US20030157117A1 - Novel method for down-regulation of amyloid - Google Patents

Novel method for down-regulation of amyloid Download PDF

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US20030157117A1
US20030157117A1 US10/223,809 US22380902A US2003157117A1 US 20030157117 A1 US20030157117 A1 US 20030157117A1 US 22380902 A US22380902 A US 22380902A US 2003157117 A1 US2003157117 A1 US 2003157117A1
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cell
app
analogue
epitope
residues
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Peter Rasmussen
Martin Jensen
Klaus Nielsen
Peter Koefoed
Florence Degan
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Definitions

  • the present invention relates to improvements in therapy and prevention of Alzheimer's disease (AD) and other diseases characterized by deposition of amyloid, e.g. characterized by amyloid deposits in the central nervous system (CNS). More specifically, the present invention provides a method for down-regulating (undesired) deposits of amyloid by enabling the production of antibodies against a relevant protein (APP or A ⁇ ) or components thereof in subjects suffering from or in danger of suffering from diseases having a pathology involving amyloid deposition. The invention also provides for methods of producing polypeptides useful in this method as well as for the modified polypeptides as such.
  • AD Alzheimer's disease
  • CNS central nervous system
  • nucleic acid fragments encoding the modified polypeptides as well as vectors incorporating these nucleic acid fragments and host cells and cell lines transformed therewith.
  • the present invention also provides for a new type of conjugate peptide immunogen.
  • Amyloidosis is the extracellular deposition of insoluble protein fibrils leading to tissue damage and disease (Pepys, 1996; Tan et al., 1995; Kelly, 1996). The fibrils form when normally soluble proteins and peptides self-associate in an abnormal manner (Kelly, 1997).
  • Amyloid is associated with serious diseases including systemic amyloidosis, AD, maturity onset diabetes, Parkinson's disease, Huntington's disease, fronto-temporal dementia and 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 instance Alzheimer's seems to be closely associated with the progression of human disease.
  • over-expression, or the expression of modified forms, of proteins found in deposits, like the ⁇ -amyloid protein has been shown to induce various symptoms of disease, e.g. Alzheimer's-like symptoms. There is no specific treatment for amyloid deposition and these diseases are usually fatal.
  • the subunits of amyloid fibrils may 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 character of the amyloidosis.
  • all amyloid fibrils are of indeterminate length, unbranched, 70 to 120 ⁇ in diameter, and display characteristic staining with Congo Red (Pepys, 1996).
  • amyloid proteins are characteristic of a cross- ⁇ structure (Pauling & Corey, 1951) in which the polypeptide chain is organized in ⁇ -sheets. Although the amyloid proteins have very different precursor structures, they can all undergo a structural conversion, perhaps along a similar pathway, to a misfolded form that is the building block of the ⁇ -sheet helix protofilament.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • symptoms first appear after the age of 60, but earlier onsets are not infrequent. The earliest symptoms often include loss of recent memory, faulty judgment, and changes in personality. Often, people in the initial stages of AD think less clearly and forget the names of familiar people and common objects. Later in the disease, they may forget how to do even simple tasks. Eventually, people with AD lose all reasoning ability and become dependent on other people for their everyday care. Ultimately, the disease becomes so debilitating that patients are bedridden and likely to develop other illnesses and infections. Most commonly, people with AD die from pneumonia.
  • AD and dementia symptoms are not a part of normal aging.
  • AD and other dementing disorders are caused by diseases that affect the brain.
  • nerve cells in the brain are not lost in large numbers.
  • AD disrupts three key processes: Nerve cell communication, metabolism, and repair. This disruption ultimately causes many nerve cells to stop functioning, lose connections with other nerve cells, and die.
  • AD destroys neurons in parts of the brain that control memory, especially in the hippocampus and related structures. As nerve cells in the hippocampus stop functioning properly, short-term memory fails, and often, a person's ability to do easy and familiar tasks begins to decline. AD also attacks the cerebral cortex, particularly the areas responsible for language and reasoning. Eventually, many other areas of the brain are involved, all these brain regions atrophy (shrink), and the AD patient becomes bedridden, incontinent, totally helpless, and unresponsive to the outside world (source: National Institute on Aging Progress Report on Alzheimer's Disease, 1999).
  • AD Alzheimer's disease
  • AD puts a heavy economic burden on society.
  • the annual national cost of caring for AD patients in the US is estimated to be slightly over $50 billion (Leon et al., 1998).
  • NFT neurofibrillary tangles
  • AD familial AD
  • sporadic AD where no obvious pattern of inheritance is seen.
  • AD is further described as early-onset (occurring in people younger than 65) or late-onset (occurring in those 65 and older).
  • Early-onset AD is rare (about 10 percent of cases) and generally affects people aged 30 to 60.
  • Some forms of early-onset AD are inherited and run in families. Early-onset AD also often progresses faster than the more common, late-onset form.
  • amyloid plaques develop first in areas of the brain used for memory and other cognitive functions. They consist of largely insoluble deposits of beta amyloid (hereinafter designated A ⁇ )—a protein fragment of a larger protein called amyloid precursor protein (APP, the amino acid sequence of which is set forth in SEQ ID NO: 2)—intermingled with portions 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.
  • a ⁇ beta amyloid
  • APP amyloid precursor protein
  • AD Alzheimer's disease
  • APP is one of many proteins that are associated with cell membranes. After it is made, APP becomes embedded in the nerve cell's membrane, partly inside and partly outside the cell. Recent studies using transgenic mice demonstrate that APP appears to play an important role in the growth and survival of neurons. For example, certain forms and amounts of APP may protect neurons against both short- and long-term damage and may render damaged neurons better able to repair themselves and help parts of neurons grow after brain injury.
  • proteases act on particular sites in APP, cleaving it into protein fragments.
  • One protease helps cleave APP to form A ⁇ , and another protease cleaves APP in the middle of the amyloid fragment so that A ⁇ cannot be formed.
  • the A ⁇ formed is of two different lengths, a shorter 40 (or 41) amino acids A ⁇ that is relatively soluble and aggregates slowly, and a slightly longer, 42 amino acids “sticky” A ⁇ that rapidly forms insoluble clumps.
  • a ⁇ is being formed, it is not yet known exactly how it moves through or around nerve cells. In the final stages of this process, the “sticky” A ⁇ aggregates into long filaments outside the cell and, along with fragments of dead and dying neurons and the microglia and astrocytes, forms the plaques that are characteristic of AD in brain tissue.
  • mutations in APP render more likely that A ⁇ will be snipped out of the APP precursor, thus causing either more total A ⁇ or relatively more of the “sticky” form to be made. It also appears that mutations in the presenilin genes may contribute to the degeneration of neurons in at least two ways: By modifying A ⁇ production or by triggering the death of cells more directly. Other researchers suggest that mutated presenilins 1 and 2 may be involved in accelerating the pace of apoptosis.
  • a ⁇ is toxic to neurons.
  • tissue culture studies researchers observed an increase in death of hippocampal neurons cells engineered to over-express mutated forms of human APP compared to neurons over-expressing the normal human APP (Luo et al., 1999).
  • presenilins also have a role in causing the generation of NFT's. Some suggest that presenilins could also have a more direct role in the degeneration of neurons and neuron death. Presenilin-1 is located at chromosome 14 while presenilin-2 is linked to chromosome 1. If a person harbours a mutated version of just one of these genes he or she is almost certain to develop early onset AD.
  • Apolipoprotein E is usually associated with cholesterol, but is also found in plaques and tangles of AD brains. While alleles 1-3 do not seem to be involved in AD there is a significant correlation between the presence of the APOE- ⁇ 4 allele and development of late AD (Strittmatter et al., 1993). It is, however, a risk factor and not a direct cause as is the case for the presenilin and APP mutations and it is not limited to familial AD.
  • Apo A1 has been shown to be amyloigenic. Intact apo A1 can itself form amyloid-like fibrils in vitro that are Congo red positive (Am J Pathol 147 (2): 238-244 (Aug 1995), Wisniewski T, Golabek A A, Kida E, Wisniewski K E, Frangione B).
  • AD tau This second hallmark of AD consists of abnormal collections of twisted threads found inside nerve cells.
  • the chief component of tangles is one form of a protein called tau ( ⁇ ).
  • tau proteins are best known for their ability to bind and help stabilize microtubules, which are one constituent of the cell's internal support structure, or skeleton.
  • microtubules which are one constituent of the cell's internal support structure, or skeleton.
  • tau is changed chemically, and this altered tau can no longer stabilize the microtubules, causing them to fall disintegrate. This collapse of the transport system may at first result in malfunctions in communication between nerve cells and may later lead to neuronal death.
  • AD chemically altered tau twists into paired helical filaments—two threads of tau that are wound around each other. These filaments are the major substance found in neurofibrillary tangles.
  • the loss of neurons was studied, a similar progression was found.
  • Evidence of this type supports the idea that the formation of tangles and the loss of neurons progress together over the course of AD. (National Institute on Aging Progress Report on Alzheimer's Disease, 1999).
  • AD neurodegenerative diseases
  • Several neurodegenerative diseases, other than AD are characterized by the aggregation of tau into insoluble filaments in neurons and glia, leading to dysfunction and death.
  • several groups of researchers, who were studying families with a variety of hereditary dementias other than AD found the first mutations 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 cause neuronal cell death and dementia.
  • FTDP-17 fronto temporal dementia and parkinsonism linked to chromosome 17
  • ALS amyotrophic lateral sclerosis
  • corticobasal degeneration progressive supranuclear palsy
  • Pick's disease and are all characterized by abnormal aggregation of tau protein.
  • 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 fronto-temporal dementia. All involve deposits 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.
  • prion diseases such as kuru, Creutzfeld-Jacob disease and bovine spongiform encephalitis
  • Parkinson's disease Huntington's disease
  • fronto-temporal dementia fronto-temporal dementia. All involve deposits 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.
  • the immune system will normally take part in the clearing of foreign protein and proteinaceous particles in the organism but the deposits associated with the above-mentioned diseases consist mainly of self-proteins, thereby rendering the role of the immune system in the control of these diseases less obvious. Further, the deposits are located in a compartment (the CNS) normally separated from the immune system, both facts suggesting that any vaccine or immunotherapeutical approach would be unsuccessful.
  • mice [0049] Nevertheless, scientists have recently attempted immunizing mice with a vaccine composed of heterologous human A ⁇ and a substance known to excite the immune system (Schenk et al., 1999 and WO 99/27944).
  • the vaccine was tested in a partial transgenic mouse model of AD with a human mutated gene for APP inserted into the DNA of the mouse.
  • the mice produced the modified APP protein and developed amyloid plaques as they grew older.
  • This mouse model was used to test whether vaccination against the modified transgenic human APP had an effect on plaque build-up.
  • one group of transgenic mice was given monthly injections of the vaccine starting at 6 weeks of age and ending at 11 months.
  • a second group of transgenic mice received no injections and served as a control group. By 13 months of age, the mice in the control group had plaques covering 2 to 6 percent of their brains. In contrast, the immunized mice had virtually no plaques.
  • mice are a preliminary study in mice and for example, scientists need to find out whether vaccinated mice remain healthy in other respects and whether memory of those vaccinated remains normal. Furthermore, because the mouse model is not a complete representation of AD (the animals do not develop neurofibrillary tangles nor do many of their neurons die), additional studies will be necessary to determine whether humans have a similar or different reaction from mice. Another issue to consider is that the method may perhaps “cure” amyloid deposition but fail to stop development of dementia.
  • the object of the present invention is to provide novel therapies against conditions characterized by deposition of amyloid, such as AD.
