine.

In fact, it is usually preferred that all the analogs of APP or Aβ used according to the present invention have in common that they include only a single methionine positioned as the C-terminal amino acid of the analog and that other methionines in either the amyloidogenic polypeptide or the the foreign Th epitope is deleted or substituted with another amino acid.

A further interesting mutation is a deletion or substitution of the phenylalanine in position 19 of Ap-43, and it is particularly preferred that the mutation is a substitution of this phenylalanine residue with a proline.

The following table shows a group of particularly preferred constructs that operate with truncations or mutations in Ap-43:

In this table, the Ap segment used in the molecule is indicated by amino acid number in relation to aa 1 in the Ap(1-42/43) molecule, i.e. 1-28 means that fragment 1-28 of Ap( 1- 42/43) are used in the molecule. If two or more different segments are used, both are indicated in the table, i.e. 1-12 (a) + 13-28 (b) means that both fragment 1-12 and fragment 13-28 of Ap(l-42/ 43) is used in the molecule.

If the same segment is present in more than one copy in the construct, it is also indicated in the table, i.e. 1-13 (x3) shows that the fragment 1-12 of Ap(1-42/43) is present in three copies in the construction.

The position of the Aβ segment in the molecule is further shown by the amino acid positions in relation to the first amino acid of the molecule, i.e. 22-49 shows that the Aβ fragment in question is positioned from amino acid 22 to amino acid 49 in the molecule where both positions is included. The P2 and P30 epitope positions are indicated similarly. If two or more different Ap fragments are used in the molecule, all their positions are shown, i.e. 1 -12 (a) + 49-64 (b) means that fragment (a) is positioned from aa 1-12 in the molecule and fragment (b) from aa 49-64. If more than one copy of the same fragment is present in the molecule, the positions for all copies are shown, i.e. 1-12, 34-45, 61-72 show that the three copies of the Ap fragment are located from respectively positions 1-12, 34-45 and 61-72 in the molecule.

Finally, the total length indication for each molecule includes both the Aβ fragment or fragments and the P2 and P30 epitopes.

Variant 42 contains two amino acid substitutions at positions 19 (phe to pro) and 35 (met to lys) as indicated in the column showing the Aβ fragments.

See Figure 1 and the tables above for details on specific points of introduction of the foreign T-cell epitopes.

A further type of construct is particularly preferred. As one aim of the present invention is to avoid destruction of the cells producing APP while removal of Aβ is desired, it seems practical to prepare autovaccine constructs comprising only Aβ moieties that are not exposed to the extracellular phase when presented in APP. Such constructs will consequently need to contain at least one B-cell epitope derived from the amino acid fragment defined at amino acids 700-714 in SEQ ID NO. NO. 2. As such a short polypeptide fragment is believed to be only weakly immunogenic, it is preferred that such an autovaccine construct contains multiple copies of the B-cell epitope, for example in the form of a construct having the structure as shown in formula I in the detailed description of the present invention, see above. In this version of Formula I, the terms amyloidei-amyloidexx B-cell epitope-containing amino acid sequences are derived from amino acids 700-714 of SEQ ID NO. NO. 2. A preferred alternative is the above-described possibility of linking the amyloidogenic (poly)peptide and the selected foreign T-helper epitope via an amide bond to a polysaccharide carrier molecule - in this way multiple presentations of the "weak" epitope constituted by the amino acids are enabled 700-714 in SEQ ID NO. NO. 2, and it will also be possible to select an optimal ratio between B-cell and T-cell epitopes.

COMPARISON EXAMPLE 2

Immunization of transgenic mice with A/ 3 and modified proteins Construction of hAB43+-34-encoding DNA. The hAB43+-34 gene was constructed in several steps. First, a PCR fragment was generated with primers ME#801 (SEQ ID NO: 10) and ME#802 (SEQ ID NO: 11) using ME#800 (SEQ ID NO: 9) as a template . ME#800 encodes the human abeta-43 fragment with E. coli optimized codons. ME#801 and -802 add appropriate restriction sites to the fragment. The PCR fragment was purified, digested with NcoI and HindIII, purified again and cloned into the NcoI-HindIII-digested and purified pET28b+ E. coli expression vector. The resulting plasmid encoding wild-type human Ap-43 is called pAB1.

