HK1127486B

HK1127486B - Therapeutic vaccine

Info

Publication number: HK1127486B
Application number: HK09105160.4A
Authority: HK
Inventors: Andrea Pfeifer; Claude Nicolau
Original assignee: Ac Immune S.A.
Priority date: 2005-12-12
Filing date: 2006-12-08
Publication date: 2015-09-25

Description

Therapeutic vaccine

[ technical field ] A method for producing a semiconductor device

The present invention relates to methods and compositions for therapeutic and diagnostic use in the treatment of diseases and disorders caused by or associated with amyloid (amyloid) or amyloid-like (amyloid-like) proteins, including amyloidosis, a group of amyloid-related disorders and abnormalities, such as alzheimer's disease.

[ background of the invention ]

Amyloidosis is not a single disease entity, but a diverse group of progressive diseases characterized by extracellular tissue deposition of waxy amyloid protein (called amyloid protein) that accumulates in one or more organs or body systems. As amyloid deposits increase, they begin to interfere with normal organ or body system function. There are at least 15 different types of amyloidosis. The major forms are primary amyloidosis with no known prodromal cause, secondary amyloidosis that occurs after certain other conditions, and hereditary amyloidosis.

Secondary amyloidosis occurs in people with chronic infections or inflammatory diseases such as tuberculosis, bacterial infections known as familial mediterranean fever, bone infections (osteomyelitis), rheumatoid arthritis, small bowel inflammation (granulomatous ileitis), hodgkin's disease and leprosy.

Amyloid deposits generally contain three components. Amyloid fibrils, which comprise approximately 90% of amyloid, comprise one of several different types of proteins. These proteins are capable of folding into so-called "beta sheet" fibrils, a unique protein configuration that displays a congo red binding site, resulting in the unique staining characteristics of amyloid proteins. In addition, amyloid deposits are closely associated with the amyloid P (pentagonal) component (AP), a glycoprotein associated with normal serum amyloid P (sap), and with sulfated glycosaminoglycans (GAGs), complex carbohydrates of connective tissues.

Many senile diseases are based on or associated with amyloid-like proteins and are characterized in part by the accumulation of extracellular deposits of amyloid or amyloid-like substances that contribute to pathogenesis and disease progression. Such diseases include, but are not limited to, neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory, such as Mild Cognitive Impairment (MCI), dementia with Lewy bodies, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome. Other diseases based on or associated with amyloid-like proteins are e.g. progressive supranuclear palsy; multiple sclerosis; Creutzfeld-Jacob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration.

While the pathogenesis of these diseases may be diverse, the characteristic deposits often contain many common molecular components. It is evident that this may be due to local activation of pro-inflammatory pathways, thereby leading to the simultaneous deposition of activated complement components, acute phase reactants, immunomodulators and other inflammatory mediators (McGeer et al, 1994).

Alzheimer's Disease (AD) is a neurological disorder that is thought to be caused primarily by amyloid plaques, an abnormal deposit of protein that accumulates in the brain. In the brain of affected individuals, the most commonly found amyloid type is composed primarily of a β fibrils. Scientific evidence confirms that increased β -amyloid production and its accumulation in plaques will lead to neuronal cell death, contributing to the development and progression of alzheimer's disease. Loss of nerve cells in critical brain regions will in turn lead to a decrease in neurotransmitters and impairment of memory. The proteins primarily responsible for the composition of plaques include the Amyloid Precursor Protein (APP) and the two presenilins (presenilins) (presenilin I and presenilin II). Sequential cleavage of the Amyloid Precursor Protein (APP), which is constitutively expressed in most cells and catabolized by the enzymes β and γ secretases, results in the release of 39 to 43 amino acids of a β peptide. Degradation of APPs may increase their propensity to accumulate in plaques. Especially fragments of A.beta.1-42, which have a high tendency to form aggregates, because of the presence of two very hydrophobic amino acid residues at their C-terminus. Thus, it is believed that the A β (1-42) fragment is primarily involved in Alzheimer's disease and is responsible for the initiation of neuritic plaque formation, and therefore has a high pathogenic potential. There is therefore a need for specific antibodies that can target and eliminate amyloid plaque formation.

The symptoms of alzheimer's disease manifest slowly, while the first symptom may be only mild amnesia. At this stage, the individual may forget the name of the most recent event, activity, familiar person or thing, and may not be able to solve the simple math problem. As the disease progresses, the symptoms will be more noticeable and severe enough for people with alzheimer's disease or their families to seek medical assistance. The mid-stage symptoms of alzheimer's disease include forgetting how to do simple things like grooming and developing questions of speaking, understanding, reading or writing. Patients with advanced alzheimer's disease may become anxious or aggressive, may be lost from home and eventually require complete care.

Currently, the only way to diagnose alzheimer's disease is to identify plaques and tangles in brain tissue at necropsy after death of the individual. Thus, a physician can only make a diagnosis of "likely" or "perhaps" Alzheimer's disease while the person is still alive. Using existing methods, a "potential" diagnosis of Alzheimer's disease is made by several tools, and physicians can diagnose Alzheimer's disease correctly up to 90%. The physician asks questions about the patient's general health, past medical questions, and a history of any difficulties the patient has with performing daily activities. Behavioral tests on memory, problem solving, attention, computing, and language provide information on cognitive deterioration, while medical tests, such as tests on blood, urine, or spinal fluid, and brain scans, provide some further information.

Management of alzheimer's disease consists of both medical-based and non-medical based treatments. Treatments that attempt to alter the underlying course of the disease (delay or reverse progression) have been largely unsuccessful to date. Drugs that restore defects or dysfunctions in the chemical messengers (neurotransmitters) of nerve cells, such as cholinesterase inhibitors (ChEIs), have been shown to improve symptoms. Also, a medicine for treating the psychotic manifestations of Alzheimer's disease is obtained.

Cholinesterase inhibitors, such as Tacrine (Tacrine) and rivastigmine (Rivastgmine), are currently the only class of agents approved by the FDA for the treatment of alzheimer's disease. These agents are drugs that restore defects or dysfunctions in chemical neurotransmission in the brain. ChEIs block enzymatic degradation of neurotransmitters, thereby increasing the amount of chemical messengers available to transmit neural signals in the brain. For some people in the early and middle stages of the disease, the drug tacrine (CoGNEX)Morris Plains, NJ), donepezil (donepezil) (Aricept)Tokyo, JP), rivastigmine (Exelon)East Hanover, NJ) or galantamine (galantamine) (Reminyl)New Brunswick, NJ), may help prevent certain symptoms from getting worse for a limited time. Another drug, memantine (memantine) (memantine hydrochloride (Namenda) has been approvedNew York, NY) for the treatment of moderate to severe alzheimer's disease. In addition, some drugs may help control behavioral symptoms of alzheimer's disease, such as insomnia, restlessness, wandering, anxiety, and depression. Treating these symptoms often makes patients more comfortable and makes it easier for caregivers to take care of them. Unfortunately, despite the apparent therapeutic progress showing that such agents consistently outperform placebo, the disease continues to progress with only a modest average effect on mental function. ChEIs also associated with side effects including gastrointestinal dysfunction, hepatotoxicity and weight loss.

It is expected that advances in understanding brain abnormalities that occur in alzheimer's disease will provide a framework for obtaining new therapeutic targets that will focus more on altering the course and progression of the disease. Many compounds, including anti-inflammatory agents, are being actively investigated. Clinical trials using specific cyclooxygenase inhibitors (COX-2), such as wecker (rofecoxib) and celecoxib (celecoxib), are also ongoing.

Other diseases based on or associated with amyloid-like protein accumulation and deposition are mild cognitive impairment, dementia with Lewy Bodies (LBD), Amyotrophic Lateral Sclerosis (ALS), Inclusion Body Myositis (IBM) and macular degeneration, particularly age-related macular degeneration (AMD).

Mild Cognitive Impairment (MCI) is a general term most often defined as a subtle, but measurable, memory impairment. People with MCI experience greater memory problems than normally expected with aging, but have not yet shown other dementia symptoms, such as impaired judgment or reasoning. MCI is a condition that often reflects the preclinical stages of AD.

It is believed that β -amyloid deposits in the Entorhinal Cortex (EC) play a critical role in the development of Mild Cognitive Impairment (MCI) in the elderly. This is consistent with the observation that CSF-A A β (1-42) levels were significantly reduced when AD became clinically significant. In contrast to CSF-Abeta (1-42), CSF-tau levels increased significantly during the MCI phase and these values subsequently continued to rise, indicating that increased levels of CSF-tau may be helpful in detecting MCI individuals who are expected to develop AD.

Dementia with Lewy Bodies (LBD) is a neurodegenerative disorder that can occur in people over the age of 65, typically causing symptoms of cognitive (thought) impairment, as well as abnormal behavioral changes. Symptoms may include cognitive impairment, neurological symptoms, sleep disorders, and autonomic failure. Cognitive impairment is in most cases a hallmark of LBD. Patients are confused and worsened periodically. Fluctuations in cognitive abilities are often related to varying degrees of attention and alertness. Cognitive impairment and fluctuations in thinking, may change within minutes, hours or days.

Lewy bodies are formed from phosphorylated and non-phosphorylated neurofilament proteins; it contains the synaptoprotein alpha-synuclein (synuclein) and ubiquitin (a protein involved in the elimination of lesions or abnormalities). In addition to lewy bodies, lewy neurites may also occur, which are inclusions in the cell process of the nerve cell. In the brain of patients suffering from DLB, amyloid plaques may form, however, they tend to be fewer in number than seen in alzheimer's patients. Neurofibrillary tangles, another microscopic pathological feature of AD, are not a major feature of DLB, but are also frequently present in addition to amyloid plaques.

Amyotrophic Lateral Sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons. In some ALS patients, dementia or aphasia (ALS-D) may occur. Dementia is most commonly frontotemporal dementia (FTD), many of which have ubiquitin-positive, tau-negative inclusions in neurons in the dentate gyrus and superficial frontotemporal lobes.

Inclusion Body Myositis (IBM) is a disabling disease, commonly found in people over the age of 50, where muscle fibers inflame and begin to atrophy-but where the brain is not involved and the patient retains all of his intelligence. Within the muscle cells of patients suffering from this most common progressive senile muscle disease, two enzymes involved in the production of amyloid-beta protein were found to be increased, wherein amyloid-beta protein was also increased.

Other diseases based on or associated with amyloid-like protein accumulation and deposition are macular degeneration.

Macular degeneration is a common eye disease that causes degeneration of the macula (a paper-like thin tissue located in the back of the eye where photoreceptor cells transmit visual signals to the brain) in the central region of the retina. The sharp, 'forward' image is processed by macular processing. Macular damage results in the development of blind spots, and blurred or distorted images. Age-related macular degeneration (AMD) is the leading cause of vision loss in the united states and is the leading cause of legal blindness in caucasians for people over the age of 65. Approximately 180 million americans aged 40 and older suffer from advanced AMD, with an additional 730 million people suffering from intermediate AMD at substantial risk of losing vision. The government estimates that by 2020, 290 thousands will be suffering from advanced AMD. Victims of AMD often find that, surprisingly and frustratingly, that the cause and treatment of this blind eye condition is known to be that little.

There are two forms of macular degeneration: dry macular degeneration and wet macular degeneration. In 85% of cases of macular degeneration, a dry form is diagnosed in which the cells of the macula slowly begin to disintegrate. Both eyes are usually affected by dry AMD, although one eye may lose vision while the other eye is unaffected. Drusen (drusen), a yellow deposit under the retina, is a common early sign of dry AMD. As the number and size of drusen increase, the risk of developing advanced dry AMD or wet AMD increases. Dry AMD can progress and lead to vision loss without turning into wet disease; however, early dry AMD can also suddenly become wet.

Wet AMD, although it accounts for only 15% of cases, but results in 90% of blind eyes, is considered advanced AMD (no early or intermediate stage wet AMD). Wet AMD is always followed by dry disease. When the dry form deteriorates, some people begin to develop abnormal blood vessel growth behind the macula. These blood vessels are very fragile and will leak fluid and blood (and thus 'wet' macular degeneration) leading to rapid damage to the macula.

Dry AMD initially often causes slight blurring of vision. The center of vision, in particular, may then become blurred, and as the disease progresses, the area becomes larger. If only one eye is affected, no symptoms may be noticed. In wet AMD, straight lines can appear wavy and loss of central vision can occur rapidly.

Diagnosis of macular degeneration typically involves mydriatic eye examination, visual acuity detection, and examination of the fundus using a procedure known as fundoscopy (funduscropy) to aid in the diagnosis of AMD, and-if wet AMD is suspected-fluorescein angiography may also be performed. If the dry form of AMD reaches an advanced stage, there is currently no means to prevent vision loss. However, particularly high doses of antioxidant and zinc formulations may delay or prevent the progression of intermediate AMD to an advanced stage. Marugen root (Macugen)(pegaptanib sodium) injections), photocoagulation and photodynamic therapy can control abnormal blood vessel growth and bleeding in the macula, which is helpful for some people with wet AMD; however, vision that has been lost cannot be recovered by these techniques. If vision has been lost, the presence of low vision aids may be synergisticHelps to improve the quality of life.

One of the earliest signs of age-related macular degeneration (AMD) is the accumulation of extracellular deposits called drusen between the basal layer of the Retinal Pigment Epithelium (RPE) and Bruch's Membrane (BM). Recent studies by Anderson et al have demonstrated that drusen contain amyloid beta protein (Experimental Eye Research 78(2004)243- "256).

Ongoing research continues to explore environmental, genetic, and dietary factors that may contribute to AMD. New therapeutic strategies are also being explored, including retinal cell transplantation, drugs that will prevent or slow the progression of the disease, radiation therapy, gene therapy, computer chips implanted in the retina (which can help stimulate vision), and agents that will prevent the growth of new blood vessels under the macula.

Thus, there is a need for effective therapeutic vaccine compositions and methods for treating complications associated with amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration. There is a particular need for a specific and highly effective therapeutic vaccine and compositions comprising the vaccine that can eliminate physiological manifestations of disease, such as plaque formation associated with amyloid or amyloid-like peptide fibroaggregations.

[ summary of the invention ]

The present invention provides novel methods and compositions for eliciting highly specific and potent immune responses in an organism, particularly in an animal, particularly in a mammal or human, which are capable of preventing or alleviating amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis including, but not limited to, neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration.

In particular, the present invention provides novel methods and compositions for maintaining or improving (particularly restoring, more particularly fully restoring) cognitive memory in a mammal exhibiting an amyloid-associated disease or condition.

It is an object of the present invention to provide therapeutic vaccine compositions, and methods of producing such compositions, for treating diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), particularly diseases or conditions characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), wherein the compositions comprise a peptide fragment from the N-terminal portion of an A β peptide, particularly an A β peptide fragment consisting of a single or repeated sequence fragment of 13 to 15 consecutive amino acid residues from the N-terminal portion of an A β peptide, and particularly residues 1-15 selected from the N-terminal portion of an A β peptide, 1-14 and 1-13, more particularly, an a β peptide fragment consisting of the amino acid residues of seq id NO: 1, including functional equivalents thereof, particularly linked to or incorporated into carrier particles/adjuvants such as liposomes, or the aforementioned a β peptide fragments reconstituted therein.

The contiguous sequence segment of 13 to 15 amino acid residues may be obtained from the N-terminal segment 1-16, 1-17, 1-18 or 1-20 of the a β peptide, in particular from the sequence as set forth in SEQ ID NO: 2 and SEQ ID NO: 5, and which can be interrupted by deletion of one to three amino acid residues to produce a sequence fragment of 13 to 15 amino acid residues, wherein the deleted amino acid residues can be contiguous amino acid residues or residues separated from each other by at least 1 amino acid residue, in particular residues which are not negatively charged if the overall net charge of the antigenic peptide molecule is desired to be negative, or residues which are not positively charged if the overall net charge of the antigenic peptide molecule is desired to be positive. According to the invention, the continuous sequence fragment of 13 to 15 amino acid residues may be repeated 2 to 50 times, in particular 2 to 30 times, more particularly between 2 and 20 times, even more particularly between 2 and 16 times, especially 2 to 10 times, in the antigenic construct.

In one embodiment of the invention, the contiguous stretch of 13 to 15 amino acid residues is used in the form of a polymer selected from the group consisting of 2-mers, 3-mers, 4-mers, 5-mers, 6-mers, 7-mers, 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers, 16-mers, 20-mers, 30-mers and 50-mers.

In another embodiment, the invention provides therapeutic vaccine compositions for the treatment of diseases and disorders and methods of producing such compositions, the disease or disorder is caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), in particular diseases or conditions characterised by a loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI) (see further details below), the compositions and methods employ A β peptide fragments from the N-terminal portion of A β peptide, in particular from 1 to 15, 2 to 15, 3 to 15, 1 to 14, 2 to 14, 1 to 13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1-15(Δ 12), 15(Δ 13), 15(Δ 14), more particularly an a β peptide fragment consisting of the amino acid residues of SEQ ID NO: 1, and SEQ ID NO: 3 (Δ 14) from 1 to 16(Δ 14).

The invention also includes peptide fragments substantially identical to the above-mentioned fragments and having substantially the same biological activity as the fragments, in particular peptide fragments which are conservatively modified variants of the fragments, wherein the alteration results in the substitution of one or more amino acids, in particular 1 to 10 amino acids, more in particular 1 to 6 amino acids, still more in particular 1 to 4 amino acids, especially 1 to 3 amino acids, with a chemically similar amino acid. Conservative substitution tables provide functionally similar amino acids, which are well known in the art and described below. Conservative substitutions are preferably made in such a way that the overall net charge of the peptide, and hence the charge distribution on the peptide molecule, remains substantially unchanged.

In one embodiment of the invention, at least one, in particular 2, more in particular 3 or even all of the negatively charged amino acid residues 1, 3, 7, 11 can be replaced with a chemically similar negatively charged amino acid. In particular, Asp at positions 1 and 7 may be replaced with Glu, respectively, and Glu at positions 9 and 11 may be replaced with Asp, respectively.

In a particular embodiment of the invention, therapeutic vaccine compositions and methods of producing such compositions are provided, the compositions comprising an A β peptide fragment from the N-terminal portion of an A β peptideIn particular from 1 to 15, 2 to 15, 3 to 15, 1 to 14, 2 to 14, 1 to 13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1-15 (. DELTA.12), 15 (. DELTA.13) and 15 (. DELTA.14) amino acid residues, and A.beta.peptide fragments_1-16(Δ15)Peptide antigens, more particularly A beta_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly A beta_1-14Peptide antigens, in particular Abeta_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 shown in SEQ ID NO: 3 (Δ 14), for treating diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), particularly diseases or conditions characterized by loss of cognitive memory capacity (e.g., Mild Cognitive Impairment (MCI)).

In a particular embodiment of the present invention, there are provided therapeutic vaccine compositions and methods of producing therapeutic vaccine compositions for maintaining or improving, particularly completely restoring, cognitive memory in an animal, particularly a mammal or a human, suffering from memory impairment, using an a β peptide fragment from the N-terminal part of the a β peptide, particularly a peptide fragment consisting of a peptide selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1-15 (. DELTA.12), 15 (. DELTA.13) and 15 (. DELTA.14) amino acid residues, particularly A.beta.peptide fragment_1-16(Δ15)Peptide antigens, more particularly A beta_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly A beta_1-14Peptide antigens, in particular Abeta_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 provided in SEQ id no: 3 (Δ 14) from amino acid residues 1-16(Δ 14).

It is a further object of the present invention to provide methods of treating diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), particularly diseases or conditions characterized by loss of cognitive memory capacity such as Mild Cognitive Impairment (MCI), by administering to an animal, particularly a mammal or human, a vaccine composition according to the present invention and as described herein.