  • a further object is to develop an autovaccine against amyloid, in order to obtain a novel treatment for AD and for other pathological disorders involving amyloid deposition.
  • Described herein is the use of an autovaccination technology for generating strong immune responses against otherwise non-immunogenic APP and A ⁇ Described is also the preparation of such vaccines for the prevention, possible cure or alleviation of the symptoms of such diseases associated with amyloid deposits.
  • the present invention relates to a method for in vivo down-regulation of amyloid precursor protein (APP) or beta amyloid (A ⁇ ) in an animal, including a human being, the method comprising effecting presentation to the animal's immune system of an immunogenically effective amount of at least one analogue of APP or A ⁇ that incorporates into the same molecule at least one B-cell epitope of APP and/or A ⁇ and at least one foreign T-helper epitope (T H epitope) so that immunization of the animal with the analogue induces production of antibodies against the animal's autologous APP or A ⁇ , wherein the analogue
  • a) is a polyamino acid that consists of at least one copy of a subsequence of residues 672-714 in SEQ ID NO: 2, wherein the foreign T H epitope is incorporated by means of amino acid addition and/or insertion and/or deletion and/or substitution, wherein the subsequence is selected from the group consisting of residues 1-42, residues 1-40, residues 1-39, residues 1-35, residues 1-34, residues 1-28, residues 1-12, residues 1-5, residues 13-28, residues 13-35, residues 17-28, residues 25-35, residues 35-40, residues 36-42 and residues 35-42 of the amino acid sequence consisting of amino acid residues 673-714 of SEQ ID NO: 2; and/or
  • b) is a polyamino acid that contains the foreign T H epitopes and a disrupted APP or A ⁇ sequence so that the analogue does not include any subsequence of SEQ ID NO: 2 that binds productively to MHC class II molecules initiating a T-cell response;
  • c) is a polyamino acid that comprises the foreign T H epitope and APP or A ⁇ derived amino acids, and comprises 1 single methionine residue located in the C-terminus of the analogue, wherein other methionine residues in APP or A ⁇ and in the foreign TH epitope have been substituted or deleted, and preferably have been substituted by leucin or isoleucine; and/or
  • d) is a conjugate comprising a polyhydroxypolymer backbone to which is separately coupled a polyamino acid as defined in a) and/or a polyamino acid as defined in b) and/or a polyamino acid as defined in c); and/or
  • e) is a conjugate comprising a polyhydroxypolymer backbone to which is separately coupled 1) the foreign T H epitope and 2) a polyamino acid selected from the group consisting of a subsequence as defined in a), a disrupted sequence of APP or A ⁇ as defined in b), and an APP or A ⁇ derived amino acid sequence that comprises 1 single methionine residue located in the C-terminus, wherein other methionine residues in APP or A ⁇ and in the foreign T H epitope have been substituted or deleted, and preferably have been substituted by leucin or isoleucine.
  • the invention also relates to analogues of the APP and A ⁇ as well as to nucleic acid fragments encoding a subset of these. Also immunogenic compositions comprising the analogues or the nucleic acid fragments are part of the invention.
  • FIG. 1 Schematic depiction of Autovac variants derived from the amyloid precursor protein with the purpose of generating antibody responses against the A ⁇ protein A ⁇ -43 (or C-100).
  • the 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 various truncations of APP.
  • the black pattern indicates the APP signal sequence
  • two-way cross-hatching is the extracellular part of APP
  • dark vertical hatching is the transmembrane domain of APP
  • light vertical hatching is the intracellular domain of APP
  • coarse cross-hatching indicates the P30 epitope
  • fine cross-hatching indicates the P2 epitope.
  • the full line box indicates A ⁇ -42/43 and the full-line box and the dotted line box together indicate C-100.
  • “Abeta” denotes A ⁇ .
  • FIG. 2 Schematic depiction of an embodiment of the synthesis of generally applicable immunogenic conjugates.
  • Peptide A any antigenic sequence, e.g. an A ⁇ sequence described herein
  • peptide B an amino acid sequence including a foreign T-helper epitope are synthesized and mixed. After that they are contacted with a suitable activated polyhydroxypolymer, peptides A and B are attached via the activation group in a ration corresponding to the initial ratio between these two substances in the peptide mixture.
  • Cf. Example 4 for details.
  • amyloid and “amyloid protein” which are used interchangeably herein denote a class of proteinaceous unbranched fibrils of indeterminate length. Amyloid fibrils display characteristic staining with Congo Red and share a cross- ⁇ structure in which the polypeptide chain is organized in ⁇ -sheets. Amyloid is generally derived from amyloidogenic proteins which have very different precursor structures but which can all undergo a structural conversion to a misfolded form that is the building block of the ⁇ -sheet helix protofilament. Normally, the diameter of amyloid fibrils varies between about 70 to about 120 ⁇ .
  • amyloidogenic protein is intended to denote a polypeptide which is involved in the formation of amyloid deposits, either by being part of the deposits as such or by being part of the biosynthetic pathway leading to the formation of the deposits.
  • amyloidogenic proteins are APP and A ⁇ , but also proteins involved in the metabolism of these may be amyloidogenic proteins.
  • amyloid polypeptide is herein intended to denote polypeptides comprising the amino acid sequence of the above-discussed amyloidogenic proteins derived from humans or other mammals (or truncates thereof sharing a substantial amount of B-cell epitopes with an intact amyloidogenic protein)—an amyloidogenic polypeptide can therefore e.g. comprise substantial parts of a precursor for the amyloidogenic polypeptide (in the case of A ⁇ , one possible amyloid polypeptide could be APP derived). Also unglycosylated forms of amyloidogenic polypeptides which are prepared in prokaryotic system are included within the boundaries of the term as are forms having varying glycosylation patterns due to the use of e.g.
  • an amyloidogenic polypeptide it is intended that the polypeptide in question is normally non-immunogenic when presented to the animal to be treated.
  • the amyloidogenic polypeptide is a self-protein or is an analogue of such a self-protein which will not normally give rise to an immune response against the amyloidogenic of the animal in question.
  • an “analogue” is an APP or A ⁇ derived molecule that incorporates one or several changes in its molecular structure. Such a change can e.g. be in the form of fusion of APP or A ⁇ poly-amino acids to a suitable fusion partner (i.e. a change in primary structure exclusively involving 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 encompassed by the term are derivatized APP or A ⁇ derived molecules, cf. the discussion below of modifications of APP or A ⁇ .
  • the analogue may be constructed so as to be less able or even unable to elicit antibodies against the normal precursor protein(s) of the amyloid, thereby avoiding undesired interference with the (physiologically normal) non-aggregated form of the polypeptide being a precursor of the amyloid protein.
  • a xeno-analogue e.g. a canine or porcine analogue
  • a human APP or A ⁇ can be imagined to produce the desired immunity against the APP or A ⁇ .
  • a xeno-analogue for immunization is also considered part of the invention.
  • polypeptide is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i.e. functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may form complexes, be covalently linked, or may be non-covalently linked.
  • the polypeptide(s) in a protein can be glycosylated and/or lipidated and/or comprise prosthetic groups. Also, the term “polyamino acid” is an equivalent to the term “polypeptide”
  • T-lymphocyte and “T-cell” will be used interchangeably for lymphocytes of thymic origin which are responsible for various cell mediated immune responses as well as for helper activity in the humoral immune response.
  • B-lymphocyte and “B-cell” will be used interchangeably for antibody-producing lymphocytes.
  • sequence means any consecutive 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.
  • animal is in the present context in general intended to denote an animal species (preferably mammalian), such as Homo sapiens, Canis domesticus , etc. and not just one single animal. However, the term also denotes a population of such an animal species, since it is important that the individuals immunized according to the method of the invention all harbour substantially the same APP or A ⁇ allowing for immunization of the animals with the same immunogen(s). It will be clear to the skilled person that an animal in the present context is a living being which has an immune system. It is preferred that the animal is a vertebrate, such as a mammal.
  • in vivo down-regulation of APP or A ⁇ is herein meant reduction in the living organism of the total amount of deposited amyloid protein (or amyloid as such) of the relevant type.
  • the down-regulation can be obtained by means of several mechanisms: of these, simple interference with amyloid by antibody binding so as to prevent misaggregation is the most simple.
  • the antibody binding results in removal of amyloid by scavenger cells (such as macrophages and other phagocytic cells) and that the antibodies interfer with other amyloidogenic polypeptides which lead to amyloid formation.
  • scavenger cells such as macrophages and other phagocytic cells
  • antibodies interfer with other amyloidogenic polypeptides which lead to amyloid formation.
  • antibodies bind A ⁇ outside the CNS, thereby effectively removing A ⁇ from the CNS via a simple mass action principle.
  • effecting presentation . . . to the immune system is intended to denote that the animal's immune system is subjected to an immunogenic challenge in a controlled manner.
  • challenge of the immune system can be effected in a number of ways of which the most important are vaccination with polypeptide containing “pharmaccines” (i.e. a vaccine which is administered to treat or ameliorate ongoing disease) or nucleic acid “pharmaccine” vaccination.
  • pharmaceutical competent cells in the animal are confronted with the antigen in an immunologically effective manner, whereas the precise mode of achieving this result is of less importance to the inventive idea underlying the present invention.
  • immunogenically effective amount has its usual meaning in the art, i.e. an amount of an immunogen, which is capable of inducing an immune response that significantly engages pathogenic agents sharing immunological features with the immunogen.
  • APP or A ⁇ has been “modified”
  • a chemical modification of the polypeptide has been performed on APP or A ⁇ .
  • Such a modification can e.g. be derivatization (e.g. alkylation) of certain amino acid residues in the sequence, but as will be appreciated from the disclosure below, the preferred modifications comprise changes of the primary structure of the amino acid sequence.
  • a “foreign T-cell epitope” is a peptide which is able to bind to an MHC molecule and which stimulates T-cells in an animal species.
  • Preferred foreign T-cell epitopes in the invention are “promiscuous” epitopes, i.e. epitopes which bind to a substantial fraction of a particular 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 detail below.
  • Promiscuous T-cell epitopes are also denoted “universal” T-cell epitopes.
  • a “foreign T helper lymphocyte epitope” (a foreign T H epitope) is a foreign T cell epitope, which binds an MHC Class II molecule and can be presented on the surface of an antigen presenting cell (APC) bound to the MHC Class II molecule.
  • APC antigen presenting cell
  • a “functional part” of a (bio)molecule is in the present context intended 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 in the art that many enzymes and other effector molecules have an active site which is responsible for the effects exerted by the molecule in question. Other parts of the molecule may serve a stabilizing or solubility enhancing purpose and can therefore be left out if these purposes are not of relevance in the context of a certain embodiment of the present invention. For instance it is possible to use certain cytokines as a modifying moiety in APP or A ⁇ (cf. the detailed discussion below), and in such a case, the issue of stability may be irrelevant since the coupling to the APP or Apmay provide the stability necessary.
  • adjuvant has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen. Or, in other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen, vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combined vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.
  • Targeting of a molecule is in the present context intended to denote the situation where a molecule upon introduction in the animal will appear preferentially in certain tissue(s) or will be preferentially associated with certain cells or cell types.
  • the effect can be accomplished in a number of ways including formulation of the molecule in composition facilitating targeting or by introduction in the molecule of groups, which facilitate targeting.
  • “Stimulation of the immune system” means that a substance or composition of matter exhibits 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 immunostimulating agent 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 use of the immunogen.
  • “Productive binding” means binding of a peptide to the MHC molecule (Class I or II) so as to be able to stimulate T-cells that engage a cell that present the peptide bound to the MHC molecule.
  • MHC molecule Class I or II
  • T-cells that engage a cell that present the peptide bound to the MHC molecule.
