CN101325972B

CN101325972B - A β 1-42 specific monoclonal antibody with therapeutic properties

Info

Publication number: CN101325972B
Application number: CN200680046466.9A
Authority: CN
Inventors: R·格里佛拉特; D·希克曼; A·穆斯; A·普法伊费尔; C·尼古劳
Original assignee: AC Immune SA
Current assignee: AC Immune SA
Priority date: 2005-12-12
Filing date: 2006-12-08
Publication date: 2020-06-16
Anticipated expiration: 2026-12-08
Also published as: UA102368C2; AR107352A2; CN101325972A

Abstract

The present invention provides novel methods and compositions comprising highly specific and highly potent antibodies having the ability to specifically recognize and bind to a series of specific epitopes of β amyloid protein the antibodies made available by the teachings of the present invention are particularly useful 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 the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological diseases such as Alzheimer's Disease (AD).

Description

A β 1-42 specific monoclonal antibody with therapeutic properties

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

Amyloidosis is not a single disease entity, but a group of progressive disease processes characterized by the deposition of waxy, amyloid-like proteins (known as amyloid proteins, which accumulate in one or more organs or body systems) extracellular tissue. As amyloid deposits accumulate, they begin to interfere with the normal function of organs or body systems. There are at least 15 different types of amyloidosis. The main forms are primary amyloidosis without a known antecedent, secondary amyloidosis and hereditary amyloidosis following other diseases.

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, inflammation of the small intestine (granulomatous ileitis), hodgkin's disease and leprosy.

Amyloid deposits usually comprise three components, the amyloid fibrils (fibrils), which constitute 90% of the amyloid material, comprise one of several different types of proteins, these proteins are capable of folding into so-called "β -folded" sheet-like fibrils, this unique protein configuration showing a binding site for Congo red (Congo red), resulting in unique staining properties of amyloid proteins.

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 material that promote disease development and disease progression. Such diseases include, but are not limited to, neurological diseases such as Alzheimer's Disease (AD), including diseases or disorders characterized by loss of cognitive memory, e.g., such as Mild Cognitive Impairment (MCI), Lewy body dementia (Lewy body dementia), Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (dutch type); guam parkinsonism-dementia syndrome. Other diseases based on or associated with amyloid-like proteins are progressive supranuclear palsy, multiple sclerosis, Creutzfeld Jacob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases, including macular degeneration.

Although the pathogenesis of these diseases may be different, their characteristic deposits often contain many common molecular components. To a significant extent, these may be attributed to promoting local activation of inflammatory pathways leading to the simultaneous deposition of activated complement components, acute phase reactants, immunomodulators and other inflammatory mediators (McGeer et al, 1994).

The most common types of amyloid found in the brain of infected individuals contain predominantly a β fibrils scientific evidence suggests that an increase in the production and accumulation of β -amyloid protein in plaques leads to neuronal cell death, which promotes the development and progression of AD, loss of neuronal cells in critical brain regions in turn leads to a decrease in neurotransmitters and a loss of memory, proteins that primarily cause plaque aggregation include Amyloid Precursor Protein (APP) and two presenilins (presenilin I and presenilin II), continuous cleavage of Amyloid Precursor Protein (APP) by enzymes β and gamma secretases (which are constitutively expressed and catabolized in most cells) leads to the release of 39 to 43 amino acid a β peptides, degradation of APPs may increase their propensity to aggregate in plaques, a fragment of a β (1-42) has a high propensity to build up amyloid-specific amyloid aggregates due to its C-terminal two extremely hydrophobic amino acid residues, thus a high propensity to form amyloid-specific amyloid Aggregates (APP) and thus may be considered to have a high propensity to form neurite plaques-42 and to initiate neurite plaques-specific amyloid aggregates (AD β).

The symptoms of AD appear slowly, the first symptom may be only mild amnesia. At this stage, individuals may forget the name of recent events, activities, familiar people or things, and fail to solve simple mathematical problems. As the disease progresses, symptoms can become more easily noticed and become severe enough to cause people with AD or their families to seek medical help. Mid-stage symptoms of AD include forgetting how to do simple tasks such as grooming, and problems with speaking, understanding, reading, or writing develop. Later AD patients may become anxious or aggressive, may be lost from home and eventually require general care.

Currently, the only definitive method of diagnosing AD is to identify plaques and tangles in brain tissue in an autopsy after death of an individual. Thus, physicians can only make a diagnosis of "likely" or "likely" AD while the person is still alive. Using current methods, physicians can diagnose AD accurately up to 90% of the time using several tools to diagnose "likely" AD. The physician asks the person's general health, past medical questions, and any history of difficulty for the person to complete daily activities. Behavioral testing of memory, problem solving, attention, computing, and language provides information about cognitive deterioration, and medical tests such as tests of blood, urine, or spinal fluid and brain scans may provide some other information.

Management of AD consists of drug-based and non-drug-based treatments. Treatments aimed at altering the underlying course of the disease (slowing or reversing the progression) have been largely unsuccessful to date. Drugs that restore insufficiency (deficiency) or dysfunction of the chemical messengers (neurotransmitters) of nerve cells, in particular cholinesterase inhibitors (cheis), such as tacrine and rivastigmine, have been shown to improve symptoms. ChEI blocks enzymatic degradation of neurotransmitters, thus increasing the amount of chemical messengers available to transmit neural signals in the brain.

For some people in early and middle stages of the disease, the drug tacrine (COGNEX)

Morris Plains, NJ), donepezil (ARICEPT)

Tokyo, JP), rivastigmine (EXELON)

East Hanover, NJ) or galantamine (REMINYL)

New Brunswick, NJ) can help prevent certain symptoms from becoming more severe for a limited period of time. Memantine (NAMENDA) as another drug

New York, NY) has been approved for the treatment of moderate to severe AD. There are also drugs used to combat the psychiatric manifestations of AD. Some drugs may also help control behavioral symptoms of AD, such as insomnia, excitement, absentmindedness, anxiety, and depression. Treating these symptoms often makes the patient more comfortable and easier for the caregiver to take care of. Unfortunately, despite significant treatment advances showing that this class of agents is reliably better than placebo, the disease continues to progress and the average effect on mental function is only limited. Many drugs used for AD pharmacotherapy, such as ChEI, for example, also have side effects, including gastrointestinal dysfunction, hepatotoxicity, and weight loss.