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

The cloning was performed by making a PCR fragment with the primers ME#178 (SEQ ID NO: 8) and ME#806 using pAB1 as template. The fragment was purified, digested with NcoI and HindIII, purified again and cloned into an NcoI-HindIII-digested and purified pET28b+ vector. The resulting plasmid was named pAB2.

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

The fragment was purified, digested with NcoI and HindIII, purified again and cloned into an NcoI-HindIII-digested and purified pET28b+ vector. The resulting plasmid was named pAB3.

In the third step, a second Ap-43 repeat portion was added C-terminally to the P2 epitope in plasmid pAB2 using primer ME#809 (SEQ ID NO: 14). ME#809 also forms a 2?amHI site immediately after the Ap-43 repeat unit. A PCR fragment was then generated with primers ME#178 and ME#809 using pAB2 as template. The fragment was purified, digested with NcoI and HindIII, purified again and cloned into an NcoI-HindIII-digested and purified pET28b+ vector. The resulting plasmid was named pAB4.

Finally, the P30 epitope - Ap-43 repeat sequence from pAB3 was cloned into pAB4 plasmid. This was done by making a PCR fragment with primers ME#811 (SEQ ID NO: 16) and ME#105 using pAB3 as template. The fragment was purified and used as primer in a subsequent PCR with ME#810 (SEQ ID NO: 15) using pAB3 as template. The resulting fragment was purified, digested with BamHI and HindIII, purified again and cloned into a BamHI-HindIII 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 edition. Ed. Cold Spring Harbor Laboratory, N.Y.

For all cloning procedures, E. coli K-12 cells, strain top-10 F'

(Stratagene, USA) applied. 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 BL21-Gold (Novagen) E. cø/z cells as described by the supplier of the pET28b+ system (Novagen).

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

Immunization study. Mice that were transgenic for human APP (Alzheimer's precursor protein) were used in the study. These mice, called TgRND8+, express a mutated form of APP that results in high concentrations of Ap-40 and Ap-42 in the mice's brains (Janus, C. et al).

The mice (8-10 mice per group) were immunized with either Ap-42 (SEQ ID NO: 2, residues 673-714, synthesized using a standard Fmoc strategy) or the hAB43+-34 variant (construct 34 in the table in Example 1, recombinantly prepared) four times at 2 week intervals. The doses were either 100 mg for Ap or 50 mg for hAB43+-34. The mice were bled at day 43 (after three injections) and after 52 (after four injections) and sera were used to determine the level of anti-Ap42 specific titers using a direct Ap-42 ELISA.

The following table shows mean relative anti-Ap-42 titers.

It is clear that the antibody titers obtained by immunization with the hAB43+-34 Ap variant are approximately 4-fold and 7.5-fold higher after 3 and 4 immunizations, respectively, than the titers obtained using the unmodified wild-type Ap-42 as an immunogen. This fact is put into further perspective when one considers that the amount of variant used for immunization was only 50% of the amount used of the wild-type sequence for immunization.

COMPARISON EXAMPLE 3

Synthesis of an A fi peptide copolymer vaccine using activated polyhydroxy polymer as cross-linking agent.

Introduction. A traditional conjugate vaccine consists of a (poly)peptide covalently linked to a carrier protein. The peptide contains the B cell epitope(s) and the carrier protein provides T helper epitopes. However, most of the carrier proteins would normally be irrelevant as a source of T-helper epitopes, as only a minor portion of the total sequence contains the relevant T-helper epitopes. Such epitopes can be defined and synthesized as peptides of, for example, 12-15 amino acids. If these peptides are covalently bound to the peptides containing the B-cell epitopes, for example via a multivalent activated polyhydroxy polymer, a vaccine molecule containing only the relevant parts can be obtained. It is further possible to provide a vaccine conjugate containing an optimal ratio between B-cell and T-cell epitopes.