  • a peptide 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 T H cell that binds to the presented peptide-MHC Class II complex.
  • the analogue used as an immunogen in the method of the invention is a modified APP or A ⁇ molecule wherein at least one change is present in the amino acid sequence of the APP or A ⁇ , since the chances of obtaining the all-important breaking of autotolerance is greatly facilitated that way—this is e.g. evident from the results presented in Example 2 herein, where immunization with wild-type A ⁇ is compared to immunization with an A ⁇ variant molecule. It has been shown (in Dalum I et al., 1996, J. Immunol. 157: 4796-4804) that potentially self-reactive B-lymphocytes recognizing self-proteins are physiologically present in normal individuals.
  • T H -cells or T H -lymphocytes cytokine producing T-helper lymphocytes
  • APCs antigen presenting cells
  • T-cells recognizing the foreign element are activated upon recognizing the foreign epitope on an APC (such as, initially, a mononuclear cell).
  • Polyclonal B-lymphocytes (which are also APCs) capable of recognising self-epitopes on the modified self-protein also internalise the antigen and subsequently presents the foreign T-cell epitope(s) thereof, and the activated T-lymphocytes subsequently 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 which are also present in the native polypeptide, an antibody cross-reactive with the non-modified self-protein is induced.
  • the T-lymphocytes can be led to act as if the population of polyclonal B-lymphocytes have recognised an entirely foreign antigen, whereas in fact only the inserted epitope(s) is/are foreign to the host. In this way, antibodies capable of cross-reacting with non-modified self-antigens are induced.
  • analogue according to the present invention includes
  • At least one first moiety is introduced which effects targeting of the modified molecule to an antigen presenting cell (APC), and/or
  • At least one second moiety is introduced which stimulates the immune system, and/or
  • At least one third moiety is introduced which optimizes presentation of the analogue to the immune system.
  • side groups in the form of the foreign T-cell epitopes or the above-mentioned first, second and third moieties
  • side groups are covalently or non-covalently introduced. This is to mean that stretches of amino acid residues derived from the APP or AD are derivatized without altering 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 utilises amino acid substitution and/or deletion and/or insertion and/or addition (which may be effected by recombinant means or by means of peptide synthesis; modifications which involves longer stretches of amino acids can give rise to fusion polypeptides).
  • WO 95/05849 discloses a method for down-regulating self-proteins by immunising with analogues of the self-proteins wherein a number of amino acid sequence(s) has been substituted with a corresponding number of amino acid sequence(s) which each comprise a foreign immunodominant T-cell epitope, while at the same time maintaining the overall tertiary structure of the self-protein in the analogue.
  • 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 substantial number of the B-cell epitopes in the APP or A ⁇ .
  • the APP or A ⁇ or fragments thereof are mutated.
  • substitution variants where the methionine in position 35 in A ⁇ -43 has been substituted, preferably with leucine or isoleucine, or simply deleted.
  • Especially preferred analogues contain one single methionine that is located in the C-terminus, either because it is naturally occurring in the amyloidogenic polypeptide or foreign T H epitope, or because it has been inserted or added.
  • the part of the analogue that includes the foreign T H epitope is free from methionine, except from the possible C-terminal location of a methionine.
  • all analogues of APP or A ⁇ that are used according to the present invention share the characteristic of merely including one single methionine that is positioned as the C-terminal amino acid in the analogue and that other methionines in either the amyloidogenic polypeptide or the foreign T H epitope are deleted or substituted for another amino acid.
  • One further interesting mutation is a deletion or substitution of the phenylalanine in position 19 in A ⁇ -43, and it is especially preferred that the mutation is a substitution of this phenylalanine residue with a proline.
  • a ⁇ -43 protein truncated parts of the A ⁇ -43 protein. These can also be employed in immunogenic analogues according to the present invention. Especially preferred are the truncates A ⁇ (1-42), A ⁇ (1-40), A ⁇ (1-39), A ⁇ (1-35), A ⁇ (1-34), A ⁇ (1-34), A ⁇ (1-28), A ⁇ (1-12), A ⁇ (1-5), A ⁇ (13-28), A ⁇ (13-35), A ⁇ (17-28), A ⁇ (25-35), A ⁇ (35-40), A ⁇ (36-42), and A ⁇ (35-42) (where the numbers in the parentheses indicate the amino acid stretches of A ⁇ -43 that constitute the relevant fragment—A ⁇ (35-40) is e.g. identical to amino acids 706-711 in SEQ ID NO: 2). All these variants with truncated parts of A ⁇ -43 can be made with the A ⁇ fragments described herein, in particular with variants 9, 10, 11,
  • amyloid e1 -amyloid ex are x B-cell epitope containing sub-sequences of APP or A ⁇ which independently are identical or non-identical and which may contain or not contain foreign side groups
  • x is an integer ⁇ 3
  • n1-nx are x integers ⁇ 0 (at least one is ⁇ 1)
  • MOD 1 -MOD x are x modifications introduced between the preserved B-cell epitopes
  • s 1 -s x are x integers ⁇ 0 (at least one is ⁇ 1 if no side groups are introduced in the amyloid ex sequences).
  • modified APP or A ⁇ obtained by omission of parts of the sequence which e.g. exhibit adverse effects in vivo or omission of parts which are normally intracellular and thus could give rise to undesired immunological reactions.
  • One preferred version of the constructs outlined above are, when applicable, those where the B-cell epitope containing subsequence of an amyloid protein is not extracellularly exposed in the precursor polypeptide from which the amyloid is derived.
  • a selection of monoclonal antibodies reactive with distinct epitopes on the APP or A ⁇ can be prepared and used as a test panel.
  • This approach has the advantage of allowing 1) an epitope mapping of the APP or A ⁇ and 2) a mapping of the epitopes which are maintained in the analogues prepared.
  • a third approach would be to resolve the 3-dimensional structure of the APP or A ⁇ or of a biologically active truncate thereof (cf. above) and compare this to the resolved three-dimensional structure of the analogues prepared.
  • Three-dimensional structure can be resolved by the aid of X-ray diffraction studies and NMR-spectroscopy. Further information relating to the tertiary structure can to some extent be obtained from circular dichroism studies which have the advantage of merely requiring the polypeptide in pure form (whereas X-ray diffraction requires the provision of crystallized polypeptide and NMR requires the provision of isotopic variants of the polypeptide) in order to provide useful information about the tertiary structure of a given molecule.
  • X-ray diffraction and/or NMR are necessary to obtain conclusive data since circular dichroism can only provide indirect evidence of correct 3-dimensional structure via information of secondary structure elements.
  • One preferred embodiment of the invention utilises multiple presentations of B-lymphocyte epitopes of APP or A ⁇ (i.e. formula I wherein at least one B-cell epitope is present in two positions).
  • This effect can be achieved in various ways, e.g. by simply preparing fusion polypeptides comprising the structure (APP or A ⁇ derived polypeptide) m , where m is an integer ⁇ 2 and then introduce the modifications discussed herein in at least one of the APP or A ⁇ sequences. It is preferred that the modifications introduced includes at least one duplication of a B-lymphocyte epitope and/or the introduction of a hapten.
  • These embodiments including multiple presentations of selected epitopes are especially preferred in situations where merely minor parts of the APP or A ⁇ are useful as constituents in a vaccine agent.
  • the introduction of a foreign T-cell epitope can be accomplished by introduction of at least one amino acid insertion, addition, deletion, or substitution.
  • the normal situation will be the introduction of more than one change in the amino acid sequence (e.g. insertion of or substitution by a complete T-cell epitope) but the important goal to reach is that the analogue, when processed by an antigen presenting cell (APC), will give rise to such a foreign immunodominant T-cell epitope being presented in context of an MCH Class II molecule on the surface of the APC.
  • APC antigen presenting cell
  • the introduction of a foreign TH epitope can be accomplished 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 in order to fulfill the purpose of the present invention.
  • 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 furthermore preferred that the number of amino acid insertions, substitutions, additions or deletions is not in excess of 150, such as at most 100, at most 90, at most 80, and at most 70. It is especially preferred that the number of substitutions, insertions, deletions, or additions does not exceed 60, and in particular the number should not exceed 50 or even 40. Most preferred is a number of not more than 30. With respect to amino acid additions, it should be noted that these, when the resulting construct is in the form of a fusion polypeptide, is often considerably higher than 150.
  • Preferred embodiments of the invention includes modification by introducing at least one foreign immunodominant T-cell epitope.
  • T-cell epitope the question of immune dominance of a T-cell epitope depends on the animal species in question.
  • immunodominance simply refers to epitopes which in the vaccinated individual/population gives rise to a significant immune response, but it is a well-known fact that a T-cell epitope which is immunodominant in one individual/population is not necessarily immunodominant in another individual of the same species, even though it may be capable of binding MHC-II molecules in the latter individual.
  • an immune dominant T-cell epitope is a T-cell epitope which will be effective in providing T-cell help when present in an antigen.
  • immune dominant T-cell epitopes has as an inherent feature that they will substantially always be presented bound to an MHC Class II molecule, irrespective of the polypeptide wherein they appear.
  • T-cell epitopes Another important point is the issue of MHC restriction of T-cell epitopes.
  • naturally occurring T-cell epitopes are MHC restricted, i.e. a certain peptides constituting a T-cell epitope will only bind effectively to a subset of MHC Class II molecules.
  • This in turn has the effect that in most cases the use of one specific T-cell epitope will result in a vaccine component which is only effective in a fraction of the population, and depending on the size of that fraction, it can be necessary to include more T-cell epitopes in the same molecule, or alternatively prepare a multi-component vaccine wherein the components are variants of APP or AP which are distinguished from each other by the nature of the T-cell epitope introduced.
  • ⁇ j is the sum of frequencies in the population of allelic haplotypes encoding MHC molecules which bind any one of the T-cell epitopes in the vaccine and which belong to the j th 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 thereafter these are listed by type (DP, DR and DQ)—then, the individual frequencies of the different listed allelic haplotypes are summed for each type, thereby yielding ⁇ 1 , ⁇ 2 , and ⁇ 3 .
  • T-cell epitopes which are active in a large proportion of individuals of an animal species or an animal population and these are preferably introduced in the vaccine thereby reducing the need for a very large number of different analogues in the same vaccine.
  • the promiscuous epitope can according to the invention be a naturally occurring human T-cell epitope such as epitopes from tetanus toxoid (e.g. the P2 and P30 epitopes), diphtheria toxoid, Influenza virus hemagluttinin (HA), and P. falciparum CS antigen.
  • tetanus toxoid e.g. the P2 and P30 epitopes
  • diphtheria toxoid e.g. the P2 and P30 epitopes
  • Influenza virus hemagluttinin (HA) Influenza virus hemagluttinin
  • P. falciparum CS antigen e.g. the P2 and P30 epitopes
  • the epitope can be any artificial T-cell epitope which is capable of binding a large proportion of MHC Class II molecules.
  • the pan DR epitope peptides (“PADRE”) described in WO 95/07707 and in the corresponding paper Alexander J et al., 1994, Immunity 1: 751-761 (both disclosures are incorporated by reference herein) are interesting candidates for epitopes to be used according to the present invention. It should be noted that the most effective PADRE peptides disclosed in these papers carry D-amino acids in the C- and N-termini in order to improve stability when administered.
  • the present invention primarily aims at incorporating the relevant epitopes as part of the analogue which should then subsequently be broken down enzymatically inside the lysosomal compartment of APCs to allow subsequent presentation in the context of an MHC-II molecule and therefore it is not expedient to incorporate D-amino acids in the epitopes used in the present invention.