In a particular embodiment of the present invention, there is provided a method for maintaining or increasing cognitive memory capacity, in particular fully restoring cognitive memory capacity, in an animal, in particular a mammal or a human suffering from memory impairment by administering to the animal, in particular the mammal or the human, a vaccine composition according to the present invention and as described herein.

It is a further object of the present invention to provide therapeutic vaccine compositions and methods of producing such compositions, and methods of treating diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, neurological disorders such as Alzheimer's Disease (AD), particularly diseases or conditions characterized by loss of cognitive memory capacity such as Mild Cognitive Impairment (MCI), using A β peptide antigens according to the present invention, particularly A β peptide fragments from the N-terminal portion of A β peptide, particularly from a member selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16 (. DELTA.12) 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1-15 (. DELTA.12), 15 (. DELTA.13) and 15 (. DELTA.14), particularly A.beta.peptide fragment_1-16(Δ15)Peptide antigens, more particularly A beta_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly A beta_1-14Peptide antigens, in particular Abeta_1-15A peptide antigen, in particular a peptide consisting of the amino acid sequence set forth in SEQ id no: 1 and amino acid residues 1-15 provided in SEQ ID NO: 3 (Δ 14), wherein said a β peptide antigen is modified, wherein said modification enables it to maintain and stabilize a defined conformation characterized by an equilibrium proportion of α -helices and/or β -sheets and/or random coil portions, and to induce a highly specific immune response in a treated animal.

According to the invention and as described in the preceding text, when administered to an animal, in particular a mammal, especially a human, antibodies of the non-inflammatory Th2 subtype, such as the IgG1 and IgG2b isotypes, and/or antibodies of the IgG subtype independent of T-cells, such as IgG3, are predominantly produced and/or do not cause a significant increase in inflammatory markers in the brain, in particular inflammatory markers selected from IL-1 β, IL-6, IFN- γ and TNF α.

In a further embodiment of the invention, the vaccine according to the invention and as described hereinbefore, when administered to an animal, in particular a mammal, especially a human, causes a significant reduction of insoluble, plaque-associated a β 1-40 and a β 1-42 in the brain.

In a further embodiment of the invention, the vaccine according to the invention and as described in the foregoing, when administered to an animal, in particular a mammal, especially a human, results in a significant reduction of the level of soluble a β 1-42 in the brain.

There is also provided a vaccine according to the invention and as described hereinbefore, which when administered to an animal, particularly a mammal or a human, suffering from an amyloid-related condition characterized by a loss of cognitive memory capacity, causes an increase in the retention of cognitive memory capacity. The present invention also relates to a vaccine according to the present invention and as described in the foregoing, which when administered to an animal, in particular a mammal or a human, suffering from an amyloid-related condition characterized by a loss of cognitive memory, results in a complete recovery of cognitive memory.

In particular, an A.beta.peptide antigen according to the invention and as described hereinbefore, in particular an A.beta.peptide fragment derived from the N-terminal part of an A.beta.peptide, in particular from a peptide selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1-15 (. DELTA.12), 15 (. DELTA.13) and 15 (. DELTA.14) amino acid residues, particularly A.beta.peptide fragment_1-16(Δ15)Peptide antigens, more particularly A beta_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly A beta_1-14Peptide antigens, in particular Abeta_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 provided in SEQ id no: 3 (Δ 14) is bound to, incorporated into, or reconstituted in a carrier, e.g., a vesicle, particle or molecule, particularly a liposome.

The immunogenic composition of the invention may comprise liposomes made by reconstituting the liposomes in the presence of purified or partially purified or modified antigenic peptides according to the invention. In addition, peptide fragments can be reconstituted within liposomes. The invention also includes antigenic peptide fragments that have been modified to increase their antigenicity. For example, the antigenic portion and adjuvant can be linked to the peptide, or mixed with the peptide. Examples of antigenic moieties and adjuvants include, but are not limited to, lipophilic muramyl dipeptide derivatives, non-ionic block polymers, aluminum hydroxide or aluminum phosphate adjuvants, and mixtures thereof.

In another embodiment of the present invention, the method of the present inventionAn a β peptide antigen according to the invention and as described hereinbefore, in particular an a β peptide fragment derived from the N-terminal part of the a β peptide, is modified with a lipophilic or hydrophobic moiety which facilitates insertion into the lipid bilayer of the liposome carrier/immunoadjuvant, in particular by a peptide fragment selected from 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1-15 (. DELTA.12), 15 (. DELTA.13) and 15 (. DELTA.14) amino acid residues, particularly A.beta.peptide fragment_1-16(Δ15)Peptide antigens, more particularly A beta_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly A beta_1-14Peptide antigens, in particular Abeta_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 provided in SEQ ID NO: 3 (Δ 14), wherein the lipophilic or hydrophobic moiety is in particular a lipophilic or hydrophobic moiety which functions as an anchor for anchoring the peptide in the liposome bilayer and has a size which results in the localization and stabilization of the peptide at a position immediately adjacent to the liposome surface.

In a further embodiment of the invention, the lipophilic or hydrophobic moiety is a fatty acid, triglyceride or phospholipid, especially a fatty acid, triglyceride or phospholipid in which the carbon backbone of the fatty acid has at least 10 carbon atoms. In particular, lipophilic or hydrophobic moieties are fatty acids having a carbon backbone of at least about 14 carbon atoms and no more than about 24 carbon atoms, wherein the number of carbon atoms per carbon atom falling within this range is also a part of the present invention. More particularly, the lipophilic or hydrophobic moiety has a carbon backbone of at least 14 carbon atoms, especially 16 carbon atoms. Examples of hydrophobic moieties include, but are not limited to, palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, and linolenic acid. In one embodiment of the invention, the lipophilic or hydrophobic moiety is palmitic acid.

In a further embodiment of the invention, the hydrophobic moiety is palmitic acid and the liposome preparation may additionally contain adjuvants, such as microcapsules of lipid a, alum, calcium phosphate, interleukin 1 and/or polysaccharides and proteins, in particular detoxified lipid a, such as monophosphoryl or diphosphoryl lipid a or alum.

It is a further object of the present invention to provide therapeutic vaccine compositions using immunogenic antigenic peptides according to the present invention and as described hereinbefore, as well as methods of producing such compositions, for the treatment of diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), particularly for maintaining, increasing or restoring cognitive memory capacity in an animal, particularly a mammal or a human, suffering from memory impairment; and methods of treating said amyloidosis wherein the β -amyloid peptide antigen is a palmitoylated a β peptide antigen, particularly a palmitoylated a β peptide fragment derived from the N-terminal portion of a β peptide, particularly a peptide fragment selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); palmitoylated a β peptide fragments consisting of amino acid residues 1-15(Δ 12), 15(Δ 13), 15(Δ 14), in particular palmitoylated a β peptide fragments_1-16(Δ15)Peptide antigens, more particularly palmitoylated a β_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly palmitoylated a β_1-14Peptide antigens, in particular palmitoylated a β_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 provided in SEQ ID NO: 3 (Δ 14) by covalently linking palmitoyl residues, particularly 2 to 4 palmitoyl residues, more particularly 4 palmitoyl residuesAnd modifications in which the palmitoyl residue is coupled to each end of the peptide antigen via one or more, particularly via one or two, suitable amino acid residues, such as lysine, glutamic acid or cysteine, or any other amino acid residue suitable for coupling a palmitoyl residue to an antigenic peptide.

In one embodiment of the invention, 2 or more palmitoylated a β peptide antigen molecules modified by covalently linking palmitoyl residues at each end of the peptide are reconstituted in a single liposome.

The present invention provides novel methods and immunogenic compositions comprising immunogenic antigenic peptides that, when administered to an animal, particularly a mammal or human, suffering from an amyloid-associated condition, particularly a condition characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), induce an immune response in that animal or human. Treatment with the therapeutic vaccine according to the invention results in the maintenance or enhancement of cognitive memory capacity, in particular the complete restoration of cognitive memory capacity.

It is another object of the present invention to provide therapeutic vaccine compositions and methods of producing such compositions using immunogenic antigenic peptides for inducing an immune response in an animal, particularly a mammal or a human; and provides a therapeutic vaccine composition comprising an A.beta.peptide antigen according to the invention and as described hereinbefore, in particular a palmitoylated fragment of an A.beta.peptide derived from the N-terminal part of the A.beta.peptide, in particular from a peptide selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13, 1-16 (. DELTA.2), 1-16 (. DELTA.4), 1-16 (. DELTA.5), 1-16 (. DELTA.6), 1-16 (. DELTA.8), 1-16 (. DELTA.9), 1-16 (. DELTA.10), 1-16 (. DELTA.12), 16 (. DELTA.13), 16 (. DELTA.14), 1-16 (. DELTA.15), 1-15 (. DELTA.2), 1-15 (. DELTA.4), 1-15 (. DELTA.5), 1-15 (. DELTA.6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); palmitoylated a β peptide fragments consisting of amino acid residues 1-15(Δ 12), 15(Δ 13), 15(Δ 14), in particular palmitoylated a β peptide fragments_1-16(Δ15)Peptide antigens, more particularly palmitoylated a β_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularlyIs palmitoylated A beta_1-14Peptide antigens, in particular palmitoylated a β_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 provided in SEQ ID NO: 3 (Δ 14) in an animal, particularly a mammal or a human, suffering from a condition associated with amyloid and characterized by a loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), said induction of an immune response allowing to maintain or increase, particularly to fully restore cognitive memory capacity of the treated animal or human.

An antigenic peptide as hereinbefore described, particularly an A.beta.peptide fragment derived from the N-terminal part of an A.beta.peptide, particularly a fragment of an A.beta.peptide selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1-15 (. DELTA.12), 15 (. DELTA.13) and 15 (. DELTA.14) amino acid residues, particularly A.beta.peptide fragment_1-16(Δ15)Peptide antigens, more particularly A beta_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly A beta_1-14Peptide antigens, in particular Abeta_1-15A peptide antigen, in particular a peptide consisting of the amino acid sequence set forth in SEQ id no: 1 and amino acid residues 1-15 provided in SEQ ID NO: 3 (Δ 14) is also part of the present invention.

Also part of the invention are palmitoylated a β peptide antigens according to the invention and as described hereinbefore, in particular palmitoylated a β peptide fragments derived from the N-terminal part of a β peptide, in particular from a peptide selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1 to 15 (. DELTA.12), 15 (. DELTA.13),15 (. DELTA.14) amino acid residue, in particular palmitoylated A.beta.peptide fragments_1-16(Δ15)Peptide antigens, more particularly palmitoylated a β_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly palmitoylated a β_1-14Peptide antigens, in particular palmitoylated a β_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 shown in SEQ ID NO: 3 (Δ 14), said peptide antigen being modified by covalently linking palmitoyl residues, in particular 2 to 4 palmitoyl residues, more in particular 4 palmitoyl residues, wherein the palmitoyl residues are coupled to each end of the peptide antigen via one or more, in particular via one or two, suitable amino acid residues, such as lysine, glutamic acid or cysteine, or any other amino acid residue suitable for coupling palmitoyl residues to antigen peptides, respectively.

The invention also includes an antigenic peptide as hereinbefore described linked to or incorporated into or reconstituted in a carrier, such as a vesicle, particle or molecule, particularly a liposome, particularly exhibiting an a β peptide fragment from the N-terminal part of an a β peptide linked to or incorporated into or reconstituted in a carrier, particularly a peptide fragment from the N-terminal part of an a β peptide selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1-15 (. DELTA.12), 15 (. DELTA.13) and 15 (. DELTA.14) amino acid residues, particularly A.beta.peptide fragment_1-16(Δ15)Peptide antigens, more particularly A beta_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly A beta_1-14Peptide antigens, peptides and peptidesIs other than A beta_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 provided in SEQ ID NO: 3 (Δ 14), wherein the carrier is, for example, a vesicle, particle or molecule as described hereinbefore.

Without intending to be bound by a particular theory, it is reasonable to speculate that the immune response induced by the therapeutic vaccine composition of the present invention may lead to stimulation of T-cells and other reactive immune cells against immunogenic agents in animals or humans, in particular to the production of highly specific and highly potent antibodies capable of specifically recognizing and binding to specific epitopes from a range of β -amyloid antigens, which antibodies, when bound to the antigen, mediate and/or induce the observable effects of maintaining, increasing and in particular fully restoring cognitive memory in the treated animals or humans.

The invention also provides a vaccine composition which, when administered to an animal, in particular a mammal or a human, induces the production of antibodies in the treated animal or human, wherein said antibodies bind specifically by targeting to epitopes located in the epitope region of beta-amyloid (in particular restricted to amino acid residue aa)_n-aa_mAn epitope region of an A.beta.polypeptide in which n is an integer between 2 and 15, in particular between 5 and 15, more in particular between 8 and 15, even more in particular between 10 and 15, and m is an integer between 3 and 17, in particular between 6 and 17, more in particular between 9 and 17, even more in particular between 11 and 17, where n and m cannot be equal numbers and n must always be a number smaller than m, and the difference between n and m is ≧ 2), directly and specifically binding to beta-amyloid fibrils, for example comprising A.beta.monomeric peptides 1-39; 1-40, 1-41, 1-42 or 1-43, in particular comprising A beta_1-42Fibres of monomeric peptides and/or capable of inducing the dissolution of pre-formed high molecular polymer amyloid fibrils or filaments by amyloid monomeric peptides, particularly β -amyloid monomeric peptides, such as a β monomeric peptides 1-39; 1-40, 1-41, 1-42 or 1-43,in particular A beta_1-42Aggregation of monomeric peptides.

In one embodiment of the invention, the antibody binds to an epitope within the epitope region of beta-amyloid comprising amino acid residues 1-10, in particular amino acid residues 1-9.

The antibodies also specifically bind to soluble amyloid monomeric and oligomeric peptides, particularly peptides selected from the group consisting of a β peptides 1-39; 1-40, 1-41, 1-42 or 1-43, especially Abeta_1-42And inhibits the aggregation of a β monomers or oligomers into high molecular polymer fibrils.

In a further embodiment, the invention provides an antibody, in particular a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which antibody has at least one property selected from the group consisting of inhibiting aggregation, disaggregation, inducing conformational transition, recognition and direct binding to an epitope in a region 4-16, in particular in a region 1-9, especially a combination of two or more of said properties. More particularly, an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional part thereof is provided, said antibody exhibiting a combined reactivity towards the 1-16 and 29-40 regions (more particularly towards the 1-16 and 22-35 regions), i.e. it is capable of recognizing and binding two of said regions, respectively the 1-16 and 29-40 regions or the 1-16 and 22-35 regions, and having at least one of the aforementioned properties, i.e. inhibiting aggregation, disaggregation, inducing conformational transition, particularly two or more of said properties.

Antibodies induced by the vaccine composition according to the invention and obtainable from an immunized animal or an antibody-producing hybridoma cell line are also part of the invention.

In one embodiment, the invention provides an antibody (including any functionally equivalent antibody or functional part thereof), in particular a monoclonal antibody (including any functionally equivalent antibody or functional part thereof), which is obtainable by administration of a vaccine composition according to the invention and as described hereinbefore, in particular comprising a β_1-16(Δ15)Peptide antigens, more particularly A beta_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly A beta_1-14Peptide antigens, especially Abeta_1-15A vaccine composition of a peptide antigen, obtained by immunizing a suitable animal, wherein said antibody is a bifunctional antibody and is useful when administered in combination with an amyloid monomeric peptide, particularly a β -amyloid monomeric peptide, such as a β monomeric peptides 1-38, 1-39; 1-40, 1-41, 1-42 or 1-43, especially Abeta_1-42Inhibit aggregation of A beta monomer into high molecular polymer fibrils when incubated with monomeric peptides, and, in addition, when combined with preformed high molecular polymer amyloid fibrils or filaments formed from amyloid monomeric peptides, particularly beta-amyloid monomeric peptides, e.g., A beta monomeric peptides 1-38, 1-39; 1-40, 1-41, 1-42 or 1-43, especially A beta monomeric peptides_1-42Aggregation of monomeric peptides) can disaggregate preformed polymer fibrils or filaments.

In one embodiment, the invention provides peptides that exhibit high specificity for A β 1-42 monomeric peptides, but exhibit high specificity for A β_1-38、Aβ_1-39、Aβ_1-40And/or Abeta_1-41An antibody, particularly a bifunctional antibody, especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which exhibits substantially no or only little cross-reactivity with the monomeric peptide; in particular antibodies, especially monoclonal antibodies, including any functionally equivalent antibody or functional parts thereof, wherein A.beta.is reacted with_1-38、Aβ_1-39、Aβ_1-40、Aβ_1-41In comparison, the antibody was directed against the amyloid peptide A β_1-42Is up to 100 times, in particular 50 to 100 times, more in particular 80 to 100 times, especially 100 times, and is sensitive to A beta_1-38In comparison, the antibody was directed against the amyloid peptide A β_1-42Is up to 1000 times, in particular 500 to 1000 times, more in particular 800 to 1000 times, especially 1000 times, and is thus capable of inhibiting amyloidogenic monomeric peptides, in particular the amyloid peptide a β, in vitro and in vivo_1-42To (3) is performed.

In another embodiment of the present inventionIn this case, the antibody, in particular the bifunctional antibody, in particular the monoclonal antibody, in particular the bifunctional monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, is directed against the amyloid peptide a β_1-42Has high binding sensitivity and can detect A beta with concentration as low as at least 0.001 microgram_1-42The fibres are, in particular, in a concentration range between 0.5 microgram and 0.001 microgram, more in particular in a concentration range between 0.1 microgram and 0.001 microgram, especially 0.001 microgram.

In a very particular embodiment of the invention, there is provided an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which is capable of detecting A.beta.at concentrations as low as 0.001. mu.g at a minimum_1-42Fibers, and as low as a minimum concentration of 0.1 microgram of Abeta_1-40Fibers, and as low as a minimum concentration of 1 microgram of Abeta_1-38A fiber.

Binding of an antibody according to the invention and as hereinbefore described to an amyloidogenic monomeric peptide, particularly the amyloid form (1-42), results in inhibition of aggregation of the amyloidogenic peptide monomer into polymeric fibrils or filaments. By inhibiting aggregation of amyloidogenic monomeric peptides, the antibodies according to the invention are capable of preventing or slowing down the formation of amyloid plaques, particularly amyloid forms (1-42), which are known to become insoluble by conformational changes and are a major part of amyloid plaques in the brain of a patient animal or human.

In a particular embodiment, the invention relates to a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, having the characteristic properties of an antibody produced by the hybridoma cell line EJ 7H3 deposited at 12/8 of 2005 with DSM ACC 2756.

More particularly, the present invention relates to monoclonal antibodies, including any functionally equivalent antibodies or functional parts thereof, produced by the hybridoma cell line EJ 7H3 deposited at 8/12/2005 with DSM ACC 2756.

It is a further object of the present invention to provide a method for preventing, treating or reducing the effects of amyloidosis by administering a supramolecular antigenic construct according to the present invention to an animal, particularly a mammal or a human, that is affected by, and thus in need of treatment for, a disease or condition associated with amyloid plaque formation, including secondary amyloidosis, as well as age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other diseases based on or associated with amyloid-like proteins, such as progressive supranuclear palsy; multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration, particularly diseases or conditions characterized by a loss of cognitive memory, such as Mild Cognitive Impairment (MCI).

In another embodiment of the invention, methods are provided for preparing vaccine compositions useful for inducing an immune response in an organism, particularly an animal or human afflicted with, and in need of treatment for, a disorder, disease or condition that prevents, treats or reduces amyloidosis, a group of diseases or disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis including but not limited to neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory such as Mild Cognitive Impairment (MCI), Lewy body dementia, Down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam Parkinson-dementia syndrome, and other diseases based on or associated with amyloid-like proteins, such as progressive supranuclear palsy; multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration, particularly diseases or conditions characterized by a loss of cognitive memory, such as Mild Cognitive Impairment (MCI)).