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

Mild Cognitive Impairment (MCI) is a general term, most often defined as an unobvious but measurable disorder of memory. People with MCI experience more severe memory problems than would be expected with normal aging, but do not show other symptoms of dementia, such as impaired judgment or reasoning. MCI is a symptom that generally reflects the preclinical stages of AD.

β deposition of amyloid in the Entorhinal Cortex (EC) is believed to play a critical role in the progression of Mild Cognitive Impairment (MCI) in the elderly consistent with the observation that CSF-A A β (1-42) levels are significantly reduced once AD becomes clinically evident, in contrast to CSF-A β (1-42), CSF-tau levels increase significantly during the MCI phase and these values continue to rise thereafter, suggesting that the development of increased CSF-tau levels is likely to aid in the detection of MCI subjects who are predicted to become AD.

Dementia with Lewy Bodies (LBD) is a neurodegenerative disease that can occur in people over the age of 65, which generally causes cognitive (thought) impairment and abnormal behavioral changes. Symptoms may include cognitive impairment, neurological signs, sleep disorders, and autonomic failure. Cognitive impairment is a characteristic exhibited by LBD in most patients. Patients have recurrent episodes of confusion and are increasingly severe. Fluctuations in cognitive abilities are often associated with the degree of distraction of attention and alertness. Cognitive impairment and fluctuations in thinking may vary with minutes, hours, or days.

In addition to Lewy bodies, there may also be Lewy nerves (Lewy nerves), which are inclusions in nerve cell neurites amyloid plaques may form in the brain of patients with DLB, however they tend to be fewer in number than seen in patients with alzheimer's disease.

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

Inclusion Body Myositis (IBM) is a disabling disease commonly found in people over the age of 50, in which muscle fibers develop inflammation and begin to atrophy, but in which the brain is not damaged and the patient retains full intelligence in muscle cells of patients suffering from this most common, progressive muscle disease in the elderly, two enzymes involved in amyloid- β protein production are found to be increased, with amyloid- β also being increased.

Another disease based on or associated with aggregation and deposition of amyloid-like proteins is macular degeneration.

Macular degeneration is a common disease that causes degeneration of the central region of the retina (the paper-like thin tissue at the back of the eye where the photoreceptor cells transmit visual signals to the brain), the macular region. Clear, distinct, "straight-ahead" vision is produced by treatment of the macular area. The damaged macular area results in the creation of blind spots and blurring or distortion of vision. Age-related macular degeneration (AMD) is the leading cause of vision loss in the united states and, for people over the age of 65, the leading cause of legal blindness in caucasians. Approximately 180 million americans aged 40 and older have advanced AMD, and 730 million others with intermediate AMD are at real risk of vision loss. Government estimates that by 2020, there will be 290 thousands of people with advanced AMD. The discovery that AMD patients are often surprised and frustrated is so little understood about the causes and treatment of this blind symptom.

There are two forms of macular degeneration: dry macular degeneration and wet macular degeneration. Eighty-five percent of patients with macular degeneration are diagnosed with a dry form in which the cells in the macular region slowly begin to deteriorate. Both eyes are typically affected by dry AMD, although one eye may lose vision while the other remains unaffected. Drusen, a yellow deposit under the retina, are an early sign of dry AMD. As the number and size of drusen increases, the risk of developing advanced dry AMD or wet AMD also increases. Dry AMD can progress further without conversion to the wet form of the disease and lead to loss of vision; however, early dry AMD also has the potential to suddenly change to the wet form.

Although only fifteen percent of the cases are accounted for, the wet form results in ninety percent of blindness and is considered advanced AMD (no early or intermediate stage wet AMD). Wet AMD is always behind the dry form of the disease. As the dry form becomes more severe, some people begin to have abnormal blood vessel growth behind the macular area. These blood vessels are very fragile and leak fluid and blood (hence "wet" macular degeneration), which results in rapid damage to the macular area.

The dry form of AMD initially often causes mild blurred vision. In particular, the center of vision may become blurred and this area becomes larger as the disease progresses. If only one eye is affected, no symptoms can be noticed. In wet AMD, straight lines may look wavy, and loss of central vision may occur quickly.

Diagnosis of macular degeneration generally involves dilated eye examination, visual sensitivity testing, and viewing the back of the eye in a method known as fundus examination to aid in the diagnosis of AMD, and possibly fluorescein angiography if wet AMD is suspected. If dry AMD reaches an advanced stage, there is no current treatment to prevent vision loss. However, certain high dose formulations of antioxidants and zinc may delay or prevent the progression of intermediate AMD to the advanced stage. Macugen

(pegaptanib sodium injection), laser photocoagulation, and photodynamic therapy may control abnormal blood vessel growth and bleeding in the macular region, which may be useful in some patients with wet AMD; however, vision that has been lost cannot be recovered by these techniques. Such asFruit vision has been lost and there are low vision aids that can help improve 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 β (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 grafts, drugs that can prevent or slow the progression of disease, radiation therapy, gene therapy, computer chips implanted in the retina that can help stimulate vision, and agents that can prevent the growth of new blood vessels under the macular area.

An important factor to consider when developing new drugs is the ease of use for the target patient. Oral drug delivery (particularly tablets, capsules and gums) accounts for 70% of all consumer dosage forms due to convenience to the patient. Drug developers agree that patients prefer oral delivery rather than receiving injections or other more invasive modes of drug administration. Formulations that result in short dosing intervals (i.e., once a day or sustained release) are also preferred. The ease of administering antibiotics in oral dosage forms has led to increased patient compliance during treatment.

What is needed are methods and compositions for producing antibodies of high specificity and high potency, which is a prerequisite if the antibodies are to be provided in an oral dosage form. Preferably, such antibodies recognize specific epitopes on different antigens (such as amyloid).

Therefore, what is also needed are effective compositions and methods for treating complications associated with or caused by amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological diseases such as Alzheimer's Disease (AD), including diseases or disorders characterized by loss of cognitive memory capacity, such as, for example, Mild Cognitive Impairment (MCI), lewy body dementia, down's syndrome, 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, creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases, including macular degeneration. In particular, what is needed are specific and highly effective antibodies that are capable of eliminating the physiological manifestations of disease, such as eliminating plaque formation associated with the aggregation of fibers of amyloid or amyloid-like peptides.

For human Alzheimer's disease, A β mixed with Freund's complete or incomplete adjuvant has been demonstrated_1-42The resulting anti-amyloid antibodies were able to reduce the amyloid burden in transgenic mice (Schenk et al, 1999).