Synthesis of the activated polyhydroxy polymer. Polyhydroxy polymers such as dextran, starch, agarose, etc. can be activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride), either by means of a homogeneous synthesis (dextran) dissolved in N-methylpyrrolidinone (NMP) or by means of a heterogeneous synthesis (starch, agarose, cross-linked dextran) in, for example, acetone.

225 mL of dry N-methylpyrrolidone (NMP) is added under dry conditions to lyophilized, water-soluble dextran (4.5 g, 83 mmol, clinical grade, molecular weight (average) 78000) in a 500 round bottom flask fitted with a magnet for stirring. The flask is placed in a 60°C oil bath with magnetic stirring. The temperature was increased to 92°C over a period of 20 minutes. When the dextran was dissolved, the flask was immediately removed from the oil bath and the temperature in the bath was lowered to 40°C. The flask was again placed in the oil bath, still with magnetic stirring, and trichloride (2.764 mL, 25 mmol) was added dropwise. After 15 minutes, dry pyridine (anhydrous, 2.020 mL, 25 mmol) was added dropwise. The flask was removed from the oil bath and stirred for 1 hour at room temperature. The product (tresyl-activated dextran, TAD) was precipitated in 1200 ml of cold ethanol (99.9%). The supernatant was poured off and the precipitate was collected in 50 ml polypropylene tubes in a centrifuge at 2000 rpm. The precipitate was dissolved in 50 ml of 0.5% acetic acid, dialyzed twice against 5000 ml of 0.5% acetic acid and freeze-dried. TAD can be stored as a freeze-dried product at -20°C.

An insoluble polyhydroxy polymer such as agarose or cross-linked dextran can be tresyl-activated by forming a suspension of the polyhydroxy polymer in, for example, acetone and carrying out the synthesis as a solid phase synthesis. The activated polyhydroxy polymer can be harvested by filtration. Suitable methods are reported in, for example, Nilsson K and Mosbach K (1987), Methods in Enzymology 135, page 67 and in Hermansson GT et al. (1992), in "Immobilized Affinity Ligand Techniques", Academic Press, Inc., page 87.

Synthesis of AP-peptide copolymer vaccines. TAD (10 mg) was dissolved in 100 µl H 2 O and 1000 µl carbonate buffer, pH 9.6, containing 5 mg Ap-42 (SEQ ID NO: 2, residues 673-714), 2.5 mg P 2 (SEQ ID NO: 2, residues 673-714). ID NO 4) and 2.5 mg of P30 (SEQ ID NO 6) were added. The Ap-42 and P2 and P30 peptides all contained protected lysine groups; these were in the form of 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protected lysine groups. The peptides were prepared using a standard Fmoc strategy where the conventional Fmoc-Lys(Boc)-OH was substituted with Fmoc-Lys(Dde)-OH (obtained from Novabiochem, catalog no. 04-12-11121), that is say that the e-amino acid group in lysine is protected with Dde instead of Boe.

The pH was measured and adjusted to 9.6 using 1 M HCl. After 2.5 hours at room temperature, hydrazine from an 80% solution was added to a final hydrazine concentration of 8% and the solution was incubated for an additional 30 minutes at room temperature and lyophilized immediately thereafter. The freeze-dried product was dissolved in H2O and thoroughly dialyzed against H2O before final freeze-drying.

The ratio of the B-cell epitopes (Ap) to the T-helper epitopes (P2 and P30) in the final product can be varied by using different concentrations of these peptides in the synthesis step. The final product can be labeled with, for example, mannose (so as to direct the conjugate to APC) by adding aminated mannose to the carbonate buffer in the synthesis step.

If an insoluble activated polyhydroxy polymer is used to combine the peptides containing the B cell epitope and the T helper epitopes, the coupling of the polymer can be performed as a solid phase synthesis and the final product harvested and purified by washing and filtration.

As mentioned in the general description, the present described approach for producing a peptide-based vaccine can be applied to any other polypeptide antigen where it is expedient to produce a pure synthetic peptide vaccine and where the polypeptide antigen of interest provides sufficient immunogenicity in a single peptide.