Thus, in a further embodiment of the invention, there is provided a method of making a therapeutic vaccine composition for preventing, treating or ameliorating the effects of amyloidosis, a group of diseases or disorders associated with amyloid plaque formation, including secondary amyloidosis, as well as age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory capacity such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other diseases based on or associated with amyloid-like proteins, such as progressive supranuclear palsy; multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration, particularly diseases or conditions characterized by a loss of cognitive memory, such as Mild Cognitive Impairment (MCI), comprising formulating an antibody according to the invention into a pharmaceutically acceptable form.

In a particular embodiment, the invention utilizes antigen presentation, resulting in improved exposure and stabilization of the preferred antigen conformation, ultimately leading to a highly specific immune response and the production of antibodies with unique properties.

In one embodiment, the invention provides an immunogenic composition comprising a supramolecular antigenic construct comprising a β -amyloid peptide antigen according to the invention and as described hereinbefore, typically the N-terminal part of a β -amyloid peptide, wherein the antigenic peptide is modified in such a way that it is capable of maintaining and stabilizing a defined conformation of the antigen, in particular a conformation characterized by a balanced ratio of random coil, α -helix and β -sheet portions. This defined conformation, when introduced into animals or humans, induces a strong, highly specific immune response.

In another embodiment of the invention, the vaccine composition according to the invention may comprise an inhibitor of complement activation in addition to the a β peptide antigen, in particular an a β peptide antigen of the invention as described in the foregoing. The present invention therefore relates to vaccine compositions and methods for producing such compositions for the treatment of diseases and conditions caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases or conditions associated with amyloid plaque formation, including secondary amyloidosis, and age-related amyloidosis, including but not limited to neurological conditions such as Alzheimer's Disease (AD), particularly diseases or conditions characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), comprising a peptide fragment from the N-terminal portion of an A β peptide, particularly an A β peptide fragment consisting of a single or repeated sequence of 7 to 16 consecutive amino acid residues, particularly 13 to 16 consecutive amino acid residues, particularly from the N-terminal portion of an A β peptide, in particular a β peptide fragments consisting of amino acid residues selected from residues 1-16, 1-15, 1-14 and 1-13 from the N-terminal part of the a β peptide, more in particular a peptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 1, including functionally equivalent fragments thereof, particularly the aforementioned a β peptide fragments linked to or incorporated into a carrier particle/adjuvant (e.g., liposome) or reconstituted therein, and inhibitors of the complement system, particularly inhibitors of the complement pathway selected from soluble forms of membrane regulatory proteins, humanized antibodies against complement proteins, small molecule inhibitors acting at various stages of the complement pathway, and human complement modulators expressed in transgenic animals.

The continuous sequence fragment of 13 to 15 amino acid residues may be repeated 2 to 50 times, in particular 2 to 30 times, more in particular 2 to 20 times, even more in particular 2 to 16 times, especially 2 to 10 times in the construct according to the invention.

In a particular embodiment of the invention, the complement activation inhibitor, which is a component of the aforementioned therapeutic vaccine composition, is a compound selected from the group consisting of soluble human complement receptor 1, anti-human complement protein C5, e.g., a humanized anti-C5 monoclonal antibody or a single chain fragment of a humanized monoclonal antibody, C1-esterase inhibitor-N, and a natural human C1 inhibitor.

The invention also includes a vaccine composition according to the invention as mentioned before, which comprises, in addition to the a β peptide fragment, in particular the a β peptide fragment according to the invention, an allosteric modulator of hemoglobin, wherein said allosteric modulator can trigger O when in erythrocytes₂A decrease in hemoglobin affinity, such that oxygen can subsequently be released to the tissue in a regulated manner.

The present invention therefore relates to vaccine compositions and methods for producing such compositions for the treatment of diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), particularly diseases or conditions characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), comprising a peptide fragment from the N-terminal portion of an A β peptide, particularly an A β peptide fragment consisting of a single or repeated sequence fragment of 7 to 16 consecutive amino acid residues, particularly 13 to 16 consecutive amino acid residues, particularly from the N-terminal portion of an A β peptide, in particular a β peptide fragments consisting of amino acid residues selected from residues 1-16, 1-15, 1-14 and 1-13 from the N-terminal part of the a β peptide, more in particular a peptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 1, including functionally equivalent fragments thereof, particularly the aforementioned a β peptide fragments modified by covalent attachment of palmitoyl residues at each end of the peptide (resulting in 2 to 4, particularly 4 palmitoyl residues), particularly attached toOr incorporated into carrier particles/adjuvants (e.g., liposomes), or reconstituted therein, as previously mentioned, a β peptide fragments, together with trigger O₂Hemoglobin affinity decreases such that oxygen is subsequently released to organ tissue compounds. The continuous sequence fragment of 13 to 15 amino acid residues may be repeated 2 to 50 times, in particular 2 to 30 times, more in particular 2 to 20 times, even more in particular 2 to 16 times, especially 2 to 10 times in the construct according to the invention.

In particular, compounds suitable for use in the compositions according to the invention are those selected from hypolipidemic agents, such as clofibric acid (clofibric acid) or bezafibrate (bezafibrate), including bezafibrate derivatives LR16 and L35, urea derivatives such as [2- [4[ [ (arylamino) carbonyl ] -amino ] phenoxy ] -2-methylpropionic acid, allosteric effectors of hemoglobin.

Adjusting O₂The hemoglobin affinity compound may also be an anionic ligand comprising an allosteric portion of hemoglobin, wherein the anionic ligand comprises an internal pyrophosphate ring, optionally together with a non-toxic cation, such as Ca²⁺And Na⁺The compound of (1).

More particularly, the present invention relates to a therapeutic vaccine composition according to the present invention as mentioned before, comprising besides the a β peptide fragment according to the present invention a phytic acid (IHP) derivative (comprising an internal pyrophosphate ring, optionally together with a non-toxic cation, such as Ca)²⁺And Na⁺)。

In another embodiment, there is provided a vaccine composition according to the invention and as mentioned before, comprising in addition to the a β peptide fragment, in particular a β peptide fragment according to the invention, an inhibitor of the complement activation system (in particular an inhibitor of the complement pathway selected from the group consisting of soluble forms of membrane regulatory proteins, humanized antibodies against complement proteins, small molecule inhibitors acting at various stages of the complement pathway, and human complement regulators expressed in transgenic animals) and an allosteric effector of hemoglobin (which reduces O₂Hemoglobin affinity, make more oxygen followAnd then released to the tissue in a regulated manner).

Thus, the invention also relates to vaccine compositions and methods for producing such compositions for the treatment of diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), particularly diseases or conditions characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), comprising a peptide fragment from the N-terminal portion of an A β peptide, particularly an A β peptide fragment consisting of a single or repeated sequence of 7 to 16 consecutive amino acid residues, particularly 13 to 16 consecutive amino acid residues, particularly from the N-terminal portion of an A β peptide, in particular a β peptide fragments consisting of amino acid residues selected from residues 1-16, 1-15, 1-14 and 1-13 from the N-terminal part of the a β peptide, more particularly the amino acid residues shown in SEQ ID NO: 1, including functionally equivalent fragments thereof, particularly the aforementioned a β peptide fragments modified by covalent attachment of palmitoyl residues at each end of the peptide (resulting in 2 to 4, particularly 4 palmitoyl residues), particularly linked or incorporated into carrier particles/adjuvants such as liposomes or as aforementioned a β peptide fragments reconstituted therein, together with inhibitors of the complement system, particularly inhibitors of complement activation (selected from soluble forms of membrane-regulated proteins, humanized antibodies against complement proteins, small molecule inhibitors acting at various stages of the complement pathway, and human complement regulators expressed in transgenic animals), and compounds, particularly allosteric inhibitors of hemoglobin, which reduce O₂Hemoglobin affinity such that more oxygen is subsequently released to the tissue in a regulated manner).

In another embodiment, there is provided a method of treating an amyloid-associated disease or condition comprising administering to an animal, particularly a mammal, especially a human, suffering from such a disease or condition a therapeutic vaccine composition according to the invention and as described hereinbefore, particularly comprising a β_1-15Peptide antigens, more particularly palmitoylated a β_1-15A vaccine composition of a peptide antigen.

In one embodiment of the invention, administration of the vaccine composition results primarily in the production of antibodies of the non-inflammatory subtype, particularly the non-inflammatory Th2 subtype, e.g. IgG1 and IgG2b isotypes.

In yet another specific embodiment, administration of the vaccine composition results primarily in the production of antibodies of the IgG subtype, particularly the IgG3 isotype, which are independent of T-cells.

In another embodiment of the invention, the administration of the vaccine composition does not result in a significant increase in inflammatory markers in the brain, in particular inflammatory markers selected from the group consisting of IL-1 β, IL-6, IFN- γ and TNF α.

In another embodiment of the invention, administration of the vaccine composition results in a significant reduction in plaque-associated A β 1-40 and A β 1-42 that are insoluble in the brain.

In another embodiment of the invention, administration of the vaccine composition results in a decrease in the level of soluble A β 1-42 in the brain.

In particular, amyloid-associated diseases or conditions are selected from the group including, but not limited to, neurological disorders (e.g., Alzheimer's Disease (AD)), including diseases or conditions characterized by loss of cognitive memory, such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration.

More particularly, the amyloid-associated disease or condition is Alzheimer's disease.

In another particular embodiment of the invention, there is provided a method according to the invention and as described above for treating an amyloid-associated disease or condition, wherein administration of the vaccine composition to an animal, particularly a mammal or a human, suffering from an amyloid-associated condition characterized by a loss of cognitive memory results in the maintenance, increase, particularly complete restoration of cognitive memory.

In another embodiment, a method of treating an amyloid-associated disease or condition is provided, comprising administering to an animal, particularly a mammal, particularly a human, suffering from such a disease or condition a therapeutic vaccine composition comprising an antigenic construct according to the invention and as described above, and an inhibitor of the complement system, wherein the vaccine composition is particularly administered in such a way that the complement inhibitor and the antigenic construct can be administered concomitantly, intermittently or sequentially.

In a particular embodiment, the complement inhibitor is administered prior to vaccination with the antigenic construct, in particular within a time window of no more than 20 hours before the start of vaccination until immediately before the end of vaccination.

In another specific embodiment, following vaccination with the antigenic construct, the complement inhibitor is administered within a time window beginning immediately after vaccination and ending one day after vaccine administration.

In another embodiment of the invention, there is provided a method of preparing a medicament for the treatment of an amyloid-associated disease or condition comprising the use of a vaccine composition according to the invention and as described hereinbefore.

These and other objects, features and advantages of the present invention will become apparent upon reading the following detailed description of the disclosed embodiments and the appended claims.

[ detailed description of the invention ]

As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably and are defined to mean a biomolecule composed of amino acids linked by peptide bonds.

The term "peptide" is a chain of amino acids (typically L-amino acids) in which the alpha carbons of the amino acids are linked via a peptide bond formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amino terminus) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminus) has a free carboxyl group. Thus, the term "amino-terminus" (abbreviated N-terminus) means the free α -amino group on an amino acid at the amino terminus of a peptide, or the α -amino group (imino group when referring to a peptide bond) of an amino acid at any other position in a peptide. Likewise, the term "carboxy terminus" (abbreviated C-terminus) means the free carboxyl group on an amino acid at the carboxy terminus of a peptide, or the carboxyl group of an amino acid at any other position in a peptide.

Generally, the amino acids that make up a peptide are numbered sequentially, beginning at the amino terminus and increasing toward the carboxy terminus of the peptide. Thus, when an amino acid is said to be "behind" another, that amino acid is closer to the carboxy terminus of the peptide than the preceding amino acid.

The term "residue", as used herein, means an amino acid that is incorporated into a peptide by means of an amide bond. The amino acid can be a naturally occurring amino acid, or can include, unless otherwise limited, known analogs (i.e., amino acid mimetics) of a natural amino acid that function in a similar manner to a naturally occurring amino acid. In addition, amide bond mimetics include peptide backbone modifications well known to those skilled in the art.

As used herein, the phrase "consisting essentially of …" excludes any component that would materially alter the basic characteristics of the peptide to which the phrase refers. Thus, the expression that a peptide "consists essentially of …" excludes any amino acid substitution, addition or deletion that would substantially alter the biological activity of the peptide.

Moreover, it will be apparent to those skilled in the art that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage (typically less than 5%, more typically less than 1%) of amino acids in the encoded sequence, as mentioned above, are conservatively modified variations, where such alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables provide functionally similar amino acids, which are well known in the art. The following six groups contain amino acids that are conservative substitutions for each other:

1) alanine (a), serine (S), threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V); and

6) phenylalanine (F), tyrosine (Y), tryptophan (W).

The phrases "isolated" or "biologically pure" mean that the material is substantially or essentially free of the components that normally accompany it as found in its natural state. Thus, the peptides described herein do not contain substances with which they normally bind in their original environment. Typically, the isolated immunogenic peptides described herein are at least about 80% pure, usually at least about 90% pure, and preferably at least about 95% pure, as measured by the intensity of the bands on a silver stained gel.

Protein purity or homogeneity can be indicated by a number of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by development with staining. For some purposes where high resolution is required, HPLC or similar purification methods may be used.

When the immunogenic peptides are relatively short in length (i.e., less than about 50 amino acids), they can generally be synthesized using standard chemical peptide synthesis techniques.

Solid phase synthesis, in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence, is a preferred chemical synthesis method for immunogenic peptides described herein. Techniques for solid phase synthesis are known to those skilled in the art.

Alternatively, immunogenic peptides described herein are synthesized using recombinant nucleic acid methodologies. Typically, this involves making a nucleic acid sequence encoding the peptide, placing the nucleic acid under the control of a particular promoter in an expression cassette, expressing the peptide in a host, isolating the expressed peptide or polypeptide, and, if desired, renaturing the peptide. Sufficient techniques to guide those skilled in the art in the implementation of such procedures can be found in the literature.

Once expressed, the recombinant peptide can be purified according to standard procedures, including ammonium sulfate precipitation, affinity chromatography, column chromatography, gel electrophoresis, and the like. Substantially pure compositions having about 50% to 95% homogeneity are preferred, with 80% to 95% or greater homogeneity being most preferred for use as therapeutic agents.

One skilled in the art will appreciate that the immunogenic peptide may have a conformation that is substantially different from the native conformation of the constituent peptide following chemical synthesis, biological expression or purification. In this case, it is generally necessary to denature and reduce the anti-proliferative peptide and then refold the peptide into the preferred conformation. Methods for reducing and denaturing proteins and inducing refolding are well known to those skilled in the art.

The antigenicity of the purified protein can be confirmed, for example, by demonstrating a reaction with an immune serum, or with an antiserum raised against the protein itself.

As used herein, "a", "an" and "the" are defined to mean "one or more" and include plural unless the context does not apply.

As used herein, "detecting" refers to methods of detecting a biological molecule using known techniques, such as immunochemistry or histology, and refers to determining, either qualitatively or quantitatively, the presence or concentration of the biological molecule under study.

By "isolated" is meant that the biological molecule does not contain at least some of the components with which it naturally occurs.

As used herein, the term "antibody" is a term recognized in the art and is understood to refer to molecules or active fragments of molecules that bind to a known antigen, particularly immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a binding site that immunospecifically binds to an antigen. The immunoglobulins according to the present invention may be immunoglobulin molecules of any class (IgG, IgM, IgD, IgE, IgA and IgY) or type (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subtype.

The term "antibody" within the scope of the present invention is intended to include monoclonal, polyclonal, chimeric, single chain, bispecific, simianized, human and humanized antibodies, and active fragments thereof. Examples of active fragments of molecules that bind to known antigens include Fab and F (ab')₂Fragments, including products of Fab immunoglobulin expression libraries, as well as epitope-binding fragments of any of the antibodies and fragments mentioned above.

These active fragments can be derived from the antibodies of the invention by a variety of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing the Fab fragments is then collected and concentrated by membrane filtration and the like. For further description of the general techniques for isolating active fragments of antibodies, see, e.g., Khaw, b.a. et al j.nuclear.med.23: 1011-; rousseaux et al Methods Enzymology, 121: 663-69, Academic Press, 1986.

"humanized antibodies" means a class of engineered antibodies having CDRs derived from a non-human donor immunoglobulin and the remaining immunoglobulin-derived portion of the molecule derived from one (or more) human immunoglobulin(s). In addition, framework support residues can be altered to retain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art. (see, e.g., Queen et al, Proc. Natl Acad Sci USA, 86: 10029-.

"humanized antibodies" can also be obtained by novel genetic engineering methods that produce affinity-matured human-like polyclonal antibodies in large animals, such as rabbits (http:// www.rctech.com/biovents/therapeutic. php).

The term "monoclonal antibody" is also well understood in the art and refers to an antibody that is produced in large quantities in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies are typically made by fusing normal short-lived antibody-producing B cells with fast-growing cells, such as cancer cells (sometimes referred to as "immortalized" cells). The resulting hybrid cells or hybridomas expand rapidly to form clones that produce large amounts of antibody.

In the context of the present invention, "functionally equivalent antibody" is understood to mean an antibody which shares substantially at least one of the main functional properties with the antibodies mentioned above and described herein, said properties including: for beta-amyloid proteins, especially for Abeta_1-42Proteins, more particularly Abeta_1-42Binding specificity of the 4-16 epitope region of the protein; immunoreactivity in vitro; inhibition of A beta_1-42Aggregation of monomers into high molecular polymer fibrils, and/or pre-formation of Abeta_1-42Polymer fibril depolymerization, and/or β -sheet failure characteristics; and alleviating disorders associated with amyloidosis when administered prophylactically or therapeuticallyEffects, the disorders are a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration. The antibody may be of any type, such as IgG, IgM, IgA, or the like, or any subtype, such as IgG1, IgG2a, or the like, as well as other subtypes mentioned above or known in the art. In addition, the antibodies can be produced by any method, such as phage display, or in any organism or cell line, including bacteria, insects, mammals or other types of cells or cell lines that can produce antibodies with the desired characteristics, such as humanized antibodies. Antibodies can also be formed by combining Fab portions and Fc regions from different species.

The term "antigen" means an entity or fragment thereof that can elicit an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term includes immunogens and regions responsible for antigenicity or antigenic determinants.

As used herein, the term "soluble" means partially or completely dissolved in an aqueous solution.

As used herein, the term "immunogenic" refers to a substance that can elicit or enhance the production of antibodies, T-cells and other reactive immune cells to the immunogenic agent and contribute to an immune response in a human or animal.

An immune response occurs when an individual produces sufficient antibodies, T-cells and other reactive immune cells to administer an immunogenic composition of the invention to alleviate or palliate the condition to be treated.

The term "hybridoma" is well known in the art and understood by those skilled in the art to mean a cell produced by fusion of an antibody-producing cell and an immortalized cell, such as a multiple myeloma cell. The hybrid cell is capable of producing a sustained supply of antibodies. For a more detailed description of the fusion method, see the definition of "monoclonal antibody" above and the examples below.

The term "carrier" as used herein means a structure into which an antigenic peptide or supramolecular construct can be incorporated or to which it can be bound, whereby the antigenic peptide or a portion of the peptide can be presented or exposed to the immune system of a human or animal. Any particle, e.g. vesicle, granule or granule, suitable for use in animal or human therapy may be used as a carrier in the context of the present invention.

The term "carrier" also includes methods of delivery wherein the supramolecular antigenic construct composition comprising the antigenic peptide may be delivered to a desired location by a delivery mechanism. One example of such a delivery system utilizes colloidal metals, such as colloidal gold.