The tetrapalmitoylated A β reconstituted in liposomes_1-16Intraperitoneal inoculation into NORBA transgenic mice elicits significant anti-amyloid antibody titers that also lyse amyloid fibers and plaques in vitro and in vivo (Nicolau et al, 2002).

Bard et al (2000) first proposed a possible mechanism for the occurrence of amyloid plaques and fibrolysis and they concluded based on their data that antibodies opsonize plaques, which are then destroyed by microglia macrophages DeMattos et al (2001) indicated that mAbs directed against the central domain of β -amyloid can bind and completely sequester plasma amyloid, they believe that the presence of these mAbs in the circulatory system alters the a β balance in brain and plasma, favoring peripheral clearance and catabolism rather than deposition in the brain.

The antibodies provided by the teachings of the present invention are particularly useful for treating diseases caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological diseases such as Alzheimer's Disease (AD), including diseases or disorders characterized by loss of cognitive memory capacity, such as, for example, Mild Cognitive Impairment (MCI), Lewy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch-type), Guapison-dementia syndrome, and other diseases based on or associated with amyloid-like proteins, such as progressive supranuclear sclerosis, multiple sclerosis, Creutzfeldt-Jakob disease, Parkinson's disease, HIV-related dementia, ALS (amyotrophic lateral sclerosis), IBM (IBM's disease), adult-related macular degeneration, including only some examples of these diseases, and other endocrine diseases, including, but not limited to, amyloid-induced by the presence of amyloid and age-associated diseases.

Furthermore, the present invention provides novel methods and compositions for maintaining or improving cognitive memory in a mammal exhibiting an amyloid-related disease or disorder comprising administering to an animal, particularly a mammal, more particularly a human, in need of such treatment a therapeutically effective amount of a monoclonal antibody according to the invention.

Brief description of the figures and sequences:

FIG. 1 peptides derived from the sequences 1-15, 1-16 and 1-16 (. DELTA.14), 22-35 and 29-40 of A β.

FIG. 2: binding of the mAb I-01-Ab7C2 monoclonal antibody to amyloid species in Western blotting (Western blot) and dot blotting;

FIG. 3: binding of the mACI-01-Ab7C2 monoclonal antibody to amyloid fibers by transmission electron microscopy;

FIG. 4: u-¹³Results of parallel experiments between Th-T fluorescence determination and solid-state NMR of the C Tyr10 and Val12 labeled β -amyloid 1-42 peptides.

1 antigenic peptide A β of SEQ ID NO_1-15

2 antigenic peptide A β of SEQ ID NO_1-16

3 antigenic peptide A β of SEQ ID NO_1-16(Δ14)

4 antigenic peptide A β of SEQ ID NO_22-35

5 antigenic peptide A β of SEQ ID NO_29-40

6 antigenic peptide A β of SEQ ID NO_1-17

SEQ ID NO: 7: amino acid sequence of mouse C2 light chain variable region

SEQ ID NO: 8: amino acid sequence of mouse C2 heavy chain variable region

SEQ ID NO: 9: nucleotide sequence of mouse C2 light chain variable region

SEQ ID NO: 10: nucleotide sequence of mouse C2 light chain variable region including signal sequence

SEQ ID NO: 11: nucleotide sequence of mouse C2 heavy chain variable region

SEQ ID NO: 12: nucleotide sequence of mouse C2 heavy chain variable region including signal sequence

Amino acid sequence variants of the epitope region on the peptide SEQ ID NO 13-20: A β

SEQ ID NO: 21: amino acid sequence of mouse C2 light chain

SEQ ID NO: 22: amino acid sequence of mouse C2 heavy chain

The present invention utilizes antigen presentation resulting in increased exposure and stabilization of preferred antigen conformations, and ultimately resulting in antibodies with unique properties.

In one embodiment of the invention, there is provided an antibody comprising any functionally equivalent antibody or functional parts thereof, or more particularly a monoclonal antibody comprising any functionally equivalent antibody or functional parts thereof, against a supramolecular antigenic construct comprising a peptide corresponding to β -amyloid, in particular β -amyloid peptide a β_1-15、Aβ_1-16And A β_1-16(Δ14)An antigenic peptide of an amino acid sequence, said antigenic peptide being modified with a hydrophobic moiety (e.g. such as palmitic acid) or a hydrophilic moiety (e.g. such as polyethylene glycol (PEG)) or a combination of both, wherein said hydrophobic and hydrophilic moietiesCovalently linked to each end of the antigenic peptide by at least one, in particular one or two amino acids, respectively, e.g. such as lysine, glutamic acid and cysteine or any other suitable amino acid or amino acid analogue capable of acting as a linking means for coupling hydrophilic and hydrophobic moieties to the peptide fragment. When PEG is used as the hydrophilic moiety, the free PEG terminus is covalently linked to phosphatidylethanolamine or any other compound suitable as an anchoring element, e.g., to embed the antigenic construct in the bilayer of the liposome.

In another embodiment of the invention, an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof is provided which recognizes the native conformation of amyloid in that it specifically binds to amyloid oligomers and fibers, but not to non-linearized amyloid species.

In another embodiment of the invention, there is provided an antibody, particularly a monoclonal antibody, according to the invention and as described above, comprising any functionally equivalent antibody or functional parts thereof, wherein the antibody or fragment is present in an amount of at least about 1x 10^-6To at least about 1X 10^-8Especially at least about 1 × 10^-6To at least about 1X 10^-7More particularly at least about 1 × 10^-7To at least about 1X 10^-8Even more particularly at least about 1 × 10^-7To at least about 4 x 10^-7Binds to the a β monomer, but preferably does not show any significant cross-reactivity with Amyloid Precursor Protein (APP).

In another embodiment of the invention, there is provided an antibody, particularly a monoclonal antibody, according to the invention and as described above, comprising any functionally equivalent antibody or functional parts thereof, wherein the antibody or fragment is present in an amount of at least about 1x 10^-7To at least about 1X 10^-9Especially at least about 1 × 10^-7To at least about 1X 10^-8More particularly at least about 1 × 10^-8To at least about 1X 10^-9Even more particularly at least about 1 × 10^-8To at least about 5X 10^-8With binding affinity of A β fibres, fibrils or fibrilsThe filament (filamentt) binds, but preferably does not show any significant cross-reactivity with Amyloid Precursor Protein (APP).