  • PADRE peptide is the one having the amino acid sequence AKFVAAWTLKAAA (SEQ ID NO: 17) or an immuologically effective subsequence thereof.
  • AKFVAAWTLKAAA amino acid sequence AKFVAAWTLKAAA
  • immuologically effective subsequence thereof is preferred.
  • T-cell epitopes which should be present in the analogues used in the inventive method.
  • Such super-promiscuous epitopes will allow for the most simple embodiments of the invention wherein only one single analogue is presented to the vaccinated animal's immune system.
  • the modification of the APP or A ⁇ can also include the introduction of a first moiety which targets the modified amyloidogenic polypeptide to an APC or a B-lymphocyte.
  • the first moiety can be a specific binding partner for a B-lymphocyte specific surface antigen or for an APC specific surface antigen.
  • the moiety can be a carbohydrate for which there is a receptor on the B-lymphocyte or the APC (e.g. mannan or mannose).
  • the second moiety can be a hapten.
  • an antibody fragment which specifically recognizes a surface molecule on APCs or lymphocytes can be used as a first moiety (the surface molecule can e.g. be an FC ⁇ receptor of macrophages and monocytes, such as FC ⁇ RI or, alternatively any other specific surface marker such as CD40 or CTLA-4). It should be noted that all these exemplary targeting molecules can be used as part of an adjuvant also, cf. below.
  • the analogue As an alternative or supplement to targeting the analogue to a certain cell type in order 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 moiety which stimulates the immune system.
  • second moieties are cytokines, and heat-shock proteins or molecular chaperones, as well as effective parts thereof.
  • Suitable cytokines to be used according to the invention are those which will normally also function as adjuvants in a vaccine composition, i.e. for instance interferon ⁇ (IFN- ⁇ ), 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 suffice as the second moiety.
  • IFN- ⁇ interferon ⁇
  • IL-1 interleukin 1
  • IL-2 interleukin 2
  • IL-4 interleukin 4
  • IL-6 interleukin 6
  • IL-12 interleukin 12
  • IL-13 interleukin 13
  • IL-15 interleukin 15
  • GM-CSF granulocyte-macrophage
  • suitable heat-shock proteins or molecular chaperones used as the second moiety can be HSP70, HSP90, HSC70, GRP94 (also known as gp96, cf. Wearsch P A et al. 1998, Biochemistry 37: 5709-19), and CRT (calreticulin).
  • the second moiety can be a toxin, such as listeriolycin (LLO), lipid A and heat-labile enterotoxin.
  • LLO listeriolycin
  • lipid A lipid A
  • heat-labile enterotoxin lipid A
  • mycobacterial derivatives such as MDP (muramyl dipeptide), CFA (complete Freund's adjuvant) and the trehalose diesters TDM and TDE are interesting possibilities.
  • the possibility of introducing a third moiety which enhances the presentation of the analogue to the immune system is an important embodiment of the invention.
  • the art has shown several examples of this principle. For instance, it is known that the palmitoyl lipidation anchor in the Borrelia burgdorferi protein OspA can be utilised so as to provide self-adjuvating polypeptides (cf. e.g. WO 96/40718)—it seems that the lipidated proteins form up micelle-like structures with a core consisting of the lipidation anchor parts of the polypeptides and the remaining parts of the molecule protruding therefrom, resulting in multiple presentations of the antigenic determinants.
  • lipidation anchors e.g. a myristyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI-anchor, and an N-acyl diglyceride group
  • lipidation anchors e.g. a myristyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI-anchor, and an N-acyl diglyceride group
  • C3d fragment of 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 invention which also results in the preferred presentation of multiple (e.g. at least 2) copies of the important epitopic regions of APP or A ⁇ to the immune system is the covalent coupling of the analogue to certain molecules, i.e. variants d and e mentioned above.
  • polymers can be used, e.g. carbohydrates such as dextran, cf. e.g. 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 e.g. 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.
  • KLH keyhole limpe
  • Preferred embodiments of covalent coupling of the APP or AP derived material to polyhydroxypolymers such as carbohydrates involve the use of at least one APP or A ⁇ derived peptide and at least one foreign T-helper epitope which are coupled separately to the polyhydroxypolymer (i.e. the foreign T-helper epitope and the APP or A ⁇ derived amino acid sequence are not fused to each other but rather bound to the polyhydroxypolymer which then serves as a carrier backbone).
  • the coupling of the foreign T-helper epitope and the APP or A ⁇ derived (poly)peptide is by means of an amide bond which can be cleaved by a peptidase.
  • This strategy has the effect that APCs will be able to take up the conjugate and at the same time be able to process the conjugate and subsequently present the foreign T-cell epitope in an MHC Class II context.
  • One way of achieving coupling of peptides is to activate a suitable polyhydroxypolymer with tresyl (trifluoroethylsulphonyl) groups or other suitable activation groups such as maleimido, p-Nitrophenyl cloroformate (for activation of OH groups and formation of a peptide bond between peptide and polyhydroxypolymer), and tosyl (p-toluenesulfonyl). It is e.g. possible to prepare activated polysaccharides as described in WO 00/05316 and U.S. Pat. No.
  • the APP or A ⁇ polypeptide discussed above is a human A ⁇ polypeptide.
  • the APP or A ⁇ polypeptide has been modified by substituting at least one amino acid sequence in SEQ ID NO: 2 with at least one amino acid sequence of equal or different length and containing a foreign T H epitope.
  • Preferred examples of modified amyloidogenic APP and A ⁇ are shown schematically in FIG. 1 using the P2 and P30 epitopes as examples. The rationale behind such constructs is discussed in detail in the examples.
  • a T H containing (or completing) amino acid sequence which is introduced into SEQ ID NO: 2 may be introduced at any amino acid in SEQ ID NO: 2. That is, the introduction is possible after any of amino acids 1-770, but preferably after any of 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.
  • Another embodiment of the present invention is the presentation of the analogues which do not include any subsequence of of SEQ ID NO: 2 that binds productively to MHC class II molecules initiating a T-cell response.
  • this embodiment of the invention also serves to ensure that the immunogen does not indlude peptide sequences of the target APP or A ⁇ that could serve as “self-stimulating T H epitopes” including sequences that merely contain conservative substitutions in a sequence of the target protein that might otherwise function as a T H epitope.
  • Preferred embodiments of the immune system presentation of the analogues of the APP or A ⁇ involve the use of a chimeric peptide comprising at least one APP or A ⁇ derived peptide, which does not bind productively to MHC class II molecules, and at least one foreign T-helper epitope. Moreover, it is preferred that the APP or A ⁇ derived peptide harbours a B-cell epitope. It is especially advantageous if the immunogenic analogue is one, wherein 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 inserts comprise foreign T-helper epitopes.
  • B-cell epitope carrying regions of the APP or A ⁇ are constituted by short peptide stretches that in no way would be able to bind productively to an MHC Class II molecule.
  • the selected B-cell epitope or epitopes of the amyloidogenic polypeptide should therefore comprise at most 9 consecutive amino acids of SEQ ID NO: 2. Shorter peptides are preferred, such as those having at most 8, 7, 6, 5, 4, or 3 consecutive amino acids from the amyloidogenic polypeptide's amino acid sequence.
  • the analogue comprises at least one subsequence of SEQ ID NO: 2 so that each such at least one subsequence independently consists of amino acid stretches from the APP or A ⁇ selected from the group consisting of 9 consecutive amino acids, 8 consecutive amino acids, 7 consecutive amino acids, 6 consecutive amino acids, 5 consecutive amino acids, 4 consecutive amino acids, and 3 consecutive amino acids.
  • the consecutive amino acids begins at an amino acid residue selected from the group consisting of residue 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: 2.
  • vaccines which contain peptide sequences as active ingredients are generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference.
  • such vaccines are prepared as injectables 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 may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients which 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.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines; cf. the detailed discussion of adjuvants below.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously, intracutaneously, or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral, buccal, sublingual, intraperitoneal, intravaginal, anal, epidural, spinal, and intracranial formulations.
  • 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 the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
  • cholera toxin is an interesting formulation partner (and also a possible conjugation partner).
  • the polypeptides may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired.
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 ⁇ g to 2,000 ⁇ g (even though higher amounts in the 1-10 mg range are contemplated), such as in the range from about 0.5 ⁇ g to 1,000 ⁇ g, preferably in the range from 1 ⁇ g to 500 ⁇ g and especially in the range from about 10 ⁇ g to 100 ⁇ g.
  • Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. 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.
  • polypeptides of 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 substance.
  • an adjuvant which can be demonstrated to facilitate breaking of the autotolerance to 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 immune modulating adjuvant such as a toxin, a cytokine, and a mycobacterial derivative; an oil formulation; a polymer; a micelle forming adjuvant; a saponin; an immunostimulating complex matrix (ISCOM matrix); a particle; DDA; aluminium adjuvants; DNA adjuvants; ⁇ -inulin; and an encapsulating adjuvant.
  • an immune targeting adjuvant an immune modulating adjuvant such as a toxin, a cytokine, and a mycobacterial derivative
  • an oil formulation a polymer
  • a micelle forming adjuvant such as a toxin, a cytokine, and a mycobacterial derivative
  • an oil formulation such as a toxin, a cytokine, and a mycobacterial derivative
  • a polymer such as a toxin, a cytokin
  • adjuvants include use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in buffered saline, admixture with synthetic polymers of sugars (e.g. Carbopol®) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 700 to 101° C. for 30 second to 2 minute periods respectively and also aggregation by means of cross-linking agents are possible. Aggregation by reactivation with pepsin treated antibodies (Fab fragments) to albumin, mixture with bacterial cells such as C.
  • agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in buffered saline, admixture with synthetic polymers of sugars (e.g. Carbopol®) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 700 to 101° C. for 30 second to 2 minute periods respectively and also aggregati
  • parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. Admixture with oils such as squalene and IFA is also preferred.
  • DDA dimethyldioctadecylammonium bromide
  • DNA and ⁇ -inulin are interesting candidates for an adjuvant as is DNA and ⁇ -inulin, but also Freund's complete and incomplete adjuvants as well as quillaja saponins such as QuilA and QS21 are interesting as is RIBI.
  • Further possibilities are monophosphoryl lipid A (MPL), the above mentioned C3 and C3d, and muramyl dipeptide (MDP).
  • Liposome formulations are also known to confer adjuvant effects, and therefore liposome adjuvants are preferred according to the invention.
  • immunostimulating complex matrix type (ISCOM® matrix) adjuvants are preferred choices according to the invention, especially since it has been shown that this type of adjuvants are capable of up-regulating MHC Class II expression by APCs.
  • An ISCOMO matrix consists of (optionally fractionated) saponins (triterpenoids) from Quillaja saponaria, cholesterol, and phospholipid. When admixed with the immunogenic protein, the resulting particulate formulation is what is known as an ISCOM particle where the saponin constitutes 60-70% w/w, the cholesterol and phospholipid 10-15% w/w, and the protein 10-15% w/w. Details relating to composition and use of immunostimulating complexes can e.g.
  • Another highly interesting (and thus, preferred) possibility of achieving adjuvant effect is to employ the technique described in Gosselin et al., 1992 (which is hereby incorporated by reference herein).
  • the presentation of a relevant antigen such as an antigen of the present invention can be enhanced by conjugating the antigen to antibodies (or antigen binding antibody fragments) against the Fc ⁇ receptors on monocytes/macrophages.
  • a relevant antigen such as an antigen of the present invention
  • conjugates between antigen and anti-Fc ⁇ RI have been demonstrated to enhance immunogenicity for the purposes of vaccination.
  • cytokines targeting and immune modulating substances (i.a. cytokines) mentioned above as candidates for the first and second moieties in the modified versions of amyloidogenic polypeptides.