Carrier proteins that may be used in the supramolecular antigenic construct compositions of the present invention include, but are not limited to, maltose binding protein "MBP"; bovine serum albumin "BSA"; keyhole limpet hemocyanin "KLH"; egg white protein; flagellin; thyroglobulin; serum albumin of any species; gamma globulin from any species; a syngeneic cell; syngeneic cells with the Ia antigen; and polymers of D-and/or L-amino acids.

In the supramolecular antigenic construct according to the invention, the liposome may have a dual function, since it may serve as a carrier comprising the aforementioned supramolecular construct and at the same time also have the function of an adjuvant, increasing or stimulating the immune response in the target animal or human to be treated with the therapeutic vaccine according to the inventionShould be used. It is also understood that the supramolecular antigenic construct composition of the invention may also contain additional adjuvants, including but not limited to Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), and other adjuvants such as lipid a, alum, calcium phosphate, interleukin 1, and/or microcapsules of polysaccharides and proteins, particularly detoxified lipid a (e.g., monophosphoryl or diphosphoryl lipid a) or alum, as well as preservatives, diluents, emulsifiers, stabilizers, and other ingredients known in the art and used in vaccines. Furthermore, any adjuvant system known in the art may be used in the compositions of the present invention. Such adjuvants include, but are not limited to, Freund's incomplete adjuvant, Freund's complete adjuvant, polydispersed beta- (1, 4) linked acetylated mannan ("Acemannan" (Acemannan)), Teckers (TITERMAX)(polyoxyethylene polyoxypropylene copolymer adjuvant from CytRx Corporation), modified lipid adjuvants from Chiron Corporation, saponin derivative adjuvants from Cambridge Biotech, killed Bordetella pertussis (Bordetella pertussis), Lipopolysaccharides (LPS) of gram-negative bacteria, large polymeric anions such as dextran sulfate, and inorganic gels such as alum, aluminum hydroxide, or aluminum phosphate.

Furthermore, the term "effective amount" means the amount of antigenic/immunogenic composition that elicits an immune response when administered to a human or animal. The effective amount can be readily determined by one skilled in the art according to routine procedures.

The term "supramolecular antigenic construct" means an antigenic construct according to the invention and as described hereinbefore. In particular, by "supramolecular antigenic construct" is meant an antigenic construct comprising an a β peptide antigen according to the invention and as described hereinbefore, in particular an a β peptide fragment derived from the N-terminal part of an a β peptide, in particular a peptide fragment derived from a β peptide selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1-15 (. DELTA.12), 15 (. DELTA.13), 15 (. DELTA.14) amino acid residues, particularly A.beta.peptide fragments_1-16(Δ15)Peptide antigens, more particularly A beta_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly A beta_1-14Peptide antigens, in particular Abeta_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 provided in SEQ ID NO: 3 (Δ 14), wherein the antigenic peptide is presented by ligation or incorporation into a carrier (e.g., a vesicle, particle or molecule, but particularly a liposome) or reconstitution in a carrier. More particularly, the antigenic peptide according to the invention is modified by means of a lipophilic or hydrophobic moiety, wherein said moiety facilitates insertion into the lipid bilayer of the liposome carrier/immunoadjuvant; the antigenic peptides of the present invention are particularly modified by means of lipophilic or hydrophobic moieties, including but not limited to fatty acids, triglycerides or phospholipids, especially fatty acids, triglycerides or phospholipids in which the carbon backbone of the fatty acid has at least 10 carbon atoms, wherein said moieties act as anchors for anchoring the peptide within the bilayer of the liposome and have a size that results in the localization and stabilization of the peptide in a position close to the surface of the liposome.

For example, the supramolecular antigenic construct composition according to the present invention may be administered parenterally, particularly intraperitoneally, intravenously, subcutaneously, and intramuscularly, in the range of about 1.0 micrograms to 10.0 milligrams per patient (which range is not intended to be limiting). The actual amount of the composition required to elicit an immune response will vary with each individual patient, depending on the immunogenicity of the composition administered and the immune response of the individual. As a result, the specific amount to be administered to an individual will be determined by routine experimentation and based on the training and experience of those skilled in the art.

The supramolecular antigenic constructs according to the invention may be used for the preparation of vaccine compositions for inducing an immune response in an organism, in particular an animal or a human, for preventing, treating or alleviating the effects of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders (such as Alzheimer's Disease (AD)), including diseases or conditions characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration, particularly diseases or conditions characterized by a loss of cognitive memory, such as Mild Cognitive Impairment (MCI).

It is therefore an object of the present invention to provide a method for preventing, treating or alleviating the effects of amyloidosis by administering a supramolecular antigenic construct according to the present invention, in particular a vaccine composition comprising a supramolecular antigenic construct according to the present invention, to an animal, in particular a mammal or a human, affected by and therefore in need of such treatment, wherein said amyloidosis is a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration, particularly diseases or conditions characterized by a loss of cognitive memory, such as Mild Cognitive Impairment (MCI).

In another embodiment of the invention, there is provided a method of preparing a vaccine composition for inducing an immune response in an organism, particularly an animal or human, suffering from a disorder, disease or condition and in need of treatment thereof, for preventing, treating or alleviating the effects of amyloidosis, a group of disorders and conditions associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis including, but not limited to, neurological disorders such as Alzheimer's Disease (AD) including diseases or conditions characterized by loss of cognitive memory capacity such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration, particularly diseases or conditions characterized by a loss of cognitive memory, such as Mild Cognitive Impairment (MCI).

In a further embodiment of the invention, there is thus provided a method of preparing a composition for preventing, treating or alleviating the effects of amyloidosis, wherein the amyloidosis is a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory capacity such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage and amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration, particularly diseases or conditions characterized by a loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), comprising formulating an antibody according to the invention into a pharmaceutically acceptable form.

In one embodiment, the present invention utilizes antigen presentation, resulting in improved exposure and stabilization of the preferred antigen conformation, ultimately leading to a highly specific immune response and the production of antibodies with unique properties.

In one embodiment, the invention provides an immunogenic composition comprising a supramolecular antigenic construct comprising a β -amyloid peptide antigen according to the invention and as described hereinbefore, typically the N-terminal part of a β -amyloid peptide, wherein the antigenic peptide is modified in such a way that it is capable of maintaining and stabilizing a defined conformation of the antigen, in particular a conformation characterized by a balanced ratio of random coil, α -helix and β -sheet portions. This defined conformation induces a powerful and highly specific immune response when introduced into an animal or human.

One way to achieve the desired conformation of the antigenic peptide to be formed and stabilized is to present the antigenic peptide linked or incorporated or reconstituted (partially or completely) in a carrier, such as a vesicle, particle or molecule, or any other means suitable as a carrier/adjuvant for the antigenic peptide. In a particular embodiment of the invention, the antigenic peptide is linked or incorporated or reconstituted in a carrier by weak interactions, such as van der waals forces, hydrophobic or electrostatic interactions, or a combination of two or more of said interactions, such that the peptide is presented in a particular conformation which is maintained and stabilized by restricting the freedom of three-dimensional movement of the antigenic peptide such that conformational changes are prevented or severely restricted.

When vesicles, particles or bodies of particles, such as liposomes, are used as carriers/adjuvants, the composition of the antigenic peptide may be selected such that its overall net charge is the same as that of the surface of the carrier/adjuvant to which the peptide is to be bound. Electrostatic repulsion effects between the same-charged carrier/adjuvant surface and the antigenic peptide, but in particular between the same-charged carrier surface and the amino acid residues constituting the antigenic peptide, and more particularly between the same-charged carrier surface and the same-charged amino acid residues comprised in the antigenic peptide, may result in the antigenic peptide adopting a well-defined, highly specific and stable conformation which guarantees its high biological activity. As a result, the antigenic peptide is exposed and presented in a conformation with high biological activity, such that it allows the immune system of the target organism to freely interact with the antigenic determinants contained in the antigenic construct in the biologically active conformation, which results in a strong and conformation-specific immune response, resulting for example in high antibody titers in the target organism.

By carefully adjusting the overall net charge of the antigenic peptide on the one hand and the carrier in which it is to be bound, incorporated or reconstituted on the other hand, the antigenic peptide exposed on or in close proximity to the carrier surface is presented in a conformation induced and stabilized by an effective electrostatic repulsion between the same-charged carrier surface and the antigenic peptide, in particular between the same-charged carrier surface and the amino acid residues constituting the antigenic peptide, more particularly between the same-charged carrier surface and the same-charged amino acid residues contained in the antigenic peptide. This results in the antigen construct being presented such that it is freely accessible to the immune defense machinery of the target organism and is therefore capable of inducing a powerful and highly specific immunogenic response when administered to an animal or human. The immunogenic response can be further enhanced by using liposomes, which can function as adjuvants, to increase or stimulate the immune response in the target animal or human to be treated with the therapeutic vaccine according to the invention, as a carrier. Optionally, in addition, the liposomes may contain other adjuvants, such as microcapsules of lipid a, alum, calcium phosphate, interleukin 1 and/or polysaccharides and proteins, in particular detoxified lipid a, such as monophosphoryl or diphosphoryl lipid a or alum.

In a particular embodiment of the invention, antigenic peptides according to the invention and as described hereinbefore, in particular antigenic peptides having a negative overall net charge, reconstituted in liposomes, in particular liposomes having components selected such that the net overall charge of the liposome head group is negative, are used. In particular, the liposomes are composed of a component selected from the group consisting of myristoylphosphatidylcholine (DMPC), myristoylphosphatidylethanolamine (DMPEA), myristoylphosphatidylglycerol (DMPG), and cholesterol, and optionally may also contain monophosphoryl lipid a or any other adjuvant suitable for use within the scope of the present invention, such as alum, calcium phosphate, interleukin 1, and/or microcapsules of polysaccharides and proteins.

In another particular embodiment of the invention, a modified peptide antigen according to the invention and as described hereinbefore is provided covalently bound to an anchor molecule capable of insertion into a carrier/adjuvant, whereby the peptide can be immobilised on the carrier/adjuvant and presented on or against the surface of the carrier/adjuvant molecule such that electrostatic forces can be effected as described hereinbefore.

When liposomes are used as carriers/adjuvants, the antigenic peptide constructs typically have a hydrophobic tail, which is inserted into the liposome membrane as it is formed. In addition, the antigenic peptide may be modified to contain a hydrophobic tail so that it can be inserted into liposomes.

The supramolecular antigenic constructs of the invention typically comprise peptides modified to enhance antigenic effect, wherein the peptides may be modified by pegylation (using polyethylene glycol or modified polyethylene glycol) or by other means, such as palmitic acid (as described above), poly-amino acids (e.g. poly-glycine, poly-histidine), poly-sugars (e.g. polygalacturonic acid, polylactic acid, polyglycolide, chitin, chitosan), synthetic polymers (polyamides, polyurethanes, polyesters) or co-polymers (e.g. poly (methacrylic acid) and N- (2-hydroxy) propylmethacrylamide), and the like.

In a particular embodiment of the invention, there is provided an antigenic peptide according to the invention and as described hereinbefore, wherein the antigenic peptide is modified to contain a hydrophobic tail such that the peptide is capable of insertion into a liposome. In particular, the β -amyloid peptide may be modified by a lipophilic or hydrophobic moiety that facilitates insertion into the lipid bilayer of the carrier/adjuvant. The lipophilic or hydrophobic moieties of the present invention can be fatty acids, triglycerides and phospholipids, particularly fatty acids, triglycerides and phospholipids wherein the fatty acid carbon backbone has at least 10 carbon atoms, particularly lipophilic moieties of fatty acids having a carbon backbone of at least about 14 carbon atoms and no more than about 24 carbon atoms, more particularly hydrophobic moieties having a carbon backbone of at least about 14 carbon atoms. Examples of hydrophobic moieties include, but are not limited to, palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, linolenic acid, and cholesterol, or DSPE. In a particular embodiment of the invention, the hydrophobic moiety is palmitic acid.

Palmitoylation, while providing an anchor for peptide immobilization in the liposome bilayer, due to C_16:0The relatively low length of the fatty acid moiety results in the presented peptide being exposed on or in close proximity to the liposome surface. Thus, cells processing the antigen will have to take up the entire liposome with the peptide. In another embodiment of the present invention, the supramolecular constructs are made using PEG, wherein the free PEG termini are covalently attached to molecules of phosphatidylethanolamine (where the fatty acid may be: myristic acid, palmitic acid, stearic acid, oleic acid, and the like, or combinations thereof). The supramolecular structure can be reconstituted in liposomes composed of phospholipids and cholesterol (phosphatidylethanolamine, phosphatidylglycerol, cholesterol in various molar ratios). Other phospholipids may be used. Lipid a was used at a concentration of approximately 40 micrograms per picomole of phospholipid.

It is another object of the present invention to provide a vaccine composition comprising a supramolecular antigenic construct comprising an antigenic peptide according to the invention and as described above, wherein the peptide is modified so as to increase the antigenic effect, whichModifying the peptide, in particular an A.beta.peptide fragment derived from the N-terminal part of the A.beta.peptide, in particular from a peptide selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(/2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); 1-15 (. DELTA.12), 15 (. DELTA.13) and 15 (. DELTA.14) amino acid residues, and A.beta.peptide fragments_1-16(Δ15)Peptide antigens, more particularly A beta_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly A beta_1-14Peptide antigens, in particular Abeta_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 provided in SEQ ID NO: 3 (Δ 14), or modified by other means, such as poly-amino acids (e.g., poly-glycine, poly-histidine), poly-saccharides (e.g., polygalacturonic acid, polylactic acid, polyglycolide, chitin, chitosan), synthetic polymers (polyamides, polyurethanes, polyesters), or co-polymers (poly (methacrylic acid) and N- (2-hydroxy) propylmethacrylamide), and the like.

In another embodiment of the invention, the β -amyloid peptide antigen according to the invention and as described hereinbefore is a palmitoylated a β peptide fragment from the N-terminal part of the a β peptide, modified by covalent attachment of palmitoyl residues (resulting in 2 to 4, especially 4 residues) at each end of the peptide, reconstituted in liposomes, especially from a peptide fragment selected from the group consisting of 1-15, 2-15, 3-15, 1-14, 2-14, 1-13; 1-16 (delta 2), 1-16 (delta 4), 1-16 (delta 5), 1-16 (delta 6), 1-16 (delta 8), 1-16 (delta 9), 1-16 (delta 10); 1-16(Δ 12), 16(Δ 13), 16(Δ 14), 1-16(Δ 15), 1-15(Δ 2), 1-15(Δ 4), 1-15(Δ 5), 1-15(Δ 6), 1-15(Δ 8), 1-15(Δ 9), 1-15(Δ 10); palmitoylated a β peptide fragments consisting of amino acid residues 1-15(Δ 12), 15(Δ 13), 15(Δ 14), in particular palmitoylated a β peptide fragments_1-16(Δ15)Peptide antigens, more particularly palmitoylated a β_1-16(Δ14)Or Abeta_1-16(Δ13)Peptide antigens, even more particularly palmitoylated A beta_1-14Peptide antigens, particularly palmitoylated A beta_1-15A peptide antigen, in particular consisting of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues 1-15 provided in SEQ ID NO: 3 (Δ 14) from amino acid residues 1-16(Δ 14). The palmitoylated antigen constructs can be used to treat amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis, to alleviate symptoms associated with the disease, or to restore a condition present in a healthy individual who is not affected by the disease.

In certain embodiments, the supramolecular antigenic constructs of the invention comprise an antigenic peptide sequence as described hereinbefore covalently linked at each end to at least one, in particular to 1 or 2 pegylated lysines at each end. The length of the PEG (polyethylene glycol) chain may vary from n-8 to n-150,000 or more, in particular from n-10 to n-80,000, more in particular from n-10 to n-10,000. In a particular embodiment of the invention, the PEG chain has a length of no more than n-45, in particular between n-5 and n-40, more in particular between n-10 and n-30, even more in particular n-10.

Liposomes useful in the compositions of the present invention include those known to those skilled in the art. Any standard lipid that can be used to make liposomes can be used. Standard bilayer and multilamellar liposomes can be used to make the compositions of the invention. Although any method of making liposomes known to those skilled in the art may be used, most preferably the liposomes are prepared according to the methods of Alving et al, infection. 2438 and 2444, 1992, which is incorporated herein by reference. The liposomes may optionally contain an adjuvant or an immunomodulator or both. Preferred immunomodulators are lipid a, especially detoxified lipid a, e.g. monophosphoryl or diphosphoryl lipid a.

Liposomes can have dual functions in that they can be used as a carrier comprising supramolecular constructs as described above, while at the same time can act as an adjuvant to increase or stimulate an immune response in a target animal or human being treated with a therapeutic vaccine according to the invention. Optionally, the liposomes may further contain additional adjuvants or/and immunomodulators or both, like lipid a, alum, calcium phosphate, interleukin 1 and/or microcapsules of polysaccharides and proteins, especially lipid a, more especially detoxified lipid a, such as monophosphoryl or diphosphoryl lipid a, or alum.

Age-related amyloidosis, including neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type), is treated, inter alia, by administering a supramolecular antigenic construct according to the invention, particularly a vaccine composition comprising such supramolecular antigenic constructs according to the invention, to an animal, particularly a mammal or a human, which is affected by, and therefore in need of treatment for, a condition; guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration, particularly diseases or conditions characterized by a loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), especially alzheimer's disease, the symptoms of which manifest as a mild amnesia to complete memory loss.

The composition of the invention comprising the supramolecular antigenic construct according to the invention and as described hereinbefore may be prepared in the form of a liquid solution or suspension for injection, or in solid form suitable for dissolution prior to injection, for example in a kit (as described hereinafter) using the composition of the invention.

The composition of the invention comprising the supramolecular antigenic construct is administered to a human or animal afflicted with an amyloid-associated disease, to induce an immune response in the human or animal, so as to alleviate symptoms associated with the disease, or to restore to a condition present in a healthy subject not affected by the disease.

The compositions of the present invention may be administered to humans or animals by any appropriate standard route of administration. Typically, the composition may be administered by the topical, oral, rectal, nasal or parenteral (e.g., intravenous, subcutaneous or intramuscular) route. In addition, the composition can be incorporated into a slow release matrix, such as a biodegradable polymer, which is implanted in the vicinity of the desired delivery site, e.g., near the tumor site. The method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and continuous administration for a predetermined period of time.

In particular, the antigenic peptide composition according to the invention is administered parenterally, in particular by intraperitoneal, intravenous, subcutaneous and intramuscular injection.

The dosage of the composition will depend on the condition to be treated, the particular composition used, and other clinical factors such as the weight, size and condition of the patient, the body surface area, the particular compound or composition to be administered, other drugs to be administered concurrently, and the route of administration.

The therapeutic vaccine compositions according to the invention can be administered in combination with other biologically active substances and methods for the treatment of diseases. The other biologically active substances may be part of the same composition already comprising the therapeutic vaccine according to the invention in the form of a mixture, wherein the therapeutic vaccine is mixed with the other biologically active substances, or they may be mixed with the same pharmaceutically acceptable solvent and/or carrier, or they may be provided separately as part of different compositions, which may be provided separately or together in component parts.

The therapeutic vaccine composition according to the invention and the other biologically active substance(s) may be administered simultaneously, intermittently or sequentially. For example, the first additional biologically active substance may be administered simultaneously with the administration of the therapeutic vaccine composition according to the invention or sequentially after or before the administration of the therapeutic vaccine. If an administration regimen is chosen in which more than one additional biologically active substance is administered together with at least one therapeutic vaccine according to the invention, these compounds or substances can be administered in various combinations, partly simultaneously and partly sequentially.