In a further embodiment, an antibody, particularly a monoclonal antibody according to the invention and as described above, including any functionally equivalent antibody or functional parts thereof, exhibits a binding affinity to a β fibers, fibrils or filaments which is at least 5 times, particularly at least 10 times, more particularly at least 15 times higher than the binding affinity to a β monomer.

The antibodies according to the invention are capable of inhibiting the formation of monomeric peptides of amyloid, in particular β -amyloid monomeric peptide, such as, for example, monomeric peptides A β 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β, in vivo and in vitro_1-42The monomeric peptides aggregate into polymeric amyloid fibrils or filaments.

In a particular embodiment of the invention there is provided an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which antibody is directed against an amyloid monomeric peptide, particularly β -amyloid monomeric peptide, such as, for example, A β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42The aggregation of the a β monomer into polymeric fibrils of macromolecules was inhibited after co-incubation of the monomeric peptides.

In another embodiment of the invention there is provided an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which antibody is directed against an amyloid monomeric peptide, particularly β -amyloid monomeric peptide, such as, for example, A β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42The aggregation of the A β monomer into high molecular polymeric fibrils is inhibited after co-incubation with monomeric peptides, in particular at a molar ratio of up to 1: 100, more in particular at a molar ratio of between 1: 30 and 1: 100, but especially at a molar ratio of 1: 100, in particular when compared to separate amyloid peptide monomers incubated with buffer (control), said inhibition is up to at least 50%, in particular up to at least 65%, more in particular up to at least 75%, even more in particular up to at least 80%, but especially up toIt is up to at least 85% -90%, or more.

In particular, the co-incubation of the antibody according to the invention and the amyloid monomeric peptide is carried out at a temperature of between 28 ℃ and 40 ℃, in particular between 32 ℃ and 38 ℃, more in particular at 37 ℃ for 24 hours to 60 hours, in particular 30 hours to 50 hours, more in particular 48 hours.

In another embodiment, the invention provides an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which antibody is conjugated to an amyloid monomeric peptide, particularly β -amyloid monomeric peptide, such as, for example, A β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β, at 37 ℃ in a molar ratio of 1: 100_1-42After 48 hours of co-incubation of the monomeric peptide, it is capable of inhibiting at least 85%, particularly at least 89% and more particularly at least 95% of amyloid monomers, particularly β -amyloid monomeric peptides, such as, for example, monomeric peptides A β 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β, when compared to the respective amyloid peptide monomers incubated with buffer (control)_1-42The monomeric peptides aggregate into polymeric fibrils or filaments.

In a particular embodiment, the invention provides an antibody, particularly a monoclonal antibody, to a β comprising any functionally equivalent antibody or functional parts thereof_1-42Monomeric peptides showed high specificity, but were specific for A β_1-38、Aβ_1-39、β_1-40And/or A β_1-41Monomeric peptides showing substantially no or only little cross-reactivity, in particular antibodies, especially monoclonal antibodies, comprising any functionally equivalent antibody or functional parts thereof, which are directed against the amyloid peptide A β_1-42Alignment A β_1-38、Aβ_1-39、Aβ_1-40、Aβ_1-41More sensitive up to 100 times, in particular 50 to 100 times, more in particular 80 to 100 times, but especially 100 times, and the antibody to amyloid peptide A β_1-42Alignment A β_1-38More sensitive up to 1000 times, in particular 500 to 1000 times, more in particular 800 to 1000 times, but especially 1000 times; and is therefore capable of inhibiting the formation of amyloid protein in vitro and in vivoMonomeric peptides of (4), but especially the amyloid peptide A β_1-42To (3) is performed.

In another embodiment of the invention, there is provided an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, having the activity against the amyloid peptide a β_1-42And is capable of detecting a concentration as low as at least 0.001 mug, but particularly in a concentration range between 0.5 mug and 0.001 mug, more particularly between 0.1 mug and 0.001 mug, but especially at a concentration of 0.001 mug of A β_1-42A fiber.

In a very particular embodiment of the invention, there is provided an antibody, in particular a monoclonal antibody, comprising any functionally equivalent antibody or functional parts thereof, which is capable of detecting a minimum concentration of A β of fiber amounts as low as 0.001. mu.g_1-42Fibres and a minimum concentration of A β as low as 0.1. mu.g_1-40Fibres and a minimum concentration of A β as low as 1 mug_1-38A fiber.

Binding of the antibodies according to the invention and as described above to monomeric peptides forming amyloid, but in particular to amyloid forms (1-42), results in inhibition of aggregation of monomeric peptides forming amyloid into high molecular fibrils or fibrils. The antibodies according to the invention are capable of preventing or slowing down the formation of amyloid plaques, in particular amyloid forms (1-42), by inhibition of aggregation of amyloid-forming monomeric peptides, which are known to become insoluble through secondary conformational changes and are a major part of amyloid plaques in the brain of a diseased animal or human.

The aggregation inhibition potential of the antibodies according to the invention may be determined by any suitable method well known in the art, in particular by density gradient ultracentrifugation followed by SDS-PAGE sedimentation analysis on preformed gradients and/or by thioflavin T (Th-T) fluorimetry.

The invention also provides antibodies which bind to monomeric peptides from amyloid, particularly β -amyloid, such as, for example, monomeric peptides A β 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42Pre-formed polymeric amyloid fibrils or filaments formed by aggregation of monomeric peptides are capable of disaggregating said polymeric amyloid fibrils or filaments after co-incubation.

In a further embodiment of the invention there is provided an antibody, in particular a monoclonal antibody including any functionally equivalent antibody or functional parts thereof, wherein the antibody binds to a peptide derived from an amyloid monomer, in particular β -amyloid monomer, e.g. such as A β monomer peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but in particular A β, in a molar concentration ratio of up to 1: 100, more in particular between 1: 30 and 1: 100, but especially in a molar concentration ratio of 1: 100_1-42The pre-formed high molecular polymeric amyloid fibrils or filaments formed by aggregation of the monomeric peptide are capable of disaggregating the pre-formed high molecular polymeric amyloid fibrils or filaments by at least 35%, particularly at least 40%, more particularly at least 50%, even particularly at least 60%, but especially at least 70% or higher after co-incubation.