  • synthetic inducers of cytokines like poly I:C are possibilities.
  • Suitable mycobacterial derivatives are selected from the group consisting of muramyl dipeptide, complete Freund's adjutant, RIBI, and a diester of trehalose such as TDM and TDE.
  • Suitable immune targeting adjuvants are selected from the group consisting of CD40 ligand and CD40 antibodies or specifically binding fragments thereof (cf. the discussion above), mannose, a Fab fragment, and CTLA-4.
  • Suitable polymer adjuvants are selected from the group consisting of a carbohydrate such as dextran, PEG, starch, mannan, and mannose; a plastic polymer such as; and latex such as latex beads.
  • VLN virtual lymph node
  • the VLN (a thin tubular device) mimics the structrue and function of a lymph node. Insertion of a VLN under the skin creates a site of sterile inflammation with an upsurge of cytokines and chemokines. T- and B-cells as well as APCs rapidly respond to the danger signals, home to the inflamed site and accumulate inside the porous matrix of the VLN.
  • Microparticle formulation of vaccines has been shown in many cases to increase the immunogenicity of protein antigens and is therefore another preferred embodiment of the invention.
  • Microparticles are made either as co-formulations of antigen with a polymer, a lipid, a carbohydrate or other molecules suitable for making the particles, or the microparticles can be homogeneous particles consisting of only the antigen itself.
  • polymer based microparticles examples include PLGA and PVP based particles (Gupta, R. K. et. al. 1998) where the polymer and the antigen are condensed into a solid particle.
  • Lipid based particles can be made as micelles of the lipid (so-called liposomes) entrapping the antigen within the micelle (Pietrobon, P. J. 1995).
  • Carbohydrate based particles are typically made of a suitable degradable carbohydrate such as starch or chitosan. The carbohydrate and the antigen are mixed and condensed into particles in a process similar to the one used for polymer particles (Kas, H. S. et. al. 1997).
  • Particles consisting only of the antigen can be made by various spraying and freeze-drying techniques.
  • the super critical fluid technology that is used to make very uniform particles of controlled size (York, P. 1999 & Shekunov, B. et. al. 1999).
  • the vaccine should be administered 1-6 times per year, such as 1, 2, 3, 4, 5, or 6 times a year to an individual in need thereof. It has previously been shown that the memory immunity induced by the use of the preferred autovaccines according to the invention is not permanent, and therefore the immune system needs to be periodically challenged with the amyloidogenic polypeptide or modified amyloidogenic polypeptides.
  • the vaccine according to the invention may comprise several different polypeptides in order to increase the immune response, cf. also the discussion above concerning the choice of foreign T-cell epitope introductions.
  • the vaccine may comprise two or more polypeptides, where all of the polypeptides are as defined above.
  • the vaccine may consequently comprise 3-20 different modified or unmodified polypeptides, such as 3-10 different polypeptides.
  • nucleic acid immunisation As an alternative to classic administration of a peptide-based vaccine, the technology of nucleic acid vaccination (also known as “nucleic acid immunisation”, “genetic immunisation”, and “gene immunisation”) offers a number of attractive features.
  • nucleic acid vaccination does not require resource consuming large-scale production of the immunogenic agent (e.g. in the form of industrial scale fermentation of microorganisms producing modified amyloidogenic polypeptides). Furthermore, there is no need to device purification and refolding schemes for the immunogen.
  • nucleic acid vaccination relies on the biochemical apparatus of the vaccinated individual in order to produce the expression product of the nucleic acid introduced, the optimum post-translational processing of the expression product is expected to occur; this is especially important in the case of autovaccination, since, as mentioned above, a significant fraction of the original B-cell epitopes should be preserved in the modified molecule, and since B-cell epitopes in principle can be constituted by parts of any (bio)molecule (e.g. carbohydrate, lipid, protein etc.). Therefore, native glycosylation and lipidation patterns of the immunogen may very well be of importance for the overall immunogenicity and this is best ensured by having the host producing the immunogen.
  • bio bio
  • a preferred embodiment of the invention's variants a-c comprises effecting presentation of the analogue to the immune system by introducing nucleic acid(s) encoding the analogue into the animal's cells and thereby obtaining in vivo expression by the cells of the nucleic acid(s) introduced.
  • the introduced nucleic acid is preferably DNA which can 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-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA encapsulated in a polymer, e.g. in PLGA (cf. the microencapsulation technology described in WO 98/31398) or in chitin or chitosan, and DNA formulated with an adjuvant.
  • DNA which can 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-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA
  • nucleic acid vaccines can suitably be administered intraveneously and intraarterially.
  • nucleic acid vaccines can be administered by use of a so-called gene gun, and hence also this and equivalent modes of administration are regarded as part of the present invention.
  • VLN a VLN in the administration of nucleic acids has been reported to yield good results, and therefore this particular mode of administration is particularly preferred.
  • the nucleic acid(s) used as an immunization agent can contain regions encoding the 1 st , 2 nd and/or 3 rd moieties, e.g. in the form of the immunomodulating substances described above such as the cytokines discussed as useful adjuvants.
  • a preferred version of this embodiment encompasses having the coding region for the analogue and the coding region for 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 to the immunomodulator.
  • two distinct nucleotide fragments can be used, but this is less preferred because of the advantage of ensured co-expression when having both coding regions included in the same molecule.
  • the invention also relates to a composition for inducing production of antibodies against APP or A ⁇ , the composition comprising
  • nucleic acid fragment or a vector of the invention cf. the discussion of vectors below
  • the variant-encoding nucleic acid is introduced in the form of a vector wherein expression is under control of a viral promoter.
  • vectors according to the invention cf. the discussion below.
  • detailed disclosures relating to the formulation and use of nucleic acid vaccines are available, cf. Donnelly J J et al, 1997, Annu. Rev. Immunol. 15: 617-648 and Donnelly J J et al., 1997, Life Sciences 60: 163-172. Both of these references are incorporated by reference herein.
  • a third alternative for effecting presentation of the analogues as these are defined in variants a-c to the immune system is the use of live vaccine technology.
  • presentation to the immune system is effected by administering, to the animal, a non-pathogenic microorganism which has been transformed with a nucleic acid fragment encoding an analogue or with a vector incorporating such a nucleic acid fragment.
  • the non-pathogenic microorganism can be any suitable attenuated bacterial strain (attenuated by means of passaging or by means of removal of pathogenic expression products by recombinant DNA technology), e.g. Mycobacterium bovis BCG., non-pathogenic Streptococcus spp., E.
  • nucleic acid fragment of the invention discussed below can be incorporated in a non-virulent viral vaccine vector such as a vaccinia strain or any other suitable poxyirus.
  • 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 in a lifetime in order to maintain protective immunity. It is even contemplated that immunization schemes as those detailed above for polypeptide vaccination will be useful when using live or virus vaccines.
  • live or virus vaccination is combined with previous or subsequent polypeptide and/or nucleic acid vaccination.
  • the microorganism or virus can be transformed with nucleic acid(s) containing regions encoding the 1 st , 2 nd and/or 3 rd moieties, e.g. in the form of the immunomodulating substances described above such as the cytokines discussed as useful adjuvants.
  • a preferred version of this embodiment encompasses having the coding region for the analogue and the coding region for 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 to the immunomodulator.
  • two distinct nucleotide fragments can be used as transforming agents.
  • having the 1 st and/or 2 nd and/or 3 rd moieties in the same reading frame can provide as an expression product, an analogue of the invention, and such an embodiment is especially preferred according to the present invention.
  • an important embodiment of the method of the invention for down-regulating amyloid activity comprises treating and/or preventing and/or ameliorating AD or other diseases characterized by amyloid deposition, the method comprising down-regulating APP or A ⁇ according to the method of the invention to such an extent that the amount of amyloid is significantly decreased.
  • the reduction in amyloid results in an inversion of the balance between amyloid formation and amyloid degradation/removal, i.e. that the rate of amyloid degradation/removal is brought to exceed the rate of amyloid formation.
  • the method of the invention can be used to obtain a clinically significant reduction in the formation of new amyloid, thereby significantly prolonging the time where the disease condition is non-debilitating. It should be possible to monitor the rate of amyloid depositing by either measuring the serum concentration of amyloid (which is believed to be in equilibrium with the deposited material), or by using positron-emission tomography (PET) scanning, cf. Small G W, et al., 1996, Ann NY Acad Sci 802: 70-78.
  • PET positron-emission tomography
  • the present invention is based on the concept of immunising individuals against the APP or A ⁇ antigen in order to obtain a reduced amount of pathology-related amyloid deposits.
  • the preferred way of obtaining such an immunization is to use the analogues described herein, thereby providing molecules which have not previously been disclosed in the art.
  • modified APP or A ⁇ molecules comprise modifications which results in a polypeptide having a sequence identity of at least 70% with APP or A ⁇ or with a subsequence thereof of at least 10 amino acids in length. Higher sequence identities are preferred, e.g. at least 75% or even at least 80, 85, 90, or 95%.
  • the sequence identity for proteins and nucleic acids can be calculated as (N ref -N dif ) ⁇ 100/N ref , wherein N dif is the total number of non-identical residues in the two sequences when aligned and wherein N ref is the number of residues in one of the sequences.
  • the invention also pertains to compositions useful in exercising the method of the invention.
  • the invention also relates to an immunogenic composition comprising an immunogenically effective amount of an analouge as described above, said composition further comprising a pharmaceutically and immunologically acceptable diluent and/or vehicle and/or carrier and/or excipient and optionally an adjuvant.
  • this part of the invention concerns formulations of analogues, essentially as described above.
  • the choice of adjuvants, carriers, and vehicles is accordingly in line with what has been discussed above when referring to formulation of modified and unmodified amyloidogenic polypeptide for use in the inventive method for the down-regulation of APP or A ⁇ .
  • polypeptides are prepared according to methods well-known in the art. Longer polypeptides are normally prepared by means of recombinant gene technology including introduction of a nucleic acid sequence encoding the analogue into a suitable vector, transformation of a suitable host cell with the vector, expression by the host cell of the nucleic acid sequence, recovery of the expression product from the host cells or their culture supernatant, and subseqeunt purification and optional further modification, e.g. refolding or derivatization.
  • Shorter peptides are preferably prepared by means of the well-known techniques of solid- or liquid-phase peptide synthesis. However, recent advances in this technology has rendered possible the production of full-length polypeptides and proteins by these means, and therefore it is also within the scope of the present invention to prepare the long constructs by synthetic means.
  • poly-amino acid analogues can be prepared by means of recombinant gene technology but also by means of chemical synthesis or semisynthesis; the latter two options are especially relevant when the modification consists in coupling to 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 comprises addition of side chains or side groups to an APP or A ⁇ derived peptide chain.
  • protein carriers such as KLH, diphtheria toxoid, tetanus toxoid, and BSA
  • non-proteinaceous molecules such as carbohydrate polymers
  • nucleic acid fragments encoding analogues are important chemical products.
  • an important part of the invention pertains to a nucleic acid fragment which encodes an analogue of the invention, i.e. an APP or A ⁇ derived polypeptide which either comprises the natural sequence to which has been added or inserted a fusion partner or, preferably an APP or AP derived polypeptide wherein has been introduced a foreign T-cell epitope by means of insertion and/or addition, preferably by means of substitution and/or deletion.
  • the nucleic acid fragments of the invention are either DNA or RNA fragments.
  • the nucleic acid fragments of the invention will normally be inserted in suitable vectors to form cloning or expression vectors carrying the nucleic acid fragments of the invention; such novel vectors are also part of the invention. Details concerning the construction of these vectors of the invention will be discussed in context of transformed cells and microorganisms below.