It is a further object of the invention to provide a mixture of a therapeutic vaccine according to the invention and optionally one or more other biologically active substances, and methods of preventing and/or treating amyloidosis and/or reducing the effects of amyloidosis using a therapeutic vaccine or mixture thereof, including compositions containing the therapeutic vaccine or mixture of therapeutic vaccines, according to the invention, the amyloidosis is a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, for example, diseases including, but not limited to, neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory, such as Mild Cognitive Impairment (MCI), lewy body dementia, down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases or conditions including macular degeneration.

The mixture according to the invention may comprise, in addition to the therapeutic vaccine according to the invention, biologically active substances, such as compounds known for use in the treatment of amyloidosis (a group of diseases and disorders associated with amyloid or amyloid-like proteins, such as the a β protein involved in alzheimer's disease), including antibodies raised against immunogenic peptide antigens, particularly immunogenic antigens presented in the form of supramolecular antigenic constructs, more particularly antibodies according to the invention and as disclosed herein.

In another embodiment of the invention, other biologically active substances or compounds may also be therapeutic agents, which may be used to treat diseases and conditions caused by or associated with amyloid or amyloid-like proteins, including amyloidosis caused by amyloid beta, or may be used in the pharmacological treatment of other neurological conditions.

The other biologically active substance or compound may exert its biological effect by the same or similar mechanism as the therapeutic vaccine according to the invention, or by an unrelated mechanism of action, or by a plurality of related and/or unrelated mechanisms of action.

In general, other biologically active compounds may include neuronal-delivery promoters, psychotherapeutic agents, acetylcholinesterase inhibitors, calcium-channel blockers, biogenic amines, benzodiazepinesA tranquilizer, an acetylcholine synthesis, storage or release enhancer, a postsynaptic receptor agonist for acetylcholine, a monoamine oxidase-A or-B inhibitor, an N-methyl-D-aspartate glutamate receptor antagonist, a non-steroidal anti-inflammatory drug, an antioxidant and a 5-hydroxytryptamine receptor antagonist.

In particular, the mixture according to the invention may comprise at least one further biologically active compound selected from the group consisting of compounds against oxidative stress, anti-apoptotic compounds, metal chelators, inhibitors of DNA repair, such as pirenzepin (pirenzepin), and metabolites, 3-amino-1-propanesulfonic acid (3APS), 1, 3-propanedisulfonate (1, 3PDS), secretase activators, beta-and gamma-secretase inhibitors, tau proteins, neurotransmitters, beta-sheet formation blockers (beta-sheet breakers), anti-inflammatory molecules or cholinesterase inhibitors (ChEI), such as tacrine inhibitors (ChEI), together with the therapeutic vaccine according to the invention, and optionally pharmaceutically acceptable carriers and/or diluents and/or excipients, Rivastigmine, donepezil and/or galantamine and other medicaments, and nutritional supplements.

In a further embodiment, the mixture according to the invention may comprise niacin or memantine, together with the therapeutic vaccine according to the invention, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In another embodiment of the present invention, there is provided a mixture comprising an "atypical antipsychotic" such as clozapine (clozapine), ziprasidone (ziprasidone), risperidone (risperidone), aripiprazole (aripiprazole) or olanzapine (olanzapine) together with a therapeutic vaccine according to the invention and optionally pharmaceutically acceptable carriers and/or diluents and/or excipients for the treatment of positive and negative psychotic symptoms including hallucinations, delusions, thought disorder (manifested as marked incoherence, misthought-out, disorganized behavior), and bizarre or disorganized behavior, as well as anhedonia, flattened emotion, apathy and social withdrawal.

In one embodiment of the invention, the compositions and mixtures according to the invention and as described hereinbefore comprise a therapeutically or prophylactically effective amount of a vaccine according to the invention and a biologically active substance, respectively.

In e.g. WO2004/058258 (see mainly pages 16 and 17), further compounds suitable for use in this mixture in combination with a vaccine according to the invention are described, including therapeutic drug targets (pages 36-39), alkanesulphonic and alkanolsulphuric acids (pages 39-51), cholinesterase inhibitors (pages 51-56), NMDA receptor antagonists (pages 56-58), estrogens (pages 58-59), non-steroidal anti-inflammatory drugs (pages 60-61), antioxidants (pages 61-62), peroxisome proliferator-activated receptor (PPAR) agonists (pages 63-67), cholesterol lowering drugs (pages 68-75); amyloid inhibitors (pages 75-77), amyloid formation inhibitors (pages 77-78), metal chelators (pages 78-79), anti-psychotic and anti-depressant drugs (pages 80-82), nutritional supplements (pages 83-89), as well as compounds that increase the availability of biologically active substances in the brain (see pages 89-93) and prodrugs (pages 93 and 94), which are incorporated herein by reference, especially the compounds mentioned in the pages indicated above.

It has long been known that immunization of animal or human hosts with normal host proteins results in the formation of auto-antibodies against the host proteins, resulting in conditions collectively known as autoimmune diseases. A β and its APP precursor protein are such normal proteins. Thus, the use of these host proteins in vaccination may produce unwanted side effects. There is some evidence in the literature that a β can activate a neuroinflammatory response, which may be due in part to over-activation of the complement system, which has been highly activated in patients with alzheimer's disease or other neurodegenerative diseases.

Human a β in its β -sheet conformation is a potent activator of the human complement system. It binds strongly to the collagen tail of human complement C1 q. This over-activation of the complement system can lead to diversion of the host's natural defense system and to self-destruction of cells and tissues, including neurons and their processes. Such as the Membrane Attack Complex (MAC), which is part of the host's natural defense system and protects the host against invading bacteria and viruses by inserting itself into the bacteria and viruses, and which, when over-activated, can insert itself into the host cell and cause self-destruction. Over-activation can also cause irritation to microglia, producing toxic compounds such as oxygen-free radicals and harmful proteases.

It is therefore a further object of the present invention to prevent possible side effects, such as neurological complications, caused by vaccination of animals or humans suffering from autoimmune diseases with autoantigens which may further stimulate the already over-activated complement system. It is within the scope of the invention to administer an A β peptide antigen, particularly a palmitoylated A β peptide antigen, more particularly palmitoylated A β peptide antigen, in combination with a complement inhibitor_1-15Peptide antigens, especially palmitoylated A beta_1-15Peptide antigen (ACI-24, Abeta)_1-15) Thereby achieving the purpose.

Accordingly, another embodiment of the invention provides a vaccine composition comprising an inhibitor of the complement system in addition to an a β peptide antigen, in particular an a β peptide antigen according to the invention and as described hereinbefore.

The complement inhibitor may be a compound selected from soluble human complement receptor 1, anti-human complement protein C5, e.g., a humanized anti-C5 monoclonal antibody or a single-chain fragment of a humanized monoclonal antibody, C1-esterase inhibitor-N, and a natural human C1 inhibitor.

Recent emphasis has been placed on comorbidities of A β and cerebrovascular disease, the association of A β and atherosclerosis, cognitive impairment associated with amyloid angiopathy, massive cerebral microvascular lesions, and the defect of clearing A β across the blood brain barrier in Alzheimer's disease, all of which suggest that vascular disorders are an important feature of chronic neurodegenerative conditions in Alzheimer's disease (ZLokovic, B.: 2005) Trends in neurosciens 28, 202-208). Thus, neurovascular dysfunction may have a major role in the pathogenesis of alzheimer's disease.

There is a great deal of evidence for a strong correlation between cognitive decline in alzheimer's disease and cerebrovascular disorders (Torre, de la, j.c.: 2004) neuron. res.26, 517-. Reduced microvascular density, increased number of ruptured vessels, significant changes in vessel diameter, and the like have been described in Alzheimer's disease (Bailey, T.L. et al (2004) neurol.Res.26, 573-.

Several studies, including the large population-based Rotterdam study (Greenberg, S.M et al (2004) Stroke 35, 2616-. Several risk factors for alzheimer's disease and vascular dementia overlap, including transient ischemic attacks, atherosclerosis, heart disease, high serum viscosity, and the like.

Vascular dementia occurs as a result of brain tissue damage following oxygen deprivation due to narrowing or obstruction of blood vessels in the brain, and is the second most common form of dementia. Patients often suffer from both alzheimer's disease and vascular dementia. It is estimated that 170 million people in EU and 55000 people in USA suffer from vascular dementia.

Normal O in the brain despite impaired blood flow₂The treatment of pressure recovery, which may have a significant effect on the evolution of alzheimer's disease, may drastically reduce vascular dementia.

Thus, according to another embodiment of the invention, there is provided a method of inducing O in addition to an A.beta.peptide antigen, in particular an A.beta.peptide antigen according to the invention and as described hereinbefore₂A compound having a reduced hemoglobin affinity such that oxygen can be subsequently released into organ tissue.

In particular, adjusting O₂The compound with hemoglobin affinity may be selected from hypolipidemic drugs (e.g. clofibric acid or bezafibrate, including bezafibrate derivatives LR16 and L35), urea derivatives (e.g. [2- [4[ [ (arylamino) carbonyl group]-amino group]Phenoxy radical]2-methylpropionic acid), allosteric effectors of hemoglobin (e.g. compounds of the group consisting of 2, 3-Diphosphoglycerate (DPG), Inositol Hexaphosphate (IHP) and pyridoxal phosphate).

More particularly, adjusting O₂The hemoglobin affinity compound may be a compound comprising an anionic ligand to the allosteric site of hemoglobin, wherein the anionic ligand comprises an internal pyrophosphate ring, optionally together with a non-toxic cation.

Even more particularly, O is adjusted₂The/hemoglobin affinity compound is a phytic acid (IHP) derivative that includes at least one internal pyrophosphate ring, optionally together with a non-toxic cation.

To remain byComplement inhibitors and modulation of O₂The invention provides vaccine compositions comprising in combination an A beta peptide antigen, particularly according to the invention and as described hereinbefore, together with an inhibitor of the complement system and a modulator of O₂Hemoglobin affinity compounds, particularly allosteric effectors of hemoglobin.

Vaccine compositions according to the invention comprising an A.beta.peptide antigen, in particular an A.beta.peptide antigen according to the invention and as described hereinbefore, concomitantly, intermittently or consecutively with a complement inhibitor and/or modulating O₂The hemoglobin affinity compounds are administered together, correspondingly mitigating the potentially deleterious effects of over-activated complement system and cerebrovascular disorders. For example, the vaccine composition according to the invention may be administered simultaneously with the complement inhibitor, or sequentially after or before administration of the vaccine. If one chooses to administer complement inhibitors and modulate O together with at least one vaccine according to the invention₂Hemoglobin affinity compounds, particularly allosteric effectors of hemoglobin, may be administered in various combinations, partially simultaneously, partially sequentially.

Another object of the invention is to provide a vaccine according to the invention together with a complement inhibitor and/or a regulatory O₂Mixtures of compounds of haemoglobin affinity, in particular allosteric effectors of haemoglobin, and use of a vaccine according to the invention or mixtures thereof, including compositions comprising said vaccine, or a vaccine according to the invention and a complement inhibitor and/or modulating O₂Mixtures of hemoglobin affinity compounds, particularly allosteric effectors of hemoglobin, for preventing and/or treating amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis, for example including but not limited to neurological disorders such as Alzheimer's Disease (AD), Lewy body dementia, Down's diseaseHereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome; and other amyloid-like protein based or associated diseases such as progressive supranuclear palsy, multiple sclerosis; coughas disease, parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and others, including macular degeneration.

Modified amyloid 1-15 peptides can be synthesized according to the methods reported in Nicolau et al (2002) Proc Natl.Acad Sci USA 99, 2332-2337. The method reported in Nicolau et al involves modification of the antigenic peptide by grafting a lipophilic or hydrophobic moiety onto the resin at the terminal amino acid residue of a pre-formed peptide. In particular, protected amino acids, in particular Fmoc-protected amino acids, are attached to the resin using known coupling chemistry. The protecting group is removed and a second protected amino acid residue is coupled. Standard automated peptide synthesis (using known protection chemistry, particularly Fmoc/tBu chemistry, and standard side chain protecting groups) was then used, whereby amyloid A β was coupled_1-42Amino acids 1 to 15 of SEQ ID NO: 1, synthesis of a β antigen peptides, in particular a β_1-15An antigenic peptide. In the final step, two additional protected amino acids are coupled to the growing peptide fragment. The Mtt group can then be selectively cleaved off and palmitic acid coupled. After washing the resin, the protecting group is removed using standard methods and the resin is simultaneously cleaved, followed by deprotection of the side chain. The final product can be obtained in high purity, the identity of which can be confirmed by methods known in the art, such as electrospray mass spectrometry.

The lipophilic or hydrophobic moiety according to the present invention may be a fatty acid, triglyceride or phospholipid in which the fatty acid carbon backbone has at least 10 carbon atoms. In particular, lipophilic or hydrophobic moieties are fatty acids having a carbon backbone of at least about 14 carbon atoms and no more than about 24 carbon atoms, the number of each individual carbon atom within this range also being part of the present invention. More particularly, the lipophilic or hydrophobic moiety has a carbon backbone of at least 14 carbon atoms, especially 16 carbon atoms. Examples of hydrophobic moieties include, but are not limited to, palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, and linolenic acid. In a particular embodiment of the invention, the lipophilic or hydrophobic moiety is palmitic acid.

The liposomal antigen according to the invention can then be prepared as described in Nicolau et al, 2002. Modified amyloid A β antigenic peptides, particularly modified A β antigenic peptides, may be reconstituted in constructs composed of liposomes, particularly liposomes made with myristoylphosphatidylcholine (DMPC), myristoylphosphatidylethanolamine (DMPEA), myristoylphosphatidylglycerol (DMPG), and cholesterol, and optionally containing monophosphoryl lipid A_1-15An antigenic peptide.

In a particular embodiment of the invention, an anti-amyloid vaccine is prepared using liposomes with lipid a as an adjuvant. In particular, myristoylphosphatidylcholine, -glycerol and cholesterol were mixed in a ratio of 0.9: 1.0: 0.7. A strong immunomodulator, such as monophosphoryl lipid a, is then added at a suitable concentration, in particular at a concentration of between 30 and 50 mg per millimole, more particularly at 40 mg per millimole of phospholipid. The modified antigenic A.beta.peptide is then added at a molar ratio of peptide to phospholipid of from 1: 30 to 1: 200, particularly from 1: 50 to 1: 120, more particularly at 1: 100. The solvent is removed, e.g., via evaporation, and the resulting film is hydrated with a sterile buffered solution, e.g., PBS.

Liposomes can also be prepared by the cross-flow injection technique described, for example, in Wagner et al (2002) Journal of lipid Research, Vol.12 (3), p.259-270. During injection of lipid solutions into aqueous buffered systems, lipids have a tendency to form "precipitates" that subsequently self-arrange into vesicles. The resulting vesicle size will depend on factors such as lipid concentration, agitation rate, injection rate, and the lipid selected. The preparation system consists of a cross-flow injection module, a container for a polar phase (e.g., PBS buffer solution), an ethanol/lipid solution container, and a pressurizing device, particularly a nitrogen pressurizing device. While pumping the aqueous or polar solution through the cross-flow injection module, varying pressures are applied to inject the ethanol/lipid solution into the polar phase.

To determine the immunogenicity of the modified a β antigen construct, suitable animals, in particular mice, especially C57BL/6 mice, selected from the group consisting of mice, rats, rabbits, pigs, birds and the like are immunized with the antigenic peptide. The immunogenicity of the antigenic construct is determined by probing serum samples with an immunoassay, such as an ELISA assay, at appropriate time intervals after immunization.

Use of modified antigenic constructs, in particular palmitoylated antigenic constructs, more particularly palmitoylated a β_1-15Constructs for immunizing animals suffering from symptoms associated with amyloidosis (a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis including, but not limited to, neurological disorders such as Alzheimer's Disease (AD) including diseases or conditions characterized by loss of cognitive memory such as Mild Cognitive Impairment (MCI), Lewy body dementia, Down's disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam Parkinson-dementia syndrome, and other diseases based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis, Cuja's disease, Parkinson's disease, dementia associated with HIV, ALS (amyotrophic lateral sclerosis), adult diabetes mellitus, senile cardiac amyloidosis, endocrine tumors and other diseases or conditions including macular degeneration), in particular a mammal or a human, wherein said amyloidosis is in particular a disease or condition characterized by a loss of cognitive memory capacity, such as Mild Cognitive Impairment (MCI), or any other amyloid-associated disorder.

The supramolecular antigenic construct according to the present invention, in particular the vaccine composition comprising such supramolecular antigenic construct according to the present invention, may be administered to an animal, in particular a mammal or a human, by any suitable standard route of administration. Typically, the composition may be administered by a topical, oral, rectal, nasal or parenteral (e.g., intravenous, subcutaneous or intramuscular) route. In addition, the composition can be incorporated into a sustained release matrix, such as a biodegradable polymer, which is implanted near the site of intended delivery, e.g., near the tumor site. The method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and continuous administration for a predetermined period of time.

In a particular embodiment of the invention, the antigenic construct according to the invention, in particular a vaccine composition comprising the antigenic construct in a pharmaceutically acceptable form, is administered in repeated doses, in particular in 1 to 15 doses, more in particular in 2 to 10 doses, more in particular in 3 to 7 doses, and even more in particular in 4 to 6 doses, at intervals of 1 to 10 weeks, in particular at intervals of 1 to 6 weeks, more in particular at intervals of 1 to 4 weeks, even more in particular at intervals of 2 to 3 weeks. The immunogenicity of the antigenic construct can be determined at a suitable time after the boost, particularly at 3 to 10 days after the boost, more particularly at 4 to 8 days after the boost, and more particularly at 5 to 6 days after the boost, using known methods, particularly one of the commonly used immunoassays, such as an ELISA assay, to monitor the immune response.

Immunization with the antigenic construct according to the invention, in particular a vaccine composition comprising the antigenic construct according to the invention in a pharmaceutically acceptable form, results in a significant and highly specific immune response in the treated animal or human.

Administration of the supramolecular antigenic construct compositions of the invention to humans or animals induces immunity to antigenic agents, such as infectious organisms, or to other pathological conditions, such as beta-amyloid aggregation (alzheimer's disease) or hyperproliferative disorders, such as antigenic material of cancer. The immunized human or animal develops circulating antibodies against the infectious organism, thereby reducing or inactivating its ability to stimulate disease.

The compositions of the invention may also be used to generate antibodies against antigenic peptides. The resulting antibodies are administered to an individual to passively immunize them against various diseases or disorders, including but not limited to amyloid-related diseases.

Thus, in a particular embodiment of the invention, a panel of monoclonal or polyclonal antibodies specific for various disorders, including, for example, alzheimer's disease, may be produced using the supramolecular antigenic construct compositions of the invention. Antibodies can be made by methods well known to those skilled in the art.

The compositions of the invention may be administered to humans or animals by any suitable means, preferably by injection. For example, modified antigenic peptides reconstituted in liposomes are administered by subcutaneous injection. Circulating antibodies, whether produced internally or provided from an external source, bind to the antigen and reduce or inactivate its ability to stimulate disease.

In certain embodiments, the supramolecular antigenic construct includes a peptide having the amino acid sequence of β -amyloid. The peptides may also include or correspond to whole amyloid beta peptides and active fragments thereof. In addition, the peptides useful in the present invention may also include a β.