In particular, the antibody according to the invention is incubated with amyloid preformed high molecular polymeric amyloid fibrils or filaments at a temperature of between 28 ℃ and 40 ℃, in particular between 32 ℃ and 38 ℃, more in particular at 37 ℃ for 12 hours to 36 hours, in particular 18 hours to 30 hours, more in particular 24 hours.

In a particular embodiment, the invention provides an antibody, particularly a monoclonal antibody, comprising any functionally equivalent antibody or functional parts thereof, wherein the antibody is conjugated to an amyloid monomeric peptide, particularly β -amyloid monomeric peptide, such as, for example, A β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β, at 37 ℃ in a molar ratio of 1: 100_1-42After 24 hours co-incubation with aggregated preformed high molecular polymeric amyloid fibrils or filaments of monomeric peptides, said high molecular polymeric amyloid fibrils or filaments can be depolymerized by at least 35%, particularly by at least 40%, more particularly by at least 50%, when compared to respective preformed high molecular polymeric amyloid fibrils or filaments incubated with a control carrier (amyloid only) (control)%, even more particularly at least 60%, but especially at least 70% or higher.

The disaggregation potential of the antibody according to the invention may be determined by any suitable method well known in the art, in particular by density gradient ultracentrifugation followed by SDS-PAGE sedimentation analysis on preformed gradients and/or by thioflavin T (Th-T) fluorimetry.

The invention also provides conformationally sensitive antibodies or functional portions thereof.

In a further embodiment of the invention there is provided an antibody, particularly a monoclonal antibody including any functionally equivalent antibody or functional parts thereof, which antibody is directed against an amyloid monomeric peptide, particularly β -amyloid monomeric peptide, e.g. such as A β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42Upon co-incubation of the aggregated preformed polymeric amyloid fibrils or filaments of the monomeric peptide, a transition of the β folded conformation to the α helix and/or random coil conformation, but especially to the random coil conformation, even more especially to the random coil conformation in a given region within the molecule, especially in the environment of Val12 of the a β protein, can be induced, which leads to an increase of the random coil conformation at the expense of the β folded conformation and an improved dissolution of the preformed polymeric amyloid fibrils or filaments, in particular a reduction of the β folded conformation of up to at least 30%, especially up to at least 35%, and more especially up to at least 40% and more when compared to the respective preformed amyloid polymeric fibrils or filaments incubated in buffer (control).

In particular, the antibody according to the invention is co-incubated with amyloid pre-formed high molecular polymeric amyloid fibrils or filaments at a temperature between 28 ℃ and 40 ℃, in particular between 32 ℃ and 38 ℃, more in particular at 37 ℃ for 12 hours to 36 hours, in particular 18 hours to 30 hours, in particular 24 hours.

In particular, the invention provides antibodies, particularly monoclonal antibodies, comprising any functionally equivalent antibody or functional parts thereof, said antibody being present at 1: (1:) at 37 ℃A molar concentration ratio of 100 to monomeric peptides derived from amyloid, particularly β -amyloid, such as, for example, monomeric peptides A β 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42Preformed polymeric amyloid fibrils or filaments formed by aggregation of monomeric peptides are capable of causing a transition of the β folded conformation to the α helix and/or random coil conformation, but especially to the random coil conformation, even more especially to the random coil conformation in a given region within the molecule, especially in the environment of Val12 of the a β protein, after 24 hours of co-incubation, which results in an increase of the random coil conformation at the expense of β folding, β folded conformation is reduced by at least 30%, especially by at least 35%, and more especially by at least 40% and more when compared to the respective preformed amyloid polymeric fibrils or filaments incubated in buffer (control).

The potential of an antibody in causing a conformational transition may be determined by any suitable method well known in the art, in particular by solid state 13C NMR spectroscopy, but in particular by determining the A β peptide, in particular the A β peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially at A β_1-42Integrated intensity of the conformation of Val 12C β in the monomeric peptide.

By disaggregation of the polymeric fibrils or filaments forming amyloid, the antibody according to the invention is able to prevent or slow down the formation of amyloid plaques, which can lead to the alleviation of the symptoms associated with the disease and the delay or reversal of its progression.

Thus, another embodiment of the invention provides an antibody, particularly a monoclonal antibody, as described above, comprising any functionally equivalent antibody or functional parts thereof, which antibody is capable of reducing the total amount of a β in an animal, particularly a mammal, but especially a human brain, suffering from a disease or disorder resulting in an increased concentration of a β in the brain.

In another embodiment of the invention there is provided an antibody, particularly a monoclonal antibody, as described above, including any functionally equivalent antibody or functional parts thereof, which antibody is capable of decomposing plaques and thus of reducing the plaque load in the brain of an animal, particularly a mammal, but especially a human suffering from a disease or condition which results in an increased plaque load in the brain. An antibody according to the invention, including any functionally equivalent antibody or functional parts thereof, reduces the plaque load in the brain by at least 20%, particularly by at least 25%, more particularly by at least 30%, even more particularly by more than 30%.

In another embodiment of the invention there is provided an antibody, particularly a monoclonal antibody, as described above, including any functionally equivalent antibody or functional parts thereof, which antibody is capable of dissolving plaques resulting in a reduction of the amount of plaques in the brain of an animal, particularly a mammal, but especially a human, suffering from a disease or condition which results in an increased plaque load in the brain. An antibody according to the invention, including any functionally equivalent antibody or functional parts thereof, reduces the amount of plaques in the brain by at least 10%, particularly by at least 15%, more particularly by at least 20%.

It will be appreciated that antibodies according to the invention are capable of exhibiting different combinations of one, two or more of the specific properties described above.

For example, in one embodiment, the invention provides antibodies, particularly monoclonal antibodies including any functionally equivalent antibody or functional parts thereof, which antibodies exhibit bifunctional in that they exhibit both aggregation inhibition properties as defined above as well as disaggregation properties, particularly in combination with a high conformational sensitivity.

In a further embodiment of the invention there is provided a bifunctional antibody comprising any functionally equivalent antibody or functional parts thereof, but especially a bifunctional monoclonal antibody comprising any functionally equivalent antibody or functional parts thereof, which antibody binds to an amyloid monomeric peptide, especially β -amyloid monomeric peptide, such as, for example, a β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially a β_1-42The aggregation of the A β monomer into high molecular polymeric fibrils or filaments upon co-incubation with monomeric peptides is inhibited and, in addition, upon co-incubation with monomeric peptides derived from amyloid, particularly β -amyloid monomeric peptides, such as, for example, A β monomeric peptides 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42Preformed height of aggregated formation of monomeric peptidesThe molecular polymeric amyloid fibrils or filaments can depolymerize preformed polymeric fibrils or filaments after co-incubation.