COMPARISON EXAMPLE 4

Synthesis of peptide copolymer vaccines

TAD (10 mg) was dissolved in 100 µl H2O and 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 of P30 (diphtheria toxoid P30 epitope) were added. The pH was measured and adjusted to 9.6 using 0.1 M HCl.

After 2.5 hours at room temperature, the solution was freeze-dried immediately thereafter. The lyophilized product was dissolved in H2O and thoroughly dialyzed against H2O or desalted on a gel filtration column prior to final lyophilization. In cases where the peptides have lysine in the sequence, the ε-amine in the lysine side chain should be protected by means of Dde using Fmoc-Lys(Dde)-OH derivative in the synthesis (Gregorius and Theisen 2001, submitted). After coupling, hydrazine from an 80% solution was added to a final hydrazine concentration of between 1 and 20% and the solution was incubated for an additional 30 min at room temperature, freeze-dried immediately thereafter and dialyzed thoroughly against H2O or desalted on a gel filtration column prior to final freeze-drying. The principle is described schematically in Figure 2.

Such immunogens have been exploited by the inventors with a short C-terminal fragment of Borrelia burgdorferi protein OspC as "peptide A" and a diphtheria toxoid epitope (P2 or P30) as a peptide B. The results of the immunization studies with this antigen revealed that only the immunogen of the invention including the OspC fragment and a foreign diphtheria epitope matching the MHC haplotype in the vaccinated mice were able to induce antibodies reactive with OspC in these mice. In contrast, a molecule containing only OspC was unable to induce antibody production, as was a mixture of two immunogens, one containing OspC and the other containing the epitope. It is therefore concluded that insertion into the same polyhydroxy polymer carrier is superior, if not essential, for inducing antibody production against a shorter peptide hapten such as OspC.

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Claims (24)