  • the vectors can, depending on purpose and type of application, be in the form of plasmids, phages, cosmids, mini-chromosomes, or virus, but also naked DNA which is only expressed transiently in certain cells is an important vector.
  • Preferred cloning and expression vectors of the invention are capable of autonomous replication, thereby enabling high copy-numbers for the purposes of high-level expression or high-level replication for subsequent cloning.
  • the general outline of a vector of the invention comprises the following features in the 5′ ⁇ 3′ direction and in operable linkage: a promoter for driving expression of the nucleic acid fragment of the invention, optionally a nucleic acid sequence encoding a leader peptide enabling secretion (to the extracellular phase or, where applicable, into the periplasma) of or integration into the membrane of the polypeptide fragment, the nucleic acid fragment of the invention, and optionally a nucleic acid sequence encoding a terminator.
  • a promoter for driving expression of the nucleic acid fragment of the invention optionally a nucleic acid sequence encoding a leader peptide enabling secretion (to the extracellular phase or, where applicable, into the periplasma) of or integration into the membrane of the polypeptide fragment, the nucleic acid fragment of the invention, and optionally a nucleic acid sequence encoding a terminator.
  • vectors to be used for effecting in vivo expression in an animal i.e. when using the vector in DNA vaccination
  • the vector is incapable of being integrated in the host cell genome; typically, naked DNA or non-integrating viral vectors are used, the choices of which are well-known to the person skilled in the art
  • the vectors of the invention are used to transform host cells to produce the analogue of 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 of the invention, or used for recombinant production of the analogues of the invention.
  • the transformed cells can be suitable live vaccine strains wherein the nucleic acid fragment (one single or multiple copies) have been inserted so as to effect secretion or integration into the bacterial membrane or cell-wall of the analogue.
  • Preferred transformed cells of the invention are microorganisms such as bacteria (such as the species Escherichia [e.g. E.coli ], Bacillus [e.g. Bacillus subtilis ], Salmonella, or Mycobacterium [preferably non-pathogenic, e.g. M. bovis BCG]), yeasts (such as Saccharomyces cerevisiae ), and protozoans.
  • the transformed cells are derived from a multi-cellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. Most preferred are cells derived from a human being, cf. the discussion of cell lines and vectors below.
  • the transformed cell is capable of replicating the nucleic acid fragment of the invention.
  • Cells expressing the nucleic fragment are preferred useful embodiments of the invention; they can be used for small-scale or large-scale preparation of the analogue of the invention or, in the case of non-pathogenic bacteria, as vaccine constituents in a live vaccine.
  • an effective producer cell When an effective producer cell has been identified it is preferred, on the basis thereof, to establish a stable cell line which carries the vector of the invention and which expresses the nucleic acid fragment encoding the modified amyloidogenic polypeptide.
  • this stable cell line secretes or carries the analogue of the invention, thereby facilitating purification thereof.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with the hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al., 1977).
  • the pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the prokaryotic microorganism for expression.
  • promoters most 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 commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (Siebwenlist et al., 1980). Certain genes from prokaryotes may be expressed efficiently in E. coli from their own promoter sequences, precluding the need for addition of another promoter by artificial means.
  • eukaryotic microbes such as yeast cultures may also be used, and here the promoter should be capable of driving expression.
  • Saccharomiyces cerevisiase or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
  • 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 for example ATCC No. 44076 or PEP4-1 (Jones, 1977).
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Suitable promoting sequences in yeast vectors include the promoters 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, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • the termination sequences associated with these genes are also ligated into the expression vector 3′ of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
  • promoters which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative 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.
  • cultures of cells derived from multicellular organisms may also be used as hosts.
  • any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • interest has been greatest in vertebrate cells, and propagation of vertebrate in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973).
  • useful 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 i.a. Protein Sciences, 1000 Research Parkway, Meriden, Conn. 06450, U.S.A. and from Invitrogen), and MDCK cell lines.
  • an especially preferred cell line is S 2 available from Invitrogen, PO Box 2312, 9704 CH Groningen, The Netherlands.
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
  • control functions on the expression vectors are often provided by viral material.
  • promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40).
  • the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication.
  • promoter or control sequences normally associated with the desired gene sequence provided such control sequences are compatible with the host cell systems.
  • An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • an exogenous origin such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • the “set of mutually distinct modified amyloidogenic polypeptides” is a collection of non-identical analouges which have e.g. been selected on the basis of the criteria discussed above (e.g: 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 is contemplated that the set may contain several hundred members.
  • test of members of the set can ultimately be performed in vivo, but a number of in vitro tests can be applied which narrow down the number of modified molecules which will serve the purpose of the invention.
  • T-cell proliferation can be tested by standardized proliferation assays in vitro.
  • a sample enriched for T-cells is obtained from a subject and subsequently kept in culture.
  • the cultured T-cells are contacted with APCs of the subject which have previously taken up the modified molecule and processed it to present its T-cell epitopes.
  • the proliferation of T-cells is monitored and compared to a suitable control (e.g. T-cells in culture contacted with APCs which have processed intact, native amyloidogenic polypeptide).
  • 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.
  • an immunogenic composition comprising at least one analouge which is capable of inducing antibodies against unmodified APP or A ⁇ in an animal species where the unmodified APP or A ⁇ is a self-protein, the method comprising admixing the member(s) of the set which significantly induces production of antibodies in the animal species which are reactive with the APP or A ⁇ with a pharmaceutically and immunologically acceptable carrier and/or vehicle and/or diluent and/or excipient, optionally in combination with at least one pharmaceutically and immunologically acceptable adjuvant.
  • nucleic acid sequences and/or vectors are prepared by methods comprising exercise of a molecular amplification technique such as PCR or by means of nucleic acid synthesis.
  • Alpha1-antichymotrypsin (ACT); Alpha2-macroglobulin; ABAD (A ⁇ -peptide binding alcohol dehydrogenase); APLP1 and 2 (amyloid precursor like protein 1 and 2); AMY117; Bax; Bcl-2; Bleomycin hydrolase; BR1/ABR1; Chromogranin A; Clusterin/apoJ; CRF (corticotropin releasing factor) binding protein; EDTF (endothelial-derived toxic factor); Heparan sulfate proteoglycans; Human collapsin response mediator protein-2; Huntingtin (Huntington's disease protein); ICAM-1; IL-6; Lysosome-associated antigen CD68; P21 ras; PLC-delta 1 (phospholipase C isoenzyme delta 1); Serum amyloid P component (SAP); Synaptophysin; Synuclein (alpha-synuclein or NACP); and TGF-b
  • cerebral amyloid angiopathy is a disease that would be a suitable target for the presently disclosed technology.
  • Molecules comprising a T helper epitope and APP or A ⁇ peptides representing or including B-cell epitopes linked covalently to a non-immunogenic polymer molecule acting as a vehicle, e.g. a multivalent activated poly-hydroxypolymer, will, as mentioned above, function as a vaccine molecule that only contains the immunologically relevant parts, can be obtained, and are interesting embodiments in variants d and e disclosed above.
  • Promiscuous or so-called universal T-helper epitopes can be used if e.g. the target for the vaccine is a self-antigen such as APP or A ⁇ .
  • elements that enhance the immunological response could be also co-coupled to the vehicle and thereby act as an adjuvant.
  • Such elements could be mannose, tuftsin, muramyl dipeptide, CpG motifs etc. In that case, subsequent adjuvant formulation of the vaccine product might be unnecessary and the product could be administered in pure water or saline.
  • CTL cytotoxic T cell
  • the ratio of B-cell epitopes and T-helper epitopes (P2 and P30) in the final product can be varied by varying the concentration of these peptides in the synthesis step.
  • the immunogenic molecule can be tagged with e.g. mannose, tuftsin, CpG-motifs or other immune stimulating substances (described herein) by adding these, if necessary by using e.g. aminated derivatives of the substances, to the carbonate buffer in the synthesis step.
  • an insoluble activated polyhydroxy polymer is used to combine the peptides containing the APP or A ⁇ B-cell epitope and the T-helper epitopes it can, as mentioned above be performed as a solid phase synthesis and the final product can be harvested and purified by wash and filtration.
  • the elements to be coupled to a tresyl activated polyhydroxypolymer (peptides, tags etc) can be added to the polyhydroxypolymer at low pH, e.g. pH 4-5, and allowed to be equally distributed in the “gel” by passive diffusion. Subsequently, the pH can be raised to pH 9-10 to start the reaction of the primary amino groups on the peptides and tags to the tresyl groups on the polyhydroxy polymer. After coupling of peptides and e.g. immune stimulating elements the gel is grinded to form particles of suitable size for immunization.
  • Such an immunogen therefore comprises
  • each of the at least first and at least second amino acid sequences are coupled to a pharmaceutically acceptable activated polyhydroxypolymer carrier.
  • polyhydroxypolymer is intended to have the same meaning as in WO 00/05316, i.e. the polyhydroxypolymer can have exactly the same characteristics as is specifically taught in that application. Hence, the polyhydroxypolymer can be water soluble or insoluble (thus requiring different synthesis steps during preparation of the immunogen).
  • the polyhydroxypolymer can be selected from naturally occurring polyhydroxy compounds and synthetic polyhydroxy compounds.
  • polyhydroxypolymers are polysaccharides selected from acetan, amylopectin, gum agar-agar, agarose, alginates, gum Arabic, carregeenan, cellulose, cyclodextrins, dextran, furcellaran, galactomannan, gelatin, ghatti, glucan, glycogen, guar, karaya, konjac/A, locust bean gum, mannan, pectin, psyllium, pullulan, starch, tamarine, tragacanth, xanthan, xylan, and xyloglucan. Dextran is especially preferred.
  • the polyhydroxypolymer can also be selected from highly branched poly(ethyleneimine)(PEI), tetrathienylene vinylene, Kevlar (long chains of poly-paraphenyl terephtalamide), Poly(urethanes), Poly(siloxanes), polydimethylsiloxane, silicone, Poly(methyl methacrylate) (PMMA), Poly(vinyl alcohol), Poly(vinyl pyrrolidone), Poly(2-hydroxy ethyl 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), Polylactides (PLA), Polyglycolides (PGA), Poly(lactide-co-glycolides) (PLGA), Polyanhydrides, and Polyorthoest
  • the (weight) average molecular weight of the polyhydroxypolymer in question is typically at least 1,000, such as at least 2,000, preferably in the range of 2,500-2,000,000, more preferably in the range of 3,000-1,000,000, in particular in the range of 5,000-500,000. It has been shown in the examples that polyhydroxypolymers having an average molecular weight in the range of 10,000-200,000 are particularly advantageous.
  • the polyhydroxypolymer 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, in particular 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, fulfils the requirements with respect to water solubility.
  • the ratio between C (carbon atoms) and OH groups (hydroxy groups) of the unactivated polyhydroxypolymers is in the range of 1.3 to 2.5, such as 1.5-2.3, preferably 1.6-2.1, in particular 1.85-2.05.
  • a C/OH ratio of the unactivated polyhydroxypolymer represents a highly advantageous level of hydrophilicity.
  • Polyvinylalcohol and polysaccharides are examples of polyhydroxypolymers which fulfil this requirement. It is believed that the above-mentioned ratio should be roughly the same for the activated polyhydroxypolymer as the activation ratio should be rather low.
  • polyhydroxypolymer carrier is intended-to denote the part of the immunogen that carries the amino acid sequences.
  • the polyhydroxypolymer carrier has its outer limits where the amino acid sequences can be cleaved of by a peptidase, e.g. in an antigen presenting cell that is processing the immunogen.