In a particular embodiment of the invention, the co-incubation of the bifunctional antibodies according to the invention, but especially of the bifunctional monoclonal antibodies according to the invention, with the amyloid monomeric peptides and the preformed polymeric amyloid fibrils or filaments is performed at a molar concentration ratio of up to 1: 100, more particularly at a molar concentration ratio of between 1: 30 and 1: 100, and more particularly at a molar concentration ratio of 1: 100, respectively.

In particular, the co-incubation of the antibody according to the invention and the amyloid monomeric peptide is carried out at a temperature of between 28 ℃ and 40 ℃, in particular between 32 ℃ and 38 ℃, more in particular at 37 ℃ for 24 hours to 60 hours, in particular 30 hours to 50 hours, more in particular 48 hours; whereas co-incubation with amyloid preformed high molecular polymeric amyloid fibrils or filaments is carried out at a temperature of between 28 ℃ and 40 ℃, particularly between 32 ℃ and 38 ℃, more particularly at 37 ℃ for 12 hours to 36 hours, particularly 18 hours to 30 hours, more particularly 24 hours.

In another embodiment of the invention, the bifunctional antibody according to the invention, in particular the bifunctional monoclonal antibody according to the invention, comprising any functionally equivalent antibody or functional parts thereof, is capable of disaggregating the preformed polymeric fibrils or filaments by at least 10%, in particular by at least 25%, more in particular by at least 35%, even more in particular by at least 50%, but especially by at least 60-70% or more.

In another particular embodiment of the invention, the bifunctional antibody according to the invention, in particular the bifunctional monoclonal antibody according to the invention, comprising any functionally equivalent antibody or functional parts thereof, confers an amyloid monomeric peptide, in particular β -amyloid monomeric peptide, such as, for example, a β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially a β_1-42Aggregation of the monomeric peptide is inhibited by at least 50%, particularly at least 65%, more particularly at least 75%, even more particularly at least 80%, but especially at least 85% -90%, or more (with incubation in a buffer solution)Relative to the respective amyloid peptide monomers (control).

In particular, the invention provides antibodies, particularly bifunctional antibodies, but especially monoclonal antibodies, particularly bifunctional monoclonal antibodies, comprising any functionally equivalent antibody or functional parts thereof, which mediate inhibition of amyloid monomeric peptides, particularly β -amyloid monomeric peptides, such as, for example, a β monomeric peptides 1-39, 1-40, 1-41, 1-42 or 1-43, but especially a β, by causing a secondary conformational transition through specific and direct binding to a β fibers_1-42Polymerisation of monomeric peptides, and/or induction of monomeric peptides from amyloid, especially β -amyloid, peptides, e.g. such as A β monomeric peptides 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42Dissolution of preformed high molecular polymeric amyloid fibrils or filaments formed by aggregation of monomeric peptides.

The invention also provides an antibody, particularly a bifunctional antibody, but especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, comprising any functionally equivalent antibody or functional parts thereof, which antibody binds specifically by targeting to the β -amyloid epitope region, particularly by amino acid residue aa_n-aa_mRestricted β -amyloid epitope region, directly and specifically binds to β -amyloid fibers, such as fibers comprising the A β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, for example, but especially to fibers comprising A β_1-42Fibres of monomeric peptides, and/or induced from monomeric amyloid peptides, particularly β -amyloid monomeric peptides, such as, for example, monomeric A β peptides 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42Solubilization of preformed high molecular polymeric amyloid fibrils or filaments formed by aggregation of monomeric peptides, wherein the amino acid residue aa_n-aa_mN in (b) is an integer between 2 and 16, in particular between 5 and 16, more in particular between 8 and 16, even more in particular between 10 and 16, and m is an integer between 3 and 25, in particular between 3 and 23, in particular between 3 and 20, in particular 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, which is a radical of hydrogenWhere n and m cannot be the same number and n must always be a number smaller than m, the difference between n and m is ≧ 2.

In one embodiment of the invention, n is an integer between 13 and 15, but especially 14, and m is an integer between 22 and 24, but especially 23.

The binding of the antibody according to the invention is capable of inducing a transition in conformation in said protein, in particular a transition of the β folded conformation to the α helical and/or random coil conformation, but in particular to the random coil conformation, even more in particular to the random coil conformation in the context of Val12 of the a β protein within a given region of the molecule.

In another embodiment, the invention provides an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which antibody incorporates at least one property which has been mentioned above and which is selected from the group consisting of aggregation inhibition, disaggregation, induction of conformational transition, recognition and direct binding to an epitope, particularly a conformationally discontinuous epitope of the 14-23 region, especially the 14-20 region, prevention or slowing of amyloid plaque formation, reduction of the total amount of soluble A β in the brain, reduction of the plaque load in the brain, maintenance or improvement of cognitive memory, but especially a combination of two or more of said properties.

In a particular embodiment, the invention relates to an antibody, particularly a monoclonal antibody, comprising any functionally equivalent antibody or functional parts thereof, which antibody incorporates at least 2, particularly at least 3, more particularly at least 4, even more particularly at least 5, 6, 7 or 8, but especially all of the above mentioned properties.

In a particular embodiment, the invention provides an antibody, particularly a bifunctional antibody, but especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, which is directed against a β, comprising any functionally equivalent antibody or functional parts thereof_1-42Monomeric peptides showed high specificity, but were specific for A β_1-38、Aβ_1-39、Aβ_1-40And/or A β_1-41Exhibit virtually no or only little cross-reactivity; in particular, there is provided a kit comprising any functionally equivalent antibody or function thereofPart of an antibody, but especially a monoclonal antibody, directed against the amyloid peptide A β_1-42Alignment A β_1-38，Aβ_1-39，Aβ_1-40，Aβ_1-41More sensitive up to 100 times, in particular 50 to 100 times, more in particular 80 to 100 times, but especially 100 times, and to the amyloid peptide A β_1-42Alignment A β_1-38More sensitive up to 1000-fold, in particular 500 to 1000-fold, more in particular 800 to 1000-fold, but especially 1000-fold, and thus capable of inhibiting in vitro and in vivo the formation of monomeric peptides of amyloid, but especially amyloid peptide A β_1-42To (3) is performed.