1. Pharmaceutical composition containing an immunogen that induces the production of antibody against the animal's autologous APP or Ap, in which the immunogen incorporates a) a polyamino acid consisting of a polyamino acid selected from the group consisting of: - amino acid residues 1-12, which is a subsequence of residues 672 -714 of SEQ.ID. NO. 2, Ap, followed by the amino acid residues of the tetanus toxoid P30 epitope, followed by the amino acid residues of the tetanus toxoid P2 epitope, followed by amino acid residues 13-28, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2 Ap, - amino acid residues 1-12, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, followed by the amino acid residues of the tetanus toxoid P30 epitope, followed by amino acid residues 1-12, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, followed by the amino acid residues of the tetanus toxoid P2 epitope, followed by amino acid residues 1-12, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, - amino acid residues 13-28, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, followed by the amino acid residues of the tetanus toxoid P30 epitope, followed by amino acid residues 13-28, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, followed by the amino acid residues of the tetanus toxoid P2 epitope, followed by amino acid residues 13-28, which is a subsequence of residues 672-714 of SEQ ID NO. NO. 2, Ap, in which all the amino acid sequences are indicated in the direction from the N- to the C-terminal end, or b) is a conjugate comprising a polyhydroxy polymer backbone to which a polyamino acid as defined in a) is separately attached; or c) is a nucleic acid encoding the polyamino acid as defined in a); or d) is a non-pathogenic microorganism or virus carrying a nucleic acid fragment that encodes and expresses the polyamino acid as defined in a), for use in the treatment, prevention or alleviation of Alzheimer's disease or other diseases characterized by amyloid deposition. 2. Pharmaceutical composition according to claim 1, wherein at least two copies of the polyamino acid or conjugate are covalently or non-covalently bound to a carrier molecule capable of effecting the presentation of multiple copies of antigenic determinants. 3. Pharmaceutical composition according to claim 1 or 2, variant b), in which the polyamino acid is bound to the polyhydroxy polymer by means of an amide bond. 4. Pharmaceutical composition according to any one of the preceding claims, variant b), in which the polyhydroxy polymer is a polysaccharide. 5. A pharmaceutical composition according to any one of the preceding claims, wherein the polyamino acid or conjugate is formulated with an adjuvant that facilitates the breaking of autotolerance against autoantigens. 6. A pharmaceutical composition according to any one of the preceding claims, wherein the pharmaceutical composition is adapted to administer an effective amount of the polyamino acid or conjugate to the animal via a route selected from the parenteral route such as the intracutaneous, the subcutaneous and the intramuscular route ; the peritoneal route; the oral route; the buccal route; the sublingual route; the epidural route; the spinal route; the anal route; and the intracranial route. 7. Pharmaceutical composition according to claim 6, wherein the effective amount is between 0.5 µg and 2000 µg of the polyamino acid or conjugate. 8. Pharmaceutical composition according to any one of claims 1-7, variant c) adapted for introducing the nucleic acid or nucleic acids encoding the polyamino acid or conjugate into the animal's cells and thereby achieving in vivo expression of the introduced nucleic acid or acids with the help of the cells . 9. Pharmaceutical composition according to claim 8, wherein the introduced nucleic acid(s) is selected from naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA included in a viral vector, DNA formulated with a transfection-deleting protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with calcium precipitating agents, DNA linked to an inert carrier molecule, DNA encapsulated in chitin or chitosan, and DNA formulated with an adjuvant. 10. Pharmaceutical composition according to any one of claims 6-9, wherein the composition is adapted for at least one administration/introduction per year, such as at least 2, at least 3, at least 4, at least 6 and at least 12 administrations/introductions. 11. Pharmaceutical composition according to any one of the preceding claims, wherein the pharmaceutical composition down-regulates APP or Ap to such an extent that the total amount of amyloid is reduced or that the rate of amyloid formation is reduced with clinical significance. 12. Polyamino acid or conjugate which is derived from an APP or Ap from animals, in which a modification has been introduced into the polyamino acid or conjugate which results in the immunization of the animal with the polyamino acid or conjugate inducing the production of antibodies against the animal's autologous APP or Ap, and wherein the polyamino acid is as defined in any of claims 1-4. 13. Immunogenic composition comprising an immunogenically effective amount of a polyamino acid or conjugate according to claim 12, wherein the composition further comprises a pharmaceutically and immunologically acceptable carrier and/or vehicle and optionally an adjuvant, in which the adjuvant can be alum. 14. Nucleic acid fragment, in which it codes for a polyamino acid according to claim 12. 15. Vector comprising the nucleic acid fragment according to claim 14, such as a vector that can replicate autonomously. 16. Vector according to claim 15, wherein it is selected from the group consisting of a plasmid, a phage, a cosmid, a minichromosome and a virus. 17. Vector according to claim 15 or 16, in which it comprises, in the 5'-»3' direction and operably linked, a promoter to drive the expression of the nucleic acid fragment according to claim 14, optionally a nucleic acid sequence which codes for a leader peptide capable of to secrete the polypeptide fragment or to integrate the polypeptide fragment into the membrane, the nucleic acid fragment according to claim 14, and optionally a terminator. 18. A vector according to any one of claims 15-17, which when introduced into a host cell is capable or incapable of integrating into the genome of the host cell. 19. Vector according to claim 17 or 18, in which the promoter drives the expression in a eukaryotic cell and/or in a prokaryotic cell. 20. A transformed cell, wherein it comprises the vector of any one of claims 15-19, such as a transformed cell capable of replicating the nucleic acid fragment of claim 14. 21. Transformed cell according to claim 20, wherein the microorganism is selected from a bacterium, a yeast, a protozoan or a cell derived from a multicellular organism selected from a fungus, an insect cell such as an S2 or an SF cell, a plant cell and a mammalian cell. 22. Transformed cell according to claim 20 or 21, wherein the cell expresses the nucleic acid fragment according to claim 14, such as a transformed cell which secretes or carries the analog according to claim 12 on its surface. 23. Pharmaceutical composition according to claim 1, variant d), in which the immunogen is adapted for the administration of a non-pathogenic microorganism or virus carrying a nucleic acid fragment that codes for and expresses the polyamino acid. 24. Composition for inducing the production of antibodies against amyloid, wherein the composition comprises a nucleic acid fragment according to claim 14 or a vector according to which preferably of claims 15-19, and a pharmaceutically and immunologically acceptable carrier and/or vehicle and/or adjuvant.