  • the polyhydroxypolymer carrier can be the polyhydroxypolymer with an activation group, where the bond between the activation group and the amino acid sequence is cleavable by a peptidase in an APC, or the polyhydroxypolymer carrier can be a polyhydroxypolymer with activation group and e.g. a linker such as a single L-amino acid or a number of D-amino acids, where the last part of the linker can bond to the amino acid sequences and be cleaved by a peptidase in an APC.
  • a linker such as a single L-amino acid or a number of D-amino
  • the polyhydroxypolymers carry functional groups (activation groups), which facilitate the anchoring of peptides to the carrier.
  • a wide range of applicable functional groups are known in the art, e.g. tresyl (trifluoroethylsulphonyl), maleimido, p-nitrophenyl cloroformate, cyanogenbromide, tosyl (p-toluenesulfonyl), triflyl (trifluoromethanesulfonyl), pentafluorobenzenesulfonyl, and vinyl sulphone groups.
  • Preferred examples of functional groups within the present invention are tresyl, maleimido, tosyl, triflyl, pentafluorobenzenesulfonyl, p-nitrophenyl cloroformate, and vinylsulphone groups, among which tresyl, maleimido, and tosyl groups are particularly relevant.
  • Tresyl activated polyhydroxypolymers 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.
  • Maleimido activated polyhydroxypolymers can be prepared using p-maleimidophenyl isocyanate as described for activation of dextran in Example 3 of WO 00/05316.
  • maleimido groups could be introduced to a polyhydroxypolymer, such as dextran, by derivatisation of a tresyl activated polyhydroxypolymer (such as tresyl activated dextran (TAD)) with a diamine compound (generally H 2 N—C,H 2 , —NH 2 , where n is 1-20, preferably 1-8), e.g.
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • sulfo-SMCC succinimidyl 4-(p-maleimidophenyl)butyrate
  • maleimide activated polyhydroxypolymers Although the different reagents and routes for activation formally results in slightly different maleimide activated products with respect to the linkage between the maleimide functionality and the remainder of the parent hydroxy group on which activation is performed, all and every are considered as “maleimide activated polyhydroxypolymers”.
  • Tosyl activated polyhydroxypolymers can be prepared using tosyl chloride as described for activation of dextran in Example 2 in WO 00/05316.
  • Triflyl and pentafluorobenzenesulfonyl activated polyhydroxypolymers are prepared as the tosyl or tresyl activated analogues, e.g. by using the corresponding acid chlorides.
  • Cyanogenbromide activated polyhydroxypolymer can be prepared by reacting the polyhydroxypolymer with cyanogenbromide using conventional methods.
  • the resulting functional groups are normally cyanate esters with two hydroxy groups of the polyhydroxypolymer.
  • the degree of activation can be expressed as the ratio between the free hydroxy groups and the activation groups (i.e. functionalised hydroxy groups). It is believed that a ratio between the free hydroxy groups of the polyhydroxypolymer and the activation groups should be between 250:1 and 4:1 in order to obtain an advantageous balance between the hydrophilicity and the reactivity of the polyhydroxypolymer. Preferably the ratio is between 100:1 and 6:1, more preferably between 60:1 and 8:1, in particular between 40:1 and 10:1.
  • Especially interesting activated polyhydroxypolymers for use in the method for producing the generally applicable immunogen according to the invention are tresyl, tosyl and maleimido activated polysaccharides, especially tresyl activated dextran (TAD), tosyl activated dextran (TosAD), and maleimido activated dextran (MAD).
  • TAD tresyl activated dextran
  • TosAD tosyl activated dextran
  • MAD maleimido activated dextran
  • the bond between the polyhydroxypolymer carrier and the amino acid sequences attached thereto are cleavable by a peptidase, e.g. as a peptidase active in the processing of antigens in an APC. It is therefore preferred that the at least first and at least second amino acid sequences are coupled to the activated polyhydroxypolymer carrier via an amide bond or a peptide bond. It is especially preferred that the at least first and at least second amino acid sequences each provide for the nitrogen moiety of their respective amide bond.
  • the polyhydroxypolymer carrier may be substantially free of amino acid residues, necessitating that the activation group provides for part of a peptidase cleavable bond, but as mentioned above, the carrier may also simply include a spacer including at least one L-amino acid. Nevertheless, the at least first and at least second amino acid sequences are normally bound to the activated version of the polyhydroxypolymer via the nitrogen at the N-terminus of the amino acid sequence.
  • the above-described generally applicable immunogen of the present invention can be used in immunization methods essentially as described herein for polypeptide vaccines. That is, all disclosures relating to dosages, mode of administration and formulation of polypeptide vaccines for down-regulating the amyloidogenic polypeptides discussed herein apply mutatis mutandis to the generally applicable immunogens.
  • one preferred embodiment of the present invention entails the use of variants of amyloidogenic polypeptides that are incapable of providing self-derived T H epitopes that may drive an immune response against the amyloidogenic polypeptide.
  • the present invention provides the means of effectively generating a strong regulated immune response towards true self-proteins which potentially could form plaques and cause serious disease in the CNS or in other compartments of the ody.
  • a safe and efficacious human A ⁇ protein therapeutic vaccine will be developed by using this technology for the treatent of AD.
  • AD Alzheimer's disease
  • a disease predicted to cripple the health care system in the next entury could be cured, or such vaccines described could at least constitute an effective therapeutical approach for treatment of the symptoms and progression of this disease.
  • This technique represents a entirely new immunological approach to blocking amyloid deposition in AD and other neurologic diseases as well.
  • Construction 34 contains three identical APP fragments separated by P30 and P2, respectively.
  • Construction 35 contains nine identical APP fragments separated by alternating P30 and P2 epitopes.
  • APP AutoVac constructions Start of End of Position of APP APP P2 epitope Position of segment segment relative to P30 epitope Var. relative to relative to aa 1 of relative to Molecule No.
  • the part of APP, against which it most interesting to generate a response is the 43 amino acid A ⁇ core peptide (A ⁇ -43, corresponding to SEQ ID NO: 2, residues 672-714) that is the main constituent of amyloid plaques in AD brains.
  • This APP fragment is part of all constructions listed above.
  • Variants 1 and 2 comprise a portion of APP upstream of A ⁇ -43 where the model epitopes P2 and P30 have been placed.
  • Variants 1 and 3-8 all comprise the C-100 fragment which has been shown to be neurotoxic—the C-100 fragment corresponds to amino acid residues 714-770 of SEQ ID NO: 2.
  • the epitopes substitutes a part of the C-100 fragment while the in variants 6-8 have been inserted into C-100.
  • Variants 9-35 contain only the core A ⁇ -43 protein.
  • P2 and P30 are fused to either end of A ⁇ -43; in 14-21 P2 and P30 substitutes part of A ⁇ -43; in 22-33 P2 and P30 are inserted into A ⁇ -43; 34 contains three identical A ⁇ -43 fragments spaced by P30 and P2, respectively; 35 contains 9 A ⁇ -43 repeats spaced by alternating P2 and P30 epitopes.
  • Truncated parts of the above-discussed A ⁇ -43 protein can also be employed in immunogenic analogues according to the present invention.
  • the numbers in the parentheses indicate the amino acid stretches of A ⁇ -43 that constitute the relevant fragment—A ⁇ (35-40) is e.g. identical to amino acids 706-711 in SEQ ID NO: 2). All these variants with truncated parts of A ⁇ -43 can be made with the A ⁇ fragments described herein, in particular with variants 9, 10, 11, 12, and 13.
  • the A ⁇ -43 or fragments thereof are mutated.
  • substitution variants where the methionine in position 35 in A ⁇ -43 has been substituted, preferably with leucine or isoleucine, or simply deleted.
  • Especially preferred analogues contain one single methionine that is located in the C-terminus, either because it is naturally occurring in the amyloidogenic polypeptide or foreign T H epitope, or because it has been inserted or added.
  • the part of the analogue that includes the foreign T H epitope is free from methionine, except from the possible C-terminal location of a methionine.
  • all analogues of APP or A ⁇ that are used according to the present invention share the characteristic of merely including one single methionine that is positioned as the C-terminal amino acid in the analogue and that other methionines in either the amyloidogenic polypeptide or the foreign T H epitope are deleted or substituted for another amino acid.
  • One further interesting mutation is a deletion or substitution of the phenylalanine in position 19 in A ⁇ -43, and it is especially preferred that the mutation is a substitution of this phenylalanine residue with a proline.
  • the A ⁇ segment used in the molecule is indicated by amino acid numbers relative to aa 1 of the A ⁇ (1-42/43) molecule, i.e. 1-28 means that fragment 1-28 of A ⁇ (1-42/43) is 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 A ⁇ (1-42/43) are used in the molecule.
  • the position of the A ⁇ segment in the molecule is shown by amino acid positions relative to the first amino acid of the molecule, i.e. 22-49 shows that the A ⁇ fragment in question is positioned from amino acid 22 to amino acid 49 in the molecule, both positions included. Positions of the P2 and P30 epitopes are indicated equivalently. If two or more different A ⁇ fragments are used in the molecule, their positions are all 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.
  • the total length indication of each molecule includes both the A ⁇ fragment(s) 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 it is indicated in the column showing the A ⁇ fragments.
  • One further type of construct is especially preferred. Since one goal of the present invention is to avoid destruction of the cells producing APP whereas removal of A ⁇ is desired, it seems feasible to prepare autovaccine constructs comprising only parts of A ⁇ which are not exposed to the extracellular phase when present in APP. Thus, such constructs would need to contain at least one B-cell epitope derived from the amino acid fragment defined by amino acids 700-714 in SEQ ID NO: 2. Since such a short polypeptide fragment is predicted to be only weakly immunogenic it is preferred that such an autovaccine construct consists of several copies of the B-cell epiope, e.g. in the form of a construct having the structure hown in Formula I in the detailed disclosure of the present nvention, cf. above.
  • amyloid e1 -amyloid ex are x B-cell epitope containing amino acid sequences derived from amino acids 700-714 of SEQ ID NO: 2.
  • a preferred alternative is the above-detailed possibility of coupling the amyloidogenic (poly)peptide and the selected foreign T-helper epitope to via an amide bond to a polysaccharide carrier molecule—in this way multiple presentaions of the “weak” epitope constituted by amino acids 700-714 of SEQ ID NO: 2 become possible, and it also becomes possible to select an optimum ratio between B-cell and T-cell epitopes.
  • 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 primer ME#800 (SEQ ID NO: 9) as template. ME#800 encodes the human abeta-43 fragment with E. coli optimised codons. ME#801 and 802 adds appropriate restriction sites to the fragment.
  • PCR fragment was purified, digested with NcoI and HindIII, purified again and cloned into NcoI-HindIII digested and purified pET28b+ E. coli expression vector.
  • the resulting plasmid encoding wildtype human A ⁇ -43 is named pAB1.
  • Primer ME#806 (SEQ ID NO: 12) contains the sequence encoding the P2 epitope, thus generating a fusion of P2 and Abeta-43 by the PCR reaction.
  • the cloning was performed by making a PCR fragment with 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 is called pAB2.
  • another plasmid was made harbouring the A ⁇ -43 encoding sequence with another T helper epitope, P30, added to the N-terminus. This was done by making 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 is called pAB3.
  • a second A ⁇ -43 repeat is added C-terminally to the P2 epitope of plasmid pAB2 by primer ME#809 (SEQ ID NO: 14).
  • ME#809 at the same time creates a BamHI site immediately after the A ⁇ -43 repeat.
  • a PCR fragment was made with primers ME#178 and ME#809 using pAB2 as template. The fragment was digested with NcoI and HindIII, purified and cloned into NcoI-HindIII digested and purified pET28b+vector. This plasmid is named pAB4.