In another embodiment of the invention there is provided an antibody, particularly a bifunctional antibody, but especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, comprising any functionally equivalent antibody or functional parts thereof, which has the activity against the amyloid peptide a β_1-42And is capable of detecting A β down to a concentration of at least 0.001. mu.g, but in particular in a concentration range between 0.5. mu.g and 0.001. mu.g, more in particular between 0.1. mu.g and 0.001. mu.g, but especially at a concentration of 0.001. mu.g_1-42A fiber.

In a very particular embodiment of the invention, there is provided an antibody, particularly a bifunctional antibody, but especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, comprising any functionally equivalent antibody or functional parts thereof, which antibody is capable of detecting a minimum concentration of A β of fiber amounts as low as 0.001. mu.g_1-42Fibres and a minimum concentration of A β as low as 0.1. mu.g_1-40Fibres and A β at minimum concentration of as low as 1. mu.g_1-38A fiber.

In a particular aspect, the present invention relates to an antibody or fragment thereof which recognizes and binds β to at least one distinct binding site, in particular at least two distinct binding sites on amyloid.

In a particular embodiment, the present invention relates to an antibody, including any functionally equivalent antibody or functional parts thereof, which antibody recognizes and binds β -at least one distinct binding site, particularly at least two distinct binding sites on the amyloid protein, wherein said at least one or said at least two distinct binding sites comprise at least one amino acid residue and at least two consecutive amino acid residues, respectively, predominantly involved in the binding of the antibody, wherein in a particular embodiment of the invention the at least one residue constituting the first distinct binding site is Leu inserted in the following core sequence and the at least two consecutive amino acid residues constituting the second distinct binding site are-Phe-inserted in the following core sequence:

-Xaa₁-Xaa₂-Xaa₃-Leu-Xaa₄-Phe-Phe-Xaa₅-Xaa₆-Xaa₇-

wherein

Xaa₁Is an amino acid residue selected from His, Asn, Gln, Lys and Arg;

Xaa₂is an amino acid residue selected from Asn and Gln;

Xaa₃is an amino acid residue selected from Lys, His, Asn, Gln and Arg;

Xaa₄is an amino acid residue selected from Ala, Val, Leu, norleucine, Met, Phe and Ile;

Xaa₅is an amino acid residue selected from Ala, Val, Leu, Ser and Ile;

Xaa₆is an amino acid residue selected from Glu and Asp;

Xaa₇is an amino acid residue selected from Glu and Asp.

In particular, an antibody or fragment thereof is provided which recognizes and binds β to at least one distinct binding site, in particular at least two distinct binding sites on the amyloid protein, wherein said at least one or said at least two distinct binding sites comprise at least one amino acid residue and at least two consecutive amino acid residues, respectively, predominantly involved in antibody binding, wherein in a particular embodiment of the invention the at least one residue constituting the first distinct binding site is Leu inserted in the following core sequence and the at least two consecutive amino acid residues constituting the second distinct binding site are-Phe-inserted in the following core sequence:

-Xaa₁-Xaa₂-Xaa₃-Leu-Xaa₄-Phe-Phe-Xaa₅-Xaa₆-Xaa₇-

wherein

Xaa₁Is an amino acid residue selected from His, Asn, Gln, Lys and Arg;

Xaa₂is an amino acid residue selected from Asn and Gln;

Xaa₃is an amino acid residue selected from Lys, His, Asn, Gln and Arg;

Xaa₅is an amino acid residue selected from Ala, Val, Leu, Ser and Ile;

Xaa₆is an amino acid residue selected from Glu and Asp;

Xaa₇is an amino acid residue selected from Glu and Asp.

In another embodiment of the invention, antibodies or fragments thereof are provided, wherein

Xaa₁Is His or Arg, but especially His;

Xaa₂is Gln or Asn, but especially Gln;

Xaa₃is Lys or Arg, but in particular Lys;

Xaa₄is Val or Leu, but especially Val;

Xaa₅is Ala or Val, but in particular Ala;

Xaa₆is Glu or Asp, but especially Glu; and

Xaa₇asp or Glu, but especially Asp.

In another aspect, the present invention relates to an antibody or fragment thereof, which recognizes and binds β -at least one distinct binding site, particularly at least two distinct binding sites, more particularly at least three distinct binding sites on the amyloid protein, wherein said one or at least two or at least three distinct binding sites each comprise at least one, particularly at least two consecutive amino acid residues predominantly involved in the binding of the antibody.

In particular, the antibody or fragment thereof according to the invention binds β to at least two distinct binding sites on the amyloid protein, wherein said at least two distinct binding sites each comprise at least two consecutive amino acid residues predominantly involved in the binding of the antibody, wherein said at least two distinct binding sites are located in close proximity to each other on the antigen, separated by at least one amino acid residue not involved in the binding of the antibody or being involved in the binding of the antibody to a significantly smaller extent than said at least two consecutive amino acid residues, thus forming a conformationally discontinuous epitope.

In another embodiment of the present invention there is provided an antibody or fragment thereof according to the present invention which recognizes and binds β -at least one distinct binding site, particularly at least two distinct binding sites, more particularly at least three distinct binding sites on the amyloid protein, wherein said distinct binding sites comprise at least one and at least two consecutive amino acid residues, respectively, predominantly involved in antibody binding, wherein the at least one and the at least two consecutive amino acids, which are separated by at least one amino acid residue not involved in antibody binding or to a significantly smaller extent than the amino acid residues predominantly involved in antibody binding, are-His-and-Lys-Leu-, respectively, inserted in the following core sequence:

-His-Xaa₂-Lys-Leu-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-

wherein

Xaa₂Is an amino acid residue selected from Asn and Gln;

Xaa₃is an amino acid residue selected from Ala, Val, Leu, norleucine, Met, Phe and Ile;

Xaa₅is an amino acid residue selected from Ala, Val, Leu, norleucine, Met, Phe and Ile;

Xaa₆is an amino acid residue selected from Ala, Val, Leu, Ser and Ile;

Xaa₇is an amino acid residue selected from Glu and Asp;

Xaa₈is an amino acid residue selected from Glu and Asp;

and wherein said amino acid residue Xaa₂、Xaa₃、Xaa₆、Xaa₇、Xaa₈Are not involved in antibody binding or are involved in antibody binding to a significantly lesser extent than the-His-and-Lys-Leu-binding sites.