The invention also provides a method for producing an antibody according to the invention, including any functionally equivalent antibody or functional parts thereof, in particular a monoclonal antibody according to the invention, including any functionally equivalent antibody or functional parts thereof, the method comprising producing the antibody, in particular the monoclonal antibody, against a supramolecular antigenic construct comprising an antigenic peptide corresponding to the amino acid sequence of an a β peptide antigen according to the invention and as described hereinbefore, in particular an a β 1-16(Δ 15) peptide antigen, more in particular an a β 1-16(Δ 14) or a β 1-16(Δ 13) peptide antigen, even more in particular a βBeta 1-14 peptide antigens, in particular the beta-amyloid peptide Abeta_1-15The antigenic peptide is modified with a hydrophobic moiety, such as palmitic acid, or a lipophilic moiety, such as polyethylene glycol (PEG), or a combination of both, wherein the hydrophobic and lipophilic moieties are covalently bound to each end of the antigenic peptide via at least one amino acid, respectively, in particular coupled to the terminal amino acid residue at each end of the antigenic peptide via 1 or 2 amino acids, wherein said amino acids for attachment are e.g. lysine, or any other suitable amino acid or amino acid analogue capable of coupling the hydrophobic and lipophilic moieties together with peptide fragments as a linking means, such as glutamic acid and cysteine.

The antibodies, particularly monoclonal antibodies, obtained by the method, when administered to an animal, particularly a mammal or human, suffering from memory impairment, are capable of maintaining or increasing cognitive memory in the treated animal, mammal or human. Another aspect of the invention provides an antibody, including any functionally equivalent antibody or functional parts thereof, or more particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, raised against a supramolecular antigenic construct comprising an antigenic peptide corresponding to the amino acid sequence of an A β peptide antigen according to the invention and as described hereinbefore, in particular an A β 1-16(Δ 15) peptide antigen, more particularly an A β 1-16(Δ 14) or A β 1-16(Δ 13) peptide antigen, even more particularly an A β 1-14 peptide antigen, especially the β -amyloid peptide A β_1-15The antigenic peptide is modified with a hydrophobic moiety, such as palmitic acid, or a lipophilic moiety, such as polyethylene glycol (PEG), or a combination of both, wherein the hydrophobic and lipophilic moieties are covalently bound to each end of the antigenic peptide via an amino acid, such as lysine, or any other suitable amino acid or amino acid analog capable of functioning as a linker molecule, respectively. When PEG is used as the lipophilic moiety, the free PEG end is covalently bound to phosphatidylethanolamine or any other compound suitable for use as an anchoring element to embed the antigenic construct in the liposome bilayer.

Examples

Example 1: synthesis of tetra (palmitoyl lysine) -A beta_1-15Peptide antigens

1.1 synthetic scheme 1:

palmitoylated amyloid 1-15 peptide was synthesized according to a modified previously reported method (Nicolau et al 2002). The novel method involves grafting palmitic acid onto the terminal Lys residue of a preformed peptide on a resin rather than incorporating the modified amino acids Fmoc-Lys (Pal) -OH by stepwise solid phase synthesis. The novel process improves coupling efficiency and provides a considerably higher purity product. Thus, orthogonally protected amino Fmoc-Lys (Mtt) -OH was attached to Wang resin using HBTU coupling chemistry. The Fmoc group was removed using 20% piperidine in DMF and the second residue Fmoc-Lys (Mtt) -OH was coupled. The next 15 amino acids were then coupled using standard automated peptide synthesis (using Fmoc/tBu chemistry and standard side chain protecting groups). Finally, the last two amino acids coupled are Fmoc-Lys (Mtt) -OH. The Mtt group was then selectively cleaved using 1% TFA in dichloromethane, followed by coupling of palmitic acid using HBTU. After washing the resin, the Fmoc group was removed with 20% piperidine in N, N-Dimethylformamide (DMF), and finally the resin cleavage and deprotection of the side chain were performed simultaneously under standard conditions using TFA. Trituration in cold ether afforded the product as a white solid. Electrospray mass spectrometry confirmed the identity of the product (expected M/z: 1097.9([ M ]3 +); experimental value: 1096.8([ M-3H ]3+), with no other tri-, di-or mono-palmitoylated peptides detected.

1.2 Synthesis scheme 2:

tetrakis (palmitoyl lysine) -A beta may be synthesized using another method_1-15A peptide antigen based on the grafting of palmitic acid onto the terminal lysine residue of a preformed peptide on a resin. Thus, the orthogonally protected amino acids Fmoc-Lys (ivDde) -OH were coupled to 2-chlorotrityl resin. After Fmoc deprotection, a second Fmoc-Lys (ivDde) -OH was coupled using Fmoc/tBu chemistry and standard amino acid side chain protecting groups, followed by 15 standard auto-peptidesAnd (5) synthesizing and circulating. After coupling the last two Fmoc-Lys (ivDde) -OH residues, the Fmoc group was removed using 20% piperidine in DMF and the N-terminus was protected with a Boc group using tert-butyl dicarbonate. The ivDde protecting group was then chemically removed by treatment with 3% hydrazine in DMF and palmitic acid was coupled to these four lysine residues using HBTU using two couplings each for 18 hours. After washing the resin, the side chain was deprotected using TFA/TIPS under standard conditions. Trituration in cold ether afforded the product as a white solid. MALDI-Tof confirmed the identity of the product and no other tri-, di-or mono-palmitoylated peptide was detected.

The liposomal vaccine was prepared using methods as described in US6843942 and EU 1337322.

Example 2: synthesis of N-and C-terminal lipid-PEG beta-amyloid peptide antigens

Palmitoylation, although providing an anchor for peptide immobilization on the liposome bilayer, because of C_16:0The relatively reduced length of the fatty acid moiety results in the peptide actually being placed on the liposome surface. Thus, cells processing the antigen will have to take up the entire liposome carrying the peptide, with the result that a relatively slow immune response may result.

To facilitate immune responses, other anchor/spacer arms have been used to reconstitute peptides in liposomes, such as polyethylene glycol (PEG). PEG was covalently linked to lysine residues attached to both ends of the peptide. At the other end of the chain (PEGn ═ 70), Phosphatidylethanolamine (PEA) is covalently attached, which functions as an anchoring element in the liposome bilayer. Thus, the liposome still functions as an adjuvant, while the peptide is far enough away from the lipid bilayer to be processed alone and thus increase its immunogenicity compared to palmitoylated antigen.

Methods for mono-pegylation of peptides at the N-alpha-position are known and widely used. Position-specific mono-pegylation of medium-sized peptides at internal, N-or C-terminal amino acid residues according to solid phase or peptide-grafting methods has also been described.

To avoid steric hindrance problems, the reaction is carried out in the solution phase. A successful approach involves the synthesis of peptide sequences using standard Fmoc/tBu amino acid side chain protection. For those peptide sequences containing internal Lys or His residues (1-16, 1-15), orthogonally protected Lys (ivDde) was added at each end. Additional Gly was added at the C-terminus to facilitate synthesis. The Fmoc group was removed with 20% piperidine in DMF and N-acetylated using acetic anhydride. The ivDde group was selectively cleaved using 3% hydrazine hydrate in DMF for 1 hour. 2-chlorotrityl resin is more advantageous than the more widely used Wang resin because its resistance to hydrazinolysis has been found to be much higher. Furthermore, 2-chlorotrityl resins are extremely sensitive to acids and therefore unlike Wang resins, they enable the isolation of protected peptides. Indeed, the coupling reaction must be carried out in solution phase, since the coupling of the resin-bound peptide with the pre-activated pegylated lipid reagent DSPE-PEG-SPA does not produce any coupling product. Thus, under mild conditions (acetic acid/trifluoroethanol/dichloromethane, 1: 8, 1 hour, room temperature), selective cleavage from the resin resulted in an internally protected peptide.

Solution phase coupling of peptides derived from sequences 1-16, 1-15 to DSPE-PEG-SPA was successfully performed in DMSO and excess base. The reaction was then quenched by addition of an excess of ethanolamine for 2 hours and the solution was freeze dried.

Purification by HPLC (semi-preparative reverse phase C)₄Column), which yielded N-and C-terminal PEG-lipid conjugates of 50-70% purity, the identity of which was confirmed by MALDI. To the extent that the coupling reaction is easy to proceed, each sequence shows considerable variation and the conditions (temperature, number of molar equivalents of DSPE-PEG-SPA, time) are adjusted accordingly. To separate the excess DSPE-PEG-SPA from the desired product, HPLC purification was applied. The mono-and di-coupled products can be separated by using cation-exchange chromatography before final side chain deprotection. After subsequent deprotection of the peptide side chain and separation of excess quenched DSPE-PEG-SPA,resulting in isolation of the desired conjugate in acceptable purity.

Pegylated and palmitoylated antigens

Aβ_1-15(ACI-24)

H2N-Lys-Lys-Asp(OtBu)-Ala-Glu(OtBu)-Phe-Arg(Pbf)-His(Trt)-Asp(OtBu)-Ser(tBu)-Gly-Tyr(tBu)-Glu(OtBu)-Val-His(Trt)-His(Trt)-Gln(Trt)-Lys(Boc)-Lys-Lys-OH

Aβ_1-16(ACI-01)

Ac-Lys-Asp(OtBu)-Ala-Glu(OtBu)-Phe-Arg(Pbf)-His(Trt)-Asp(OtBu)-Ser(tBu)-Gly-Tyr(tBu)-Glu(OtBu)-Val-His(Trt)-His(Trt)-Gln(Trt)-Lys(Boc)-Lys-Gly-OH

Aβ_1-16(Δ14)(ACI-02)

Ac-Lys-Asp(OtBu)-Ala-Glu(OtBu)-Phe-Arg(Pbf)-His(Trt)-Asp(OtBu)-Ser(tBu)-Gly-Tyr(tBu)-Glu(OtBu)-Val-His(Trt)-Gln(Trt)-Lys(Boc)-Lys-Gly-OH

Aβ_22-35(ACI-11)

Ac-Lys-Glu(OtBu)-Asp(OtBu)-Val-Gly-Ser(tBu)-Asn(Trt)-Lys(Boc)-Gly-Ala-Ile-Ile-Gly-Leu-Met-Lys-Gly-OH

Aβ_29-40(ACI-12)

Ac-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Lys-Gly-OH

Example 3: structural and conformational analysis

3.1 analysis of the conformation of the reconstituted antigen

To anchor antigen A β 1-15 to the liposome surface, palmitoylated lysine was used in tandem at each end of the peptide, as previously described (Nicolau, C. et al, 2002).

The fatty acids of palmitic acid contain 16 carbon atoms and have been shown to be of a length suitable for stable insertion into the bilayer of liposomes. In this construct, because of the length of the C16 fatty acid moiety, the peptide actually lies on the surface of the liposome. In order to bind the antigenic peptide to liposome-lipid A in different conformations, other anchor/spacer arms, namely polyethylene glycol (PEG with 77 repeating units), have been used to reconstitute peptide A β 1-16(ACI-01) in liposomes. The effect of the spacer arm between the liposome anchor and the a β peptide on the secondary conformation of the amyloid sequence reconstituted in the liposome was measured by circular dichroism (fig. 1 a). The pegylated a β 1-16 appeared to be in a random coil or loose protein conformation (negative signal at 210nm and slowly approached the 0 axis by 260 nm), while the palmitoylated peptide a β 1-15 contained a substantial fraction of the β -sheet conformation (positive signal by 210nm, then crossed the 0 axis and again approached the 0 axis by 260 nm). Thus, it appears that the closer proximity of palmitoylated peptides to the liposome surface may force a defined secondary conformation. This is probably due to electrostatic interactions of the peptide with the liposome surface (which is clearly not possible with pegylated peptides).

3.2 structural analysis of palmitoylated β -amyloid 1-15 reconstituted in liposomes

To analyze the effect of different linker molecules on the conformation of the beta-amyloid 1-15 peptide reconstituted in liposomes, NMR analysis was performed (FIGS. 1b and 1 c). Palmitic acid and polyethylene glycol (PEG, n 77) are used herein as linker molecules or anchors, respectively, attached to liposomes.

For NMR studies, palmitoylated amyloid 1-15(ACI-24) and pegylated A β reconstituted in liposomes by vortexing_1-16A sample of the antigen (ACI-01) peptide was homogenized and the concentration of the solution was increased by centrifugation (3 x 90 minutes at 3000rpm at 4 ℃) and the resulting wet pellet was transferred to the MAS rotor. Other samples were prepared by suspending the ACI-01 and ACI-24 peptide preparations at a concentration of 1mM in PBS buffer at pH 7.2, and preparing 4mM solutions of the linker-free peptide sequences in the same buffer. 10% D was added to each sample₂O。

Operating at a frequency of 500.13MHz (11.4T) and equipped with a 4mm triple resonance (¹H/¹³C/²H) Recording on a Bruker Avance 500 Spectroscopy of HR-MAS Probe¹HHR-MAS NMR spectra. Each sample was introduced into a 4mm ZrO equipped with a 50. mu.l cylindrical insert₂Inside the rotor. For all NMR experiments, the sample was rotated at a frequency equal to the spectral width (6250Hz), which cleared the rotational sidebands from the spectrum. One-dimensional proton NMR spectra were obtained using pre-saturation and Watergate sequences (Piotto, M. et al (1992); Piotto, M. et al (2005)) and by accumulating 1000-. The temperature of the bearing air flowing into the probe was set at 295K to ensure 298K in the sample.

FIGS. 1b and 1c demonstrate the difference in one-dimensional proton NMR spectra of palmitoylated and pegylated beta-amyloid peptide. Two significant differences were observed at 8.00 and 8.25 ppm. Since both peptides have identical amino acid sequences except for lysine at position 16, these differences at 8.00 and 8.25ppm indicate differences in secondary structure, since lysine should not produce a positive signal in this spectral region of aromatic amino acid residues.

The particular design of the supramolecular constructs according to the invention results in a unique, highly specific and important secondary structure of the amyloid antigenic peptide when reconstituted in liposomes, which structure differs from linker molecule to linker molecule, as confirmed by one-dimensional proton NMR spectroscopy in the region of aromatic amino acid residues. This may mean that the linker/anchor forces the peptide to be immobilised in a certain or defined secondary structure, depending on the linker molecule used. In the case of active immunization using these molecules as vaccines, it is likely that antibodies raised against these structurally different antigens will be antigen-and conformation-specific.

Abeta in palmitoylation_1-15And pegylated Abeta_1-16After immunization of APP XPS-1 mice with antigen (see examples below), previous data obtained by ELISA and ORT (ORT, object recognition task, a cognitive memory test) showed that onlyPalmitoylated antigens can restore memory impairment in disease patterns of Alzheimer's disease, although both exhibit the same immunogenicity. The possible mechanisms by which these two antigens presenting the same peptide elicit two different functional antibodies in vivo are most likely related to the different secondary structures of the presented peptides due to the linker technology.

Example 4: quantifying exo-and endo-oriented reconstituted peptides

The amount of peptide reconstituted in ACI-01 and ACI-24 was estimated by a Fluorescamine (FLA) -based assay, which reacts specifically with primary amines to form highly fluorescent covalent adducts (Udenfriend, S. et al, 1972). FLA is expected to react with the N-terminus of the Pal1-15 peptide in ACI-24, and with Lys-16 in ACI-01.

To separate free peptides from those in liposomes, the samples were subjected to ultracentrifugation and the resulting supernatants were analyzed for peptide content using the FLA assay. No free peptide was detected in both the supernatant of ACI-01 and the supernatant of ACI-24. For both ACI-24 and ACI-01, the fraction precipitated with FLA tag showed very high selectivity for reaction with the peptide in the liposomes. To determine the total amount of peptide present on the surface of the liposomes, the assay was repeated in the presence of Triton X-100 (2% in PBS) to disrupt the lipid bilayer. This results in a significant increase in labeling; indicating that approximately 63% of the peptide was exposed on the outer membrane surface. On the other hand, FLA-labeled ACI-01 only plateaus at 1.2mM FLA, at which concentration the emission was the same when the assay was performed with or without Triton X-100. This confirms that all peptides are exposed on the surface of the PEGylated vaccine ACI-01.

Example 5: comparison of the immunogenicity (ELISA) of Pegylated and palmitoylated antigens in wild-type C57BL/6 mice

Liposomal antigens were prepared as described (Nicolau et al, 2002). Reconstitution of antigen pegylated A beta in a construct consisting of liposomes_1-16(Δ14)、Aβ_4-11And palmitoylated Abeta_1-15The liposomes were manufactured from myristoylphosphatidylcholine (DMPC), myristoylphosphatidylethanolamine (DMPEA), myristoylphosphatidylglycerol (DMPG), and cholesterol (0.9: 0.1: 0.7 molar ratio), and contained monophosphoryl lipid A (Sigma-Aldrich, Stlouis, MO, USA) at 40 mg/mM phospholipid.

At 2 week intervals, pegylated Abeta was used_1-16(Δ14)、Aβ_4-11And palmitoylated Abeta_1-15(ACI-24) antigen immunization of C57BL/6 mice. 10-12 animals were immunized with each antigen. Sera were taken 5 days after the boost and ELISA was performed with several dilutions of sera. The results of the comparison show the immunogenicity of the different antigens.

ELISA data show that liposome PEG-Abeta_1-16(Δ14)Significantly better than palmitoylated Abeta_1-15It is more immunogenic. Additional alum did not increase PEG-Abeta in mice_1-16(Δ14)The immunogenicity of (a). With PEG-Abeta_1-16(Δ14)By comparison, with PEG-Abeta_4-11The induced antibody response is slower.

Because of the problem of converting a faster immune response to higher memory, pegylated antigens were compared to palmitoylated antigens in a dual transgenic Alzheimer's mouse model.

Another method may be used as described in US6843942 and EP 1337322.

Example 6: comparison of immunogenicity of Pegylated versus palmitoylated antigens in the Alzheimer's mouse model (ELISA)

6.1 for in vivo immunization studies, APP 717C 57BL/6 XPS-1A 246E FVB mice (APP XPS-1 mice) were housed as single individuals, randomized double blindly, age-matched, and genotyped by PCR

Young (3-4 months) female mice were used, which were double transgenic mouse strains expressing a mutant human amyloid precursor protein (APP-V717I) and a mutant human presenilin-1 (PS1-a246E), both under the control of the mouse thy1 gene promoter and in the F1(FVB × C57BI) genetic background. All mice were genotyped by Polymerase Chain Reaction (PCR) at 3 weeks of age and given a unique marker for each mouse. A second PCR was performed at the beginning of the study and before blinded randomization into different experiments, whereby all mice were genotyped twice during their lifetime. Mice were allowed free access to water and standard mouse chow (Muracon-G, Trouw Nutrition, Gent, Belgium). Mice were housed in standard metal cages under reversed day-night law according to local animal welfare regulations. Mice were housed in macrolon2 type cages 5 days before the start of behavioral testing and transported to a behavioral laboratory where they were acclimated and habituated to the testing laboratory.

6.2 immunization

Anti-amyloid vaccines were prepared using liposomes with lipid A as an adjuvant (Nicolau et al, 2002). Myristica fragrans-phosphatidyl-choline, -glycerol, and cholesterol were mixed in a molar ratio of 0.9: 1.0: 0.7. Monophosphoryl lipid a, a potent immunomodulator, was added at a concentration of 40 mg per mmol of phospholipid. Palmitoylated and pegylated peptides were added at a molar ratio of 1: 100 peptide to phospholipid. The solvent was evaporated and the resulting film was hydrated with sterile PBS (pH 7.3) to a final phospholipid concentration of 4 millimolar.

Palmitoylated (ACI-24, A. beta.) was used at 2-week intervals_1-15) And pegylated (ACI-01, Abeta)_1-16) Antigen immunization of APP × PS-1 mice (5 total times every 2 weeks, i.p. vaccination). In each experimental group, 10 animals were immunized with each antigen by intraperitoneal injection (200 microliters each containing 8 nanomoles of peptide). Empty liposomes were used as controls. Sera were taken periodically and 5 days after the boost (once every 2 weeks) and anti-amyloid ELISA was performed with several dilutions of sera. The results of the comparison show the immunogenicity of the different antigens.