  • the P30 epitope—A ⁇ -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 and cloned into BamHI-HindIII digested and purified pAB4 plasmid. The resulting plasmid, pAB5, encodes the hAB43+-34 molecule.
  • E. coli K-12 cells strain Top-10 F′ (Stratagene, USA), were used.
  • the pET28b+vector was purchased from Novagen, USA. All primers were synthesised at DNA Technology, Denmark.
  • hAB43+-34 Expression and purification of hAB43+-34.
  • the hAB43+-34 protein encoded by pAB5 was expressed in BL21-Gold (Novagen) E. coli cells as described by the suppliers of the pET28b+system (Novagen).
  • the expressed hAB43+-34 protein was purified to more than 85% purity by washing of 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 hereafter removed by stepwise dialysis against a solution containing decreasing amounts of urea.
  • the final buffer was 10 mM Tris, pH 8.5.
  • mice transgenic for human APP (Alzheimer's precursor protein) were used for the study. These mice, called TgRND8+, express a mutated form of APP that results in high concentration of A ⁇ -40 and A ⁇ -42 in the mouse brains (Janus, C. et. al.)
  • mice The mice (8-10 mice per group) were immunised with either Abeta-42 (SEQ ID NO: 2, residues 673-714, synthesised by means of a standard Fmoc strategy) or the hAB43+-34 variant (construct 34 in the table in Example 1, recombinantly produced) four times at two-week intervals. Doses were either 100 mg for A ⁇ or 50 mg for hAB43+-34. Mice were bled at day 43 (after three injections) and after day 52 (after four injections) and the sera were used to determine the level of anti-A ⁇ -42 specific titres using a direct A ⁇ -42 ELISA.
  • Abeta-42 SEQ ID NO: 2, residues 673-714, synthesised by means of a standard Fmoc strategy
  • the hAB43+-34 variant construct 34 in the table in Example 1, recombinantly produced
  • the antibody titers obtained when immunizing with the hAB43+-34 A ⁇ variant are approximately 4 times and 7.5 times higher after 3 and 4 immunizations, respectively, than the titers obtained when using the unaltered wild-type A ⁇ -42 as an immunogen. This fact is put further in perspective, when considering the fact that the amount of variant used for immunization was only 50% of the amount of wild-type sequence used for immunization.
  • a traditional conjugate vaccine consists of a (poly)peptide coupled covalently to a carrier protein.
  • the peptide contains the B-cell epitope(s) and the carrier protein provides T-helper epitopes.
  • most of the carrier protein will normally be irrelevant as a source for T-helper epitopes, since only aminor part of the total sequence contains the relevant T-helper epitopes.
  • Such epitopes can be defined and synthesized as peptides of e.g. 12-15 amino acids. If these peptides are linked covalently to peptides containing the B-cell epitopes, e.g. via a multivalent activated poly-hydroxypolymer, a vaccine molecule that only contains the relevant parts can be obtained. It is further possible to provide a vaccine conjugate that contains an optimized ratio between B-cell and T-cell epitopes.
  • Poly-hydroxypolymers such as dextran, starch, agarose etc. can be activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride), either by means of a homogenous synthesis (dextran) dissolved in N-methylpyrrolidinone (NMP) or by means of a heterogeneous synthesis (starch, agarose, cross-linked dextran) in e.g. acetone.
  • tresyl chloride 2,2,2-trifluoroethanesulfonyl chloride
  • NMP dry N-methylpyrrolidinone
  • the flask is placed into the oil bath agaom, still with magnetic stirring, and tresyl chloride (2.764 ml, 25 mmol) is added dropwise. After 15 min, dry pyridine (anhydrous, 2.020 ml, 25 mmol) is added drop-wise. The flask is removed from the oil bath and stirred for 1 hour at room temperature.
  • the product Tesyl Activated Dextran, TAD
  • the supernatant is decanted and the precipitate is harvested in 50 ml polypropylene tubes in a centrifuge at 2000 rpm. The precipitate is dissolved in 50 ml 0.5% acetic acid, dialyzed 2 times against 5000 ml 0.5% acetic acid and freeze dried.
  • TAD can be stored as a freeze dried powder at ⁇ 20° C.
  • An insoluble poly-hydroxypolymer such as agarose or croos-linked dextran can be tresyl activated by making a suspension of the poly-hydroxypolymer in e.g. acetone and perform the synthesis as a solid phase synthesis.
  • the activated poly-hydroxypolymer can be harvested by filtration. Suitable methods are reported in e.g. Nilsson K and Mosbach K (1987), Methods in Enzymology 135, p. 67, and in Hermansson G T et al. (1992), in “Immobilized Affinity Ligand Techniques”, Academic Press, Inc., p. 87.
  • the peptides are prepared by means of a standard Fmoc strategy, where the conventional Fmoc-Lys(Boc)-OH has been substituted with Fmoc-Lys(Dde)-OH (obtained from Novabiochem, cat. no. 04-12-1121), i.e. the F-amino group in lysine is protected with Dde instead of Boc.
  • the ratio between B-cell epitopes (A ⁇ ) and 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 tagged with e.g. mannose (so as to target the conjugate to APCs) by adding aminated mannose to the carbonate buffer in the synthesis step.
  • the coupling to the polymer can be performed as a solid phase synthesis and the final product is harvested and purified by wash and filtration.
  • TAD (10 mg) is dissolved in 100 ⁇ l H 2 O and 1000 ⁇ l carbonate buffer, pH 9.6, containing 1-5 mg peptide A (any immunogenic peptide of interest!), 1-5 mg P2 (diphtheria toxoid P2 epitope) and 1-5 mg P30 (diphtheria toxoid P30 epitope) is added.
  • the pH value is measured and adjusted to 9.6 using 0.1 M HCl. After 2.5 hours at room temperature the solution is freeze dried immediately hereafter.
  • the freeze-dried product is dissolved in H 2 O and dialysed extensively against H 2 O or desalted on a gelfiltration column before the final freeze-drying.
  • Such immunogens have been utilised by the inventors with a short C-terminal fragment of the Borrelia burgdorferi protein OspC as “peptide A” and a diptheria toxoid epitope (P2 or P30) as a peptide B.
  • the results of immunization studies with this antigen revealed that only the immunogen of the invention including the OspC fragment and a foreign diptheria epitope matching the MHC haplotype of the vaccinated mice were capable of inducing antibodies reactive with OspC in these mice.
  • Apolipoprotein E High-Avidity Binding to A ⁇ and Increased Frequency of Type 4 Allele in Late-Onset Familial Alzheimer Disease. Proceedings of the National Academy of Sciences U.S.A., 90, 1977-1981.
  • a) is a polyamino acid that consists of at least one copy of a subsequence of residues 672-714 in SEQ ID NO: 2, wherein the foreign T H epitope is incorporated by means of amino acid addition and/or insertion and/or deletion and/or substitution, wherein the subsequence is selected from the group consisting of residues 1-42, residues 1-40, residues 1-39, residues 1-35, residues 1-34, residues 1-28, residues 1-12, residues 1-5, residues 13-28, residues 13-35, residues 17-28, residues 25-35, residues 35-40, residues 36-42 and residues 35-42 of the amino acid sequence consisting of amino acid residues 673-714 of SEQ ID NO: 2; and/or
  • b) is a polyamino acid that contains the foreign TH epitopes and a disrupted APP or AP sequence so that the analogue does not include any subsequence of SEQ ID NO: 2 that binds productively to MHC class II molecules initiating a T-cell response;
  • c) is a polyamino acid that comprises the foreign T H epitope and APP or A ⁇ derived amino acids, and comprises 1 single methionine residue located in the C-terminus of the analogue, wherein other methionine residues in APP or A ⁇ and in the foreign T H epitope have been substituted or deleted, and preferably have been substituted by leucin or isoleucine; and/or
  • d) is a conjugate comprising a polyhydroxypolymer backbone to which is separately coupled a polyamino acid as defined in a) and/or a polyamino acid as defined in b) and/or a polyamino acid as defined in c); and/or
  • e) is a conjugate comprising a polyhydroxypolymer backbone to which is separately coupled 1) the foreign T H epitope and 2) a polyamino acid selected from the group consisting of a subsequence as defined in a), a disrupted sequence of APP or A ⁇ as defined in b), and an APP or A ⁇ derived amino acid sequence that comprises 1 single methionine residue located in the C-terminus, wherein other methionine residues in APP or A ⁇ and in the foreign T H epitope have been substituted or deleted, and preferably have been substituted by leucin or isoleucine.
  • At least one first moiety is introduced which effects targeting of the analogue to an antigen presenting cell (APC) or a B-lymphocyte, and/or
  • At least one second moiety is introduced which stimulates the immune system, and/or
  • At least one third moiety is introduced which optimizes presentation of the analogue to the immune system.
  • the foreign T-cell epitope is promiscuous, such as a foreign T-cell epitope which is selected from a natural promiscuous T-cell epitope and an artificial MHC-II binding peptide sequence.
  • T-cell epitope is selected from a Tetanus toxoid epitope such as P2 or P30, a diphtheria toxoid epitope, an influenza virus hemagluttinin epitope, and a P. falciparum CS epitope.
  • analogue comprises at most 9 consecutive amino acids of SEQ ID NO: 2., such as at most 8, at most 7, at most 6, at most 5, at most 4, and at most 3 consecutive amino acids.
  • the analogue comprises at least one subsequence of SEQ ID NO: 2 so that each such at least one subsequence of SEQ ID NO: 2 independently consists of amino acid stretches selected from the group consisting of 9 consecutive amino acids of SEQ ID NO: 2, 8 consecutive amino acids of SEQ ID NO: 2, 7 consecutive amino acids of SEQ ID NO: 2, 6 consecutive amino acids of SEQ ID NO: 2, 5 consecutive amino acids of SEQ ID NO: 2, 4 consecutive amino acids of SEQ ID NO: 2, and 3 consecutive amino acids of SEQ ID NO: 2.
  • presentation to the immune system is effected by having at least two copies of an A ⁇ derived fragment or the analogue covalently of non-covalently linked to a carrier molecule capable of effecting presentation of multiple copies of antigenic determinants.
  • an effective amount of the analogue is administered to the animal via a route selected from the parenteral route such as the intracutaneous, the subcutaneous, and the intramuscular routes; the peritoneal route; the oral route; the buccal route; the sublinqual route; the epidural route; the spinal route; the anal route; and the intracranial route.
  • parenteral route such as the intracutaneous, the subcutaneous, and the intramuscular routes; the peritoneal route; the oral route; the buccal route; the sublinqual route; the epidural route; the spinal route; the anal route; and the intracranial route.
  • nucleic acid(s) introduced is/are 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-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA encapsulated in chitin or chitosan, and DNA formulated with an adjuvant.
  • [0379] 24 A method for treating and/or preventing and/or ameliorating Alzheimer's disease or other diseases and conditions characterized by amyloid deposits, the method comprising down-regulating APP or A ⁇ according to the method of any one of the preceding paragraphs to such an extent that the total amount of amyloid is decreased or that the rate of amyloid formation is reduced with clinical significance.
  • An immunogenic composition comprising an immunogenically effective amount of an analogue according to paragraph 25, the composition further comprising a pharmaceutically and immunologically acceptable carrier and/or vehicle and optionally an adjuvant.
  • the vector according to paragraph 28 which is selected from the group consisting of a plasmid, a phage, a cosmid, a mini-chromosome, and a virus.
  • the transformed cell according to paragraph 33 which is a microorganism 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 S 2 or an SF cell, a plant cell, and a mammalian cell.
  • composition for inducing production of antibodies against amyloid comprising

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