Significant immune responses were achieved in palmitoylated and in APP x PS-1 mice immunized with pegylated liposome/Α β antigen 5 days after the sixth antigen vaccination. However, unlike the immune response in healthy C57BL/6 mice, pegylated antigens do not elicit higher antibody titers than palmitoylated antigens in the disease model.

With ACI-24, the anti- Α β -specific IgG immune response increased relatively rapidly, peaking after 5 weeks. The two vaccines induce significantly different immunoglobulin types and isotypes, palmitoylated ACI-24 antigen results in higher titers of IgG, while pegylated ACI-01 induces more IgM type antibodies. The final blood samples from all animals were analyzed for IgG isotype.

Identification of Abeta by ELISA_1-42Specific IgG and IgM antibodies. With 10. mu.g/ml amyloid beta_1-42The coated plates were kept at 4 ℃ overnight. After washing each well with PBS-0.05% tween 20 and blocking with 1% BSA, serially diluted sera were added to the plates and incubated at 37 ℃ for 2 hours. After washing, the plates were incubated with phosphatase-conjugated anti-mouse Ig (IgG, whole antibody, Sigma-Aldrich st. louis, MO, USA) or isotype specific antibodies (IgM, IgG1, IgG2a and IgG3, purchased from Pharmingen BD, San Diego, CA, USA and IgG2b from zymed laboratories, San Francisco, CA) for 2 hours at 37 ℃. After the final wash, the plate was incubated with PNPP (p-nitro-phenyl-phosphate), phosphatase substrate and the plate was read at 405nm using an ELISA plate reader. Results are expressed with reference to serial dilutions of titrated serum pools from immunized adult mice, or serial dilutions of commercially available antibodies (6E10, chemicon international, Temecula, CA, USA). Alternatively, results are expressed as OD values at the dilution at which no serum is at saturation level (table 1).

Table 1.

	IgG1 control	ACI-01	ACI-24	IgG2a control	ACI-01	ACI-24	IgG2b control	ACI-01	ACI-24
										Average value	0.1	0.11	1.33	0.15	0.22	0.55	0.59	1.81	2.88
SD	0.01	0.02	0.98	0.03	0.03	0.77	0.12	1.23	0.82

	IgG3 control	ACI-01	ACI-24
				Average value	0.1	0.63	2.05
SD	0.00	0.22	0.39

ACI-24 predominantly leads to IgG1 and IgG2b isotypes, both of which are the major non-inflammatory Th2 subtype, and ACI-24 also leads to IgG3, which is a T-cell independent IgG subtype. Both vaccines induced only very low levels of IgG2a (Th1) except for one animal vaccinated with the ACI-24 vaccine.

Epitope mapping of the resulting antibodies was performed by ELISA using a peptide library covering the complete amino acid sequence of Α β 1-42, comprising a total of 33 biotinylated peptides, and using the biotinylated complete β peptide as a positive control. Immunization with both vaccines ACI-01 and ACI-24 resulted in anti-A β antibodies with the same epitope, defined by amino acids 1-9 of A β (peptide 1). Furthermore, we analyzed the final conformation dependence by measuring the specific binding of the resulting anti-A β antisera to polymeric A β, by performing an adaptive ELISA assay on A β 1-42 fibers. ACI-24 immunization resulted in significantly higher titers of anti-A β antibodies recognizing A β 1-42 fibers compared to antisera produced by mice immunized with ACI-01 (Table 2). From the results obtained, it was found that the immune responses generated by immunization with ACI-01 and ACI-24 differ not only in their potency, subtype and Ig-isotype, but also in their conformational specificity.

Table 2.

	Control	ACI-01	ACI-24
				SEM	2049.0± 46.7	3426.2± 221.9	7770.6± 2090.1
Statistical ANOVA		p＜0.05	p＜0.01

Example 7: comparison of recognition Capacity (ORT) of Pegylated versus palmitoylated antigen in a mouse model of Alzheimer's disease

7.1 Effect on the improvement of non-spatial, Hippocampus-dependent memory Capacity in a mouse model of APP XPS-1 Alzheimer's disease

Analysis was performed in a mouse model of APP XPS-1 Alzheimer's disease by using palmitoylation (ACI-24, A β)_1-15) And pegylation (ACI-01, Abeta)_1-16) The effect of active anti- Α β 1-16/1-15 immunization with antigen on the improvement of non-spatial hippocampal-dependent memory capacity within 3 months of immunization was essentially as described (Tang et al 1999; rampon et al 2000) performed an object identification test (ORT). Statistical analysis was performed using the ANOVA Turkey-Kramer multiple comparison test, as described (Moechars, D. et al (1999) and (1996)). The test was performed using GraphPad insstat version 3.06, GraphPad software, San diego california usa.

Briefly, a three month immunization schedule was as follows: ACI-01 and ACI-24 were inoculated every 2 weeks for a total of six times. One group of mice received empty liposomes as a control. Mice were habituated to a Plexiglas (Plexiglas) open field box (52X 40cm) with black vertical walls and a translucent floor, which was weakly illuminated by a lamp placed under the box, for 1 hour. The following day the animals were placed in the same box and subjected to 10 minutes of skill training. During training, mice were individually placed in an open field with an a object (marbles or dice) and the time spent exploring the a object (the time the snout and mouth of the animal were facing the object and the distance was <1 centimeter) was measured. During a 10 minute holding session (second session) which was carried out after 3 hours, a new object (B object: marble or dice) was placed in the open field together with the already familiar object (A object). The time the animal spent exploring both objects (tA and tB) was recorded. Non-spatial memory is measured using an identification index (RI), defined as the ratio of the time spent exploring a new object to the time spent exploring two objects [ (tB/(tA + tB)) × 100 ]. Statistical analysis was performed using ANOVA single factor as described ((Moechars et al 1999; Moechars et al 1996)).

APPXPS-1 mice were immunized with palmitoylated (ACI-24) and pegylated (ACI-01) antigens at 2-week intervals. Animals of 10 ages 3 months were immunized intraperitoneally with each antigen (200 microliters, 100 micrograms of peptide per intraperitoneal injection) and empty liposomes were used as controls. Sera were taken 5 days after the boost and ELISA was performed with several dilutions of sera. The results of the comparison show the immunogenicity of the different antigens.

In a typical experiment for non-spatial visual recognition of memory, APP × PS-1 transgenic mice immunized with palmitoylated (ACI-24) and pegylated (ACI-01) Abeta antigens were evaluated for cognitive ability by subjecting the animals to an object recognition task known to be dependent on hippocampal activity ((Tang et al 1999), (Rampon et al 2000)). Basically, 3 hours after training all mice familiar with a given object, their memory retention was tested by facing them with a new object located beside the familiar object, outside the familiar object.

Palmitoylated A β compared to control-treated APP × PS-1 mice_1-15Immunization with antigen (ACI-24) significantly increased memory retention or cognitive memory capacity in APPxPS-1 mice (76.1 + -3.9% vs. 49.1 + -4.5% of control; viscosity). This confirms that ACI-24 immunized mice recognize and remember the original object for at least 3 hours, thereby causing their motivation and their exploratory ability to be as intact as healthy, age, sex, and strain matched mice when compared to healthy, non-treated and non-transgenic wild type mice (61.8 ± 5.1%). Although the ACI-01 peptide is only one C-terminal amino acid (lysine 16) longer than the ACI-24 peptide, and only the linker technology differs between these vaccines, PEGylated A β is used_1-16Immunization with antigen (ACI-01) did not show any memory recovery comparable to ACI-24 (45.6)±6.2％)。

TABLE 3

	Control	ACI-01	ACI-24	Healthy and healthy
					SEM	49.1±4.5	45.6±6.2	76.1±3.9	61.8±5.1
Statistics of		n.s.^*Relative to control	p < 0.05 vs control	n.s.^*Relative to control

n.s.^*: is not significant

7.2 possible contributions of different antibody classes IgM and IgG to cognitive function

To analyze the possible contribution of different antibody types IgM and IgG to cognitive function, correlation analysis was performed.

IgM antibodies and conjugatesMemory ability independence (r)²0.2333), but the resulting IgG-type antibody roughly correlates with the grade of memory ability (ORT index) in two stages (r)²0.857). Between the ORT indices of 0 to 20, a more linear relationship is observed, whereas at ORT indices exceeding 20, the correlation enters the saturation phase. This may indicate that IgM antibodies that fail to pass through the blood-brain barrier do not contribute to memory recovery. In contrast, IgG antibodies can cross the blood brain barrier, depending on their subtype, and are associated with memory improvement.

To assess the ability of ACI-24 immunization to alter the levels of soluble and insoluble amyloid peptides in the brain of APP XPS-1 mice, human A β 1-40 and A β 1-42 were measured by specific ELISA in the soluble fraction of brain homogenates. A commercially available ELISA kit (amyloid. beta.40 or. beta.42 ELISA, The Genetics Company, Zurich, Switzerland) was used. ELISA was performed according to the manufacturer's protocol. The A.beta.content of the sample was quantified by comparing the absorbance with a standard curve prepared using the synthesized A.beta.1-40 or A.beta.1-42 (Table 4).

Table 4.

	Soluble Abeta	Soluble Abeta 42	Insoluble Abeta 40	Insoluble Abeta 42
					Control	2.6±0.6	3.1±1.0	3.0±0.1	3.0±0.04
ACI-24	2.1±0.8	2.1±0.9	2.0±0.1.	2.0±0.07
					Statistical ANOVA	n.s.	p＜0.01	p＜0.05	p＜0.05

n.s.^*: is not significant

Data are presented as mean (A β ng/g brain homogenate. + -. SEM)

Immunization with ACI-24 resulted in a significant reduction in insoluble, plaque-associated A β 1-40 and A β 1-42. The amount of soluble A.beta.1-42 was also significantly reduced, while the amount of soluble A.beta.1-40 showed only a tendency to decrease.

Example 8: immunization with ACI-01 and-24 did not induce inflammatory responses

The safety of two liposome vaccines, ACI-01 and ACI-24, was evaluated by measuring the local production of inflammatory cytokines IL-1 β, IL-6, IFN- γ and TNF α by specific ELISA. Levels of TNF- α, IFN- γ, IL-6 and IL-1 β in total brain homogenates were measured using a sandwich ELISA according to the manufacturer's manual (both R & D Systems, Minneapolis. MN, USA). Results are expressed in pg/ml for serial dilutions of the reference recombinant cytokine. The extent of activated Microglia (MHCII) and astroglia (GFAP) in the hypothalamic region of the brain was assessed by quantitative immunohistochemistry.

Immunization with ACI-01 or ACI-24 did not significantly increase the levels of IL-1 β, IL-6, IFN- γ, and TNF α in the brain. Similarly, when immunised with ACI-24, no difference in astrogliosis was observed, whereas after a 3 month immunisation period the range of activated microglia showed a decreasing tendency.

Example 9: production of mAbs:

palmitoylated antigen (ACI-24, Abeta.) was used at 2-week intervals_1-15) C57BL/6 mice were immunized. 10-12 animals were immunized with each antigen (injection volume: 200. mu.l, containing 8 nmol of peptide). The last injection was performed 4 days before the animals were sacrificed. After 5 boosts, mice with therapeutic titers (when 1: 5,000 diluted sera were positive in the ELISA) were selected for fusion. Spleen cells are harvested from the immunized animal and hybridomas are produced by fusing the primed spleen cells with a myeloma cell line. Fusion of spleen-derived mouse B-lymphocytes with cells of the myeloma cell line SP2-0(ATCC, Manassas, Va.) was performed using the well-known methods of Kohler and Milstein (Nature 256: 495-497(1975)) and Harlow and Lane (Antibodies: A Laboratory Manual (Cold spring harbor Laboratory, New York 1988)).

Cell fusion was induced by the addition of polyethylene glycol. The resulting hybrid cells are then cloned in a conventional manner, e.g., using limiting dilution, and IgG-producing hybridoma clonal lines are selected and tested for A β by ELISA_1-42Specific binding of the peptide, and culturing the resulting clone producing the desired monoclonal antibody.

The hybridomas thus obtained are chemically selected by plating the cells in a selection medium containing hypoxanthine, methotrexate and thymidine (HAT).

The hybridomas are then screened for their ability to produce monoclonal antibodies against a particular amyloid-associated disease or disorder. Hybridomas producing the antibody of interest are cloned, expanded and frozen for future production. Preferred hybridomas produce monoclonal antibodies having the IgG isotype, more preferably the IgG2 isotype.

Example 10: specificity determination of antibody mACI-24-Ab4

To analyze the specificity of antibody ACI-24-Ab4, different concentrations of pre-formed amyloid 1-42, 1-40, and 1-38 fibrils were spotted on Hybond ECL nitrocellulose membranes (Amersham biosciences). After blocking with 10% milk powder and 0.7% tween 20, the membrane was incubated with 20 μ g/ml of primary antibody for 2 hours at room temperature. After rinsing, the membrane was incubated with horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (Amersham Biosciences) for 1 hour at room temperature, rinsed and incubated with a chemiluminescent solution, followed by exposure of an X-ray film with the membrane.

To measure the binding of mAb (mACI-24-Ab4) to amyloid β 1-42 fibers, A β 1-42, 1-40 and 1-38 fibers were preformed at 37 ℃ for 7 days and spotted onto the membrane. Binding capacity was measured using 20 μ g/ml antibody and bound antibody was detected by exposure to horseradish peroxidase conjugated sheep anti-mouse IgG antibody for 20 minutes.

As can be demonstrated by dot blot analysis, antibody mACI-24-Ab4 bound with different sensitivities and different preformed Α β fibers. Than Abeta_1-40Or Abeta_1-38Antibody pair A beta_1-42The fibers showed the highest binding sensitivity. It is capable of detecting at least 0.001 microgram of A beta_1-42Fiber, and antibody pair A beta_1-40The limit of detection of the fiber is at least 0.1 microgram for Abeta_1-38The fibres were 1 microgram, which means that the sensitivity to these types of amyloid fibres was 100 to 1000 times lower. These data demonstrate that the antibody ACI-24-Ab4 has at least 100-fold higher sensitivity to the amyloid form (1-42), which is known to become insoluble by changing secondary conformation and is a major part of amyloid plaques in the brain of Alzheimer's disease patients.

Example 11: fractionation by density-gradient ultracentrifugation

Monoclonal antibodies were studied for inhibition of A β by density-gradient ultracentrifugation (Rzepecki et al, 2004)_1-42Polymerization of fibers and production of Abeta_1-42Properties on fiber depolymerization, the density-gradient ultracentrifugation is based on the following principle: the resulting peptide fibers, distributed in different sizes after incubation with or without antibodies, are then subjected to a preformed gradient (OptiPrep)^TM) SDS-PAGE sedimentation analysis was performed. It is a clear advantage of the method that a pre-formed population of A β -fibers, the disaggregation and aggregation inhibiting properties of the co-incubated antibody, and the binding of the antibody to the fibers can be analyzed simultaneously.

All targets were analyzed for A β in disaggregation and inhibition assays_1-15The monoclonal antibody produced (mACI-24-Ab 4).

For A beta_1-42Inhibition of aggregation in two different molar ratios (monomeric Abeta)_1-4230 or 100 times higher molar ratio than MAb) is added to the mixture, and a β is added_1-42Monomers were incubated with mAbs at a final concentration of 50. mu.M of A.beta.. After incubation at 37 ℃ for 24 hours, the samples were overlaid with a discontinuous gradient of Optiprep^TMAnd the tube was centrifuged at 259000g for 3 hours at 4 ℃.15 fractions (140 microliters each) were harvested, fraction 1 being the lowest density fraction from the top of the gradient, and fraction 15 being the highest density fraction from the bottom of the gradient. The precipitate was also collected. The collected fractions were analyzed by SDS-PAGE and stained with silver. Abeta for inhibition assay_1-42The concentration is five times lower than that used for the disaggregation assay, which can reduce the aggregation movement of amyloid and ensure measurement in the linear phase.

Without mAb addition, a β peptide aggregated after 24 hours incubation time and most of the protein was found in fraction 13 to the pellet (pellet, few in 12), demonstrating complete polymerization of a β peptide monomers. Successful and significant inhibition of aggregation should result in small fibers or oligomers, which should be found in the lower density fraction. In the aggregation assay, machi-24-Ab 4 caused the major bands (the strongest bands) to move from 13 to 11 and 12, and significant dissolution of bands in fraction 13 to the pellet. These mean that machi-24-Ab 4 exhibited a strong ability to inhibit polymerization of a β peptide monomers into fibers and showed specific binding to a β fibers (in fractions 11 and 12).

As for preformed Abeta by co-incubation with MAbs_1-42Depolymerization of fibrils (MAb + monomer A β in two different molar ratios 1: 30 and 1: 100_1-42Final concentration of a β 246uM), the samples were incubated at 37 ℃ for 24 hours. After 24 hours, the samples were fractionated by ultracentrifugation and separated by SDS-PAGE as described above and before (Rzepecki et al, 2004).

Analogously to the aggregation test, with the aid of Abeta_1-42The distribution of fibrils alone in fractions 12 to P (precipitate) can demonstrate complete fiber aggregation. Here, when incubated with pre-formed fibers, the fibers migrate toward the lower density fraction, indicating the disaggregation activity of the antibody. The addition of mACI-24-Ab4 at a molar ratio of 1: 100 showed that most of the amyloid fibrils moved from 12 to 11. Thus, mACI-24-Ab4 also showed strong depolymerization activity.

Example 12: combined use of palmitoylated antigens and complement activation inhibitors in a retention of recognition ability assay in a mouse model of Alzheimer's disease

To prevent possible side effects, such as neurological complications, caused by further stimulation of the already over-activated complement system by vaccination, palmitoylated antigen (ACI-24, A β)_1-15) Administered in combination with a complement inhibitor selected from the group consisting of TP10 (soluble human complement receptor 1), Eculizumab (anti-human complement protein C5), pexizumab (Pexelizumab) (anti-C5 complement), Seatt (Cetor), a natural C1 inhibitorC1-esterseremmer-N) and natural human C1 inhibitors.

In palmitoylation of (ACI-24, Abeta)_1-15) Antigen pairThe complement inhibitor is administered to the human patient prior to, or immediately after, vaccination.

In palmitoylation of (ACI-24, Abeta)_1-15) In the application regimen of the complement inhibitor administered prior to antigen vaccination, the inhibitor compound is administered within a time window of no more than 20 hours before the start of vaccination until just before the end of vaccination (application regimen 1).

In palmitoylation of (ACI-24, Abeta)_1-15) In the application regimen of administration of the complement inhibitor after antigen vaccination, the complement inhibitor is administered within a time window beginning immediately after vaccination and ending one day after vaccine administration.

12.1TP10 (soluble human complement receptor 1)

In human trials with TP10, it was found to be preferable to maintain the concentration of TP10 after CPB in the range between 100. mu.g/ml and 160. mu.g/ml for 24 hours. To achieve such a concentration range, a starting dose of 10 mg/kg administered over 0.5 hours followed by 10 mg/kg over 23.5 hours is most appropriate (Li JS, Am Heart J.20041 months; 147 (1): 173-80).

Palmitoylated (ACI-24, A β) was performed after the desired concentration of TP10 had been reached according to application scheme 1, or before the initial dose of 10 mg/kg TP10 was administered according to application scheme 2_1-15) Vaccination of antigens.

12.2 Aikelizumab (anti-human complement protein C5)

Eculizumab (600 mg) was administered by infusion weekly for 4 weeks, followed by 900-mg doses after one week, and then 900 mg doses every other week up to week 12 (Hillmen P, N EnglJ med.20042 month 5 days; 350 (6): 552-9).

For long-term treatment, eculizumab may be administered at a dose of 900 mg every 12 to 14 days (HillA, blood.200510, month 1; 106 (7): 2559-65.Epub 20056, month 28).

Palmitoylated (ACI-24, A β) was administered after the first 600 mg dose of eculizumab had been administered according to application scheme 1, or before the initial dose of 600 mg eculizumab was administered according to application scheme 2_1-15) Vaccination of antigens.

In some cases, it may be more appropriate to use application protocol 1 only after week 4, when the first 4 rounds of eculizumab administration have ended and a stable steady state concentration is reached in humans.

12.3 mu xi Li MAb (anti-C5 complement)

Perilimumab was administered intravenously over 10 minutes at a 2.0 mg/kg bolus (bolus) which could be followed by an infusion of 1.0 mg/kg (http:// circuit. ahajournals. org/cgi/content/full/106/23/2986-a) or 0.05 mg/kg/h for 24 hours over 20 hours.

Palmitoylation was performed after the first 2.0 mg/kg shock dose of Pezilizumab according to application protocol 1, or before the initial 2.0 mg/kg shock dose of Pezilizumab according to application protocol 2 (ACI-24, A β. beta.) -was administered_1-15) Vaccination of antigens.

In some cases, it may be more appropriate to use application protocol 1 only after the 2 nd application by infusion has been completed and a stable steady state concentration is reached in humans.

12.4 Natural human C1 inhibitor

The C1 inhibitor may be administered at a dose of 6.25 to 100 units/kg (van Doorn MB, Allergy Clin Immunol.200510 months; 116 (4): 876-83. Epub 20058, 8 days).

Alternatively, pasteurized C1 esterase inhibitor concentrate (De Serres J, Transfus Apher Sci.200312 months; 29 (3): 247-54) may be administered in a dosage of 500 to 1000 International units; (Bork K, Arch Intern Med.20013.12 months; 161 (5): 714-8).

The C1-inhibitor may also be administered intravenously in 1 hour infusion starting at 6000 International units and at 12 hour intervals followed by 3000 International units, 2000 International units and 1000 International units (Caliezi C, Crit Care Med.2002, 8 months; 30 (8): 1722-8).

Finally, C1-inhibitor in the form of a steam-heated inhibitor concentrate can be administered intravenously once every three days AT a concentration of 25 plasma units per kg body weight (Waytes AT, N Engl J Med.1996, 6 months and 20 days; 334 (25): 1630-4).

Palmitoylated (ACI-24, A β) after the C1-inhibitor has been administered according to application scheme 1, or before the initial dose of C1 inhibitor is administered according to application scheme 2_1-15) Vaccination of antigens.

12.5 Natural C1 inhibitor Sauter(C1-esteraseremmer-N)

C1-esterserem mer-N or ceter at 1000 units, 1500 units or 2000 unitsAnd the same dose of the other product is administered later.

According to application protocol 1, after a second dose has been administered, or according to application protocol 2, 1000 units, 1500 units or 2000 units of C1-esterserem mer-N or serter are givenIs preceded by an initial dose of palmitoylated (ACI-24, A β)_1-15) Vaccination of antigens.

Applicant or agency volume No.: l3018PCT BS

International application No.: new PCT application

With instructions for the deposited microorganisms or other biological materials

(PCT bar 13 bis)

PCT/RO/134 Table (1998 month 7; 2004 month 1 redeployed)

[ brief description of the drawings ]

FIG. 1 a: design and biophysical characterization of two liposomal vaccines containing peptide immunogens of the first 15(ACI-24, A β 1-15) and 16(ACI-01, A β 1-16) amino acids of the full-length amyloid β 1-42 peptide. b) ACI-01 contains A β 1-16, a pegylated lysine residue on each side of the peptide, with DSPE at the other end of the PEG chain (a) as a liposome anchor. As for ACI-24(b), two terminal palmitoylated lysine residues are covalently linked at each end of A β 1-15 to allow the antigen to be reconstituted within and anchored in the liposome (a). c) CD profile of two antigens reconstituted in liposomes. ACI-01 shows a spectrum indicating a random coil or loose protein conformation (negative signal from 210nm and slowly reaching the 0-axis to 260 nm), while the ACI-24 spectrum contains a significant portion of beta structure (positive signal from 210nm, then crossing the 0-axis, then approaching the 0-axis again to 260 nm). For CD profiling, beta-amyloid samples (ACI-01 and-24) were reconstituted in liposomes and sonicated at a peptide concentration of 0.9865 mg/ml (1 ml in PBS) by using a probe sonicator. CD spectra were recorded on a Dichrograph (JASCO J-810) of a quartz cuvette with an optical path length of 0.1 cm. The spectral window was 190-260nm at 25 ℃ at a scanning speed of 20 nm/min, and the raw data were expressed in terms of ellipticity in units of θ (mdeg).

FIG. 1 b:¹magic angle spinning NMR spectra of peptide amide protons and aromatic side chains of the H spectral region including A.ACI-01 vaccine, B.ACI-24 vaccine, C.1mM ACI-01, D.1mM ACI-24 and E.4mM Ab1-15 peptide (in PBS buffer, pH 7.2).

FIG. 1 c: one-dimensional 1H NMR spectra from 9 to 5.5ppm of pegylated (black) and palmitoylated (blue) amyloid-1-15. Peptides were synthesized, covalently linked to palmitic acid or polyethylene glycol, respectively, and reconstituted in PBS. For NMR analysis, the sample was centrifuged and the total spectrum was recorded from 9 to 0.2 ppm.

FIG. 2: amyloid-specific titers in serum of APP XPS-1 mice immunized with pegylated (ACI-01) or palmitoylated (ACI-24) antigens in liposomes were analyzed, compared to mice immunized with empty liposomes (control). a) Immunization with ACI-24 produced high levels of amyloid-specific IgG antibodies only after two immunizations and 3 weeks after the first (a, left) and reached a maximum after 5 weeks. Immunization with ACI-01, however, produced high levels of amyloid-specific IgM antibodies (a, right), which were maximal after 7 weeks, but only low IgG levels (a, left, p < 0.5) compared to ACI-24.

And (4) preservation:

according to the Budapest treaty, in "German Collection of microorganisms and cultures (DSMZ)" (Braunschweig, Mascherder Weg 1B, 38124 Braunschweig), the following hybridoma cell lines were deposited:

name of hybridoma cell line	Name of antibody	Date of storage	Accession number
				EJ 7H3	mACI-24-Ab4	12/8/2005	DSM ACC2756

Reference to the literature

Alving et al, infection. immun.60: 2438-2444, 1992

Bork K，Arch Intern Med.2001 Mar 12；161(5)：714-8

Caliezi C，Crit Care Med.2002 Aug；30(8)：1722-8.

De Serres J，Transfus Apher Sci.2003 Dec；29(3)：247-54

Doorn MB van，，Allergy Clin Immunol.2005 Oct；116(4)：876-83.Epub2005 Aug 8

Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring harbor Laboratory, New York 1988)

FYLAKTAKIDOU，K.C.，LEHN，J.-M.GREFERATH，R.NICOLAU，C.，

"Inositol tripropophosphate: a new membrane permanent inductive effector haemoglobin ", bioorg.Med.chem.Lett., 15, 1605-: 421(1991)

Khaw, b.a. et al j.nuclear.med.23: 1011-1019(1982)

Kohler and Milstein (Nature 256: 495-

Moechars, d., Dewachter, i., Lorent, k., Reverse, d., baekeladt, v., Naidu, a., Tesseur, i., Spittaels, k., Haute, c.v., cheler, f., Godaux, e., corell, b., and Van Leuven, f.: 1999, J.biol.chem.274, 6483-.

Moechars，D.，Lorent，K.，De Strooper，B.，Dewachter，I.，& Van Leuven，F.Expression in brain of amyloid precursor protein mutated in thealpha-secretase site causes disturbed behavior，neuronal degeneration andpremature death in transgenic mice.EMBO J.15，1265-1274(1996).

Hill A，Blood.2005 Oct 1；106(7)：2559-65.Epub 2005 Jun 28.

Hillmen P，N Engl J Med.2004 Feb 5；350(6)：552-9.

Li JS，Am Heart J.2004Jan；147(1)：173-80

Moechars, d., Lorent, k., De Strooper, b., Dewachter, i., and Van Leuven, f.: 1996, EMBO J.15, 1265-.

Nicolau, C., Greferath, R., Balaban, T.S., Lazarte, J.E., and Hopkins, R.J. (2002). Proc Natl Acad Sci USA 99, 2332-.

Piotto，M.，Saudek，V.，& Sklenar，V.Gradient-tailored excitation forsingle-quantum NMR spectroscopy of aqueous solutions.J.Biomol.NMR2，661-665(1992).

Piotto, m., el bayer, k., Wieruszeski, j.m., and Lippens, g.practical aspectsof shifting a high resolution map indexing probe.j.magn. reson.173, 84-89(2005).

Queen et al, proc. natl Acad Sci USA, 86: 10029-10032(1989)

Rampon, c., Tang, y.p., Goodhouse, j., Shimizu, e., Kyin, m., and Tsien, j.z.: 2000, nat. neurosci.3, 238-244.

Rzepecki et al, 2004

Rousseaux et al Methods Enzymology, 121: 663-69, Academic Press, 1986

Tang, y.p., Shimizu, e., Dube, g.r., Rampon, c., Kerchner, g.a., Zhuo, m., Liu, g., and Tsien, j.z.: 1999, Nature 401, 63-69.

B Teisseire, C Ropars, M C Viller eal, and C Nicolau, < < Long-termphysiology effects of enhanced O2 release by osteoinositolphosphates-loaded erythrocytes, "Proc Natl Acad Sci USA.1987 October; 84(19): 6894-6898

Teisseire B，Ropars C，Villeréal MC，Nicolau C.Long-term physiologicaleffects of enhanced O2 release by inositol hexaphosphate-loadederythrocytes.Proc Natl Acad Sci U S A.1987 Oct；84(19)6894-6898.

Udenfriend, s. et al fluoroscamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range, science 178, 871-.

Wagner et al (2002) Journal of lipid Research Vol 12(3), pp 259-

Waytes AT，N Engl J Med.1996 Jun 20；334(25)：1630-4

US-P 6843942

EPA 1337322

Sequence listing

<110> AC immune Co

<120> therapeutic vaccine

<130>L3028PCT

<150>EP 05027091.7

<151>2005-12-12

<150>EP 06009098.2

<151>2006-05-02

<160>6

<170>PatentIn version 3.1

<210>1

<211>15

<212>PRT

<213> human (Homo sapiens)

<220>

<223>Antigenic peptide AB_1-15

<400>1

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

15

<210>2

<211>16

<212>PRT

<213> human

<220>

<223>Antigenic peptide AB_1-16

<400>2

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

16

<210>3

<211>15

<212>PRT

<213> human

<220>

<223>Antigenic peptide AB_1-16(A14)

<400>3

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

15

<210>4

<211>8

<212>PRT

<213> human

<220>

<223>Antigenic peptide AB_4-11

<400>4

Phe Arg His Asp Ser Gly Tyr Glu 8

<210>5

<211>13

<212>PRT

<213> human

<220>

<223>Antigenic peptide AB_22-35

<400>5

Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu 13

<210>6

<211>12

<212>PRT

<213> human

<220>

<223>Antigenic peptide AB_29-40

<400>6

Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val 12

Claims

1. A method for the preparation of a therapeutic vaccine composition comprising the use of a β modified with a lipophilic or hydrophobic moiety_1-15An antigenic peptide antigen, wherein the a β peptide antigen is prepared as follows: (a) pre-forming a peptide on a resin using standard automated peptide synthesis, (b) then modifying by grafting a lipophilic or hydrophobic moiety onto the resin onto a terminal amino acid residue of the pre-formed peptide, wherein the lipophilic or hydrophobic moiety is a fatty acid having a carbon backbone of at least 10 carbon atoms.

2. The method of claim 1, wherein the therapeutic vaccine composition comprises a single or repeated SEQ ID NO: 1 is A beta_1-15A peptide antigen.

3. The method of claim 1, wherein the A β peptide antigen is reconstituted in a carrier.

4. The method of claim 3, wherein the A β peptide antigen is reconstituted in liposomes.

5. The method of claim 1, wherein the hydrophobic moiety is palmitic acid.

6. The method of claim 4, wherein the liposome preparation contains an adjuvant.

7. The method of claim 6, wherein the adjuvant is monophosphoryl lipid A.

8. A method of preparing a therapeutic vaccine composition comprising the use of immunogenic A β_1-15An antigenic peptide, wherein said A β_1-15The peptide antigen is palmitoylated A beta modified by covalently linked palmitoylated amino acid residues and reconstituted in liposomes_1-15A peptide antigen wherein the A β_1-15Peptide antigens were prepared as follows: (a) pre-forming a peptide on a resin using standard automated peptide synthesis, (b) then modifying by grafting a palmitic acid moiety onto a terminal amino acid residue of the pre-formed peptide on the resin.

9. The method of any one of claims 1-5 and 8, wherein the a β_1-15The peptide antigen is modified by 2 palmitoylated amino acid residues covalently linked to the N-and C-terminus of the peptide, respectively.

10. The method of any one of claims 1-5 and 8, wherein the a β_1-15The peptide antigen is modified with 4 palmitoylated amino acid residues, wherein the N-and C-termini of the peptide are covalently linked to 2 palmitoylated amino acid residues, respectively.

11. The method of any one of claims 1-5 and 8, wherein 2 or more than 2 palmitoylated a β modified by covalently linking palmitoyl residues at each end of the peptide_1-15The peptide antigen molecules are reconstituted in a single liposome.

12. The method of claim 9 or 10, wherein the vaccine composition is used for the treatment of an animal or human suffering from alzheimer's disease, which treatment results in an increase in the retention of cognitive memory.

13. A process for the preparation of a pharmaceutical composition comprising A beta modified with a lipophilic or hydrophobic moiety_1-15A method of antigenic construction of an antigenic peptide comprising the steps of:

(a) the A.beta.was preformed by standard automated peptide synthesis on resin_1-15A peptide antigen, and

(b) by grafting lipophilic or hydrophobic moieties onto the preformed Ass on resins_1-15Modifying the preformed A.beta.at the terminal amino acid residue of the peptide_1-15A peptide antigen, wherein the peptide antigen is selected from the group consisting of,

wherein the lipophilic or hydrophobic moiety is a fatty acid having a carbon backbone of at least 10 carbon atoms.

14. An antigenic construct made by the method of claim 13.

15. The antigenic construct of claim 14, comprising a modified a β_1-15An antigenic peptide, wherein said A β_1-15Antigenic peptides modified by lipophilic or hydrophobic moieties, useful for the treatment of Alzheimer's disease, whereinThe a β peptide antigen was prepared as follows: standard automated peptide synthesis on resin to pre-form the A beta_1-15And modified by grafting onto the resin a lipophilic or hydrophobic moiety onto the terminal amino acid residue of the preformed a β peptide, wherein the lipophilic or hydrophobic moiety is a fatty acid having a carbon backbone of at least 10 carbon atoms.

16. The antigenic construct of claim 15, comprising a single or repeated SEQ ID NO: 1 is a beta_1-15A peptide antigen.

17. An antigenic construct according to any one of claims 14 to 16, wherein the a β 1-15 peptide antigen is presented in a form conjugated to or reconstituted in a carrier or adjuvant.

18. The antigenic construct of claim 17, wherein said a β_1-15The peptide antigen is presented in a reconstituted form in liposomes.

19. The antigenic construct of claim 14, wherein the hydrophobic moiety is palmitic acid.

20. The antigenic construct of claim 18, wherein the liposome preparation contains an adjuvant or an immunomodulator or both an adjuvant and an immunomodulator.

21. An antigenic construct according to claim 20, wherein the immunomodulator is lipid a.

22. A vaccine composition comprising the antigenic construct of any one of claims 14-21.

23. The vaccine composition of claim 22, wherein the liposome preparation contains an adjuvant or an immunomodulator or both an adjuvant and an immunomodulator.

24. The vaccine composition of claim 23, wherein the immunomodulator is detoxified lipid a.

25. Vaccine compositions for the treatment of alzheimer's disease comprising immunogenic a β_1-15An antigenic peptide, wherein said A β_1-15The peptide antigen is palmitoylated a β modified by covalently linked palmitoyl residues at each end of the peptide and reconstituted in liposomes_1-15A peptide antigen.

26. The vaccine composition of claim 25, wherein 2 or more than 2 palmitoylated a β modified by covalently linking palmitoyl residues at each end of the peptide_1-15Peptide antigens are reconstituted in a single liposome.

27. The vaccine composition of any one of claims 22-26, which when administered to an animal or human, results in the production of antibodies of the IgG subtype that are independent of T-cells.

28. The vaccine composition of claim 27, wherein the antibody is an IgG3 isotype.

29. The vaccine composition of any one of claims 22-26, which, when administered to an animal or human selected from a mammal, does not cause a significant increase in inflammatory markers in the brain.

30. The vaccine composition of claim 29, wherein the marker is selected from the group consisting of IL-1 β, IL-6, IFN- γ, and TNF α.

31. The vaccine composition of any one of claims 22 to 26, which when administered to an animal or human results in plaque-associated A β that is insoluble in the brain_1-40And Abeta_1-42A significant reduction in.

32. The vaccine composition of any one of claims 22 to 26, which when administered to an animal or human results in soluble a β in the brain_1-42The level of (c) is significantly reduced.

33. A vaccine composition according to any one of claims 22 to 26 for use in the treatment of alzheimer's disease.

34. The vaccine composition of any one of claims 22 to 26, which when administered to an animal or human suffering from alzheimer's disease, results in an increase in the retention of cognitive memory.

35. The vaccine composition of any one of claims 22 to 26, which when administered to an animal or human selected from mammals suffering from alzheimer's disease, results in a complete recovery of cognitive memory capacity.

36. Use of an antigenic construct according to claim 19 or a vaccine composition according to any of claims 25 to 26 for the manufacture of a medicament for the treatment of alzheimer's disease, wherein said medicament is administered to an animal or a human suffering from alzheimer's disease.

37. The use of claim 36, wherein administration of said antigenic construct or said vaccine composition results in the production of antibodies of the IgG subtype independent of T-cells.

38. The use of claim 37, wherein the antibody is an IgG3 isotype.

39. The use of any one of claims 36-38, wherein said antibody is the antibody produced by hybridoma cell line EJ 7H3 deposited at 8.12.2005 with accession number DSM ACC 2756.

40. The use of claim 36 or 37, wherein the medicament administration does not cause a significant increase in inflammatory markers in the brain.

41. The use of claim 40, wherein said marker is selected from the group consisting of IL-1 β, IL-6, IFN- γ and TNF α.

42. The use of any one of claims 36-38, wherein administration of the medicament results in insoluble, plaque-associated a β in the brain_1-40And Abeta_1-42A significant reduction in.

43. The use of any one of claims 36-38, wherein administration of the medicament results in soluble a β in the brain_1-42The level of which is significantly reduced.

44. The use of any one of claims 36-38, wherein administration of the medicament to an animal or human afflicted with alzheimer's disease results in an increase in the retention of cognitive memory.

45. The use of any one of claims 36-38, wherein administration of the medicament to an animal or human afflicted with alzheimer's disease results in a complete restoration of cognitive memory capacity.

46. Use of an antigenic construct according to any one of claims 14 to 21 or a vaccine composition according to any one of claims 22 to 26 for the manufacture of a medicament for inducing an immune response when administered in an animal or human suffering from alzheimer's disease.

47. An antibody or mixture of antibodies obtainable from an animal immunized with an antigenic construct according to any one of claims 14 to 21 or a vaccine composition according to any one of claims 22 to 26,

wherein the antibody is produced by hybridoma cell line EJ 7H3 deposited at 8.12.2005 with accession number DSM ACC 2756.

48. Use of an antibody of claim 47 in the manufacture of a medicament for the treatment of Alzheimer's disease, wherein the medicament is administered to an animal or human afflicted with Alzheimer's disease.