In another embodiment, an antibody or fragment thereof is provided that recognizes and binds β to at least one distinct binding site, particularly to at least two distinct binding sites, more particularly to at least three distinct binding sites on the amyloid protein, wherein said distinct binding sites comprise at least one and at least two consecutive amino acid residues, respectively, predominantly involved in antibody binding, wherein the at least two consecutive amino acid residues representing the first binding site are-Phe-inserted in the following core sequence and the at least one amino acid residue is-His-inserted in the following core sequence:

-Xaa₁-His-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Phe-Phe-Xaa₇-Xaa₈-Xaa₉-

wherein,

Xaa₁is an amino acid residue selected from His, Asn, Gln, Lys and Arg;

Xaa₃is an amino acid residue selected from Asn and Gln;

Xaa₄is an amino acid residue selected from His, Asn, Gln, Lys and Arg;

Xaa₅is an amino acid residue selected from Ala, Val, Leu, Ser and Ile;

Xaa₆is an amino acid residue selected from Ala, Val, Leu and Ile;

Xaa₇is an amino acid residue selected from Ala, Val, Leu and Ile;

Xaa₈is an amino acid residue selected from Glu and Asp;

Xaa₉is an amino acid residue selected from Glu and Asp;

and wherein said amino acid residue Xaa₁、Xaa₃、Xaa₆、Xaa₇、Xaa₈And Xaa₉Are not involved in antibody binding or are involved in antibody binding to a significantly lesser extent than the-His-and-Phe-Phe-binding sites.

In a particular embodiment of the invention, the first at least two consecutive amino acid residues predominantly involved in antibody binding comprise-Lys-and-Leu-inserted in the following core sequence, and the second at least two consecutive amino acid residues comprise-Phe-inserted in the following core sequence:

-Xaa₁-Xaa₂-Lys-Leu-Xaa₄-Phe-Phe-Xaa₅-Xaa₆-Xaa₇-

wherein

Xaa₁Is an amino acid residue selected from His, Asn, Gln, Lys and Arg;

Xaa₂is an amino acid residue selected from Asn and Gln;

Xaa₅is an amino acid residue selected from Ala, Val, Leu, Ser and Ile;

Xaa₆is an amino acid residue selected from Glu and Asp;

Xaa₇is an amino acid residue selected from Glu and Asp;

and wherein said amino acid residue Xaa₂、Xaa₃、Xaa₄、Xaa₅、Xaa₆、Xaa₇Are not involved in antibody binding or are involved in antibody binding to a significantly smaller extent than the Lys-Leu and-Phe-Phe-binding sites.

In another embodiment of the invention, there is provided an antibody or fragment thereof, wherein

Xaa₁Is His or Arg, but especially His;

Xaa₂is Gln or Asn, but especially Gln;

Xaa₄is Val or Leu, but especially Val;

Xaa₅is Ala or Val, butIn particular Ala;

Xaa₆is Glu or Asp, but especially Glu; and

Xaa₇asp or Glu, but especially Asp.

In a further embodiment of the invention, the antibody or fragment thereof according to the invention binds β at least three distinct binding sites on the amyloid protein, wherein said at least three distinct binding sites comprise at least one amino acid residue and at least two consecutive amino acid residues, respectively, said residues being primarily involved in the binding of the antibody, wherein said at least three distinct binding sites are located antigenically in close proximity to each other, separated by at least one amino acid residue not involved in the binding of the antibody or being significantly less involved in the binding of the antibody than said at least one amino acid residue and said at least two consecutive amino acid residues, respectively, thereby forming a conformationally discontinuous epitope.

In a particular embodiment of the invention, the first at least two consecutive amino acid residues predominantly involved in antibody binding comprise-Lys-Leu-inserted in the following core sequence, and the second at least two consecutive amino acid residues comprise-Phe-inserted in the following core sequence; and the third at least one amino acid residue comprises-His-inserted in the following core sequence:

-His-Xaa₂-Lys-Leu-Xaa₄-Phe-Phe-Xaa₅-Xaa₆-Xaa₇

wherein,

Xaa₂is an amino acid residue selected from Asn and Gln;

Xaa₅is an amino acid residue selected from Ala, Val, Leu, Ser and Ile;

Xaa₆is an amino acid residue selected from Glu and Asp;

Xaa₇is an amino acid residue selected from Glu and Asp;

and wherein said amino acid residue Xaa₂、Xaa₃、Xaa₄、Xaa₅、Xaa₆And Xaa₇Are not involved in antibody binding or are involved in antibody binding to a significantly lesser extent than-His-, -Lys-Leu and-Phe-Phe-binding sites.

Xaa₂Is Gln or Asn, but especially Gln;

Xaa₄is Val or Leu, but especially Val;

Xaa₅is Ala or Val, but in particular Ala;

Xaa₆is Glu or Asp, but especially Glu; and

Xaa₇glu or Asp, but especially Asp.

In a particular embodiment of the invention, the first at least two consecutive amino acid residues predominantly involved in antibody binding comprise-Lys-Leu-inserted in the following core sequence, and the second at least two consecutive amino acid residues comprise-Phe-inserted in the following core sequence; and the third at least one amino acid residue comprises-Asp-inserted in the following core sequence:

-Xaa₁-Xaa₂-Lys-Leu-Xaa₄-Phe-Phe-Xaa₅-Xaa₆-Asp.-

wherein,

Xaa₁is an amino acid residue selected from His, Asn, Gln, Lys and Arg;

Xaa₂is an amino acid residue selected from Asn and Gln;

Xaa₅is an amino acid residue selected from Ala, Val, Leu, Ser and Ile;

Xaa₆is an amino acid residue selected from Glu and Asp;

and wherein said amino acid residue Xaa₂、Xaa₃、Xaa₄、Xaa₅、Xaa₆And Xaa₇Not involved in antibody binding or involved in antibody binding compared to-Asp-, -Lys-Leu and-Phe-Phe-binding sitesThe degree of synthesis is significantly less.

Xaa₁Is His or Arg, but especially His;

Xaa₂is Gln or Asn, but especially Gln;

Xaa₄is Val or Leu, but especially Val;

Xaa₅is Ala or Val, but in particular Ala; and

Xaa₆is Glu or Asp, but especially Glu;

in a further embodiment of the invention there is provided an antibody or fragment thereof according to the invention which binds β -4 distinct binding sites on the amyloid protein, wherein said 4 distinct binding sites each comprise one amino acid residue and two consecutive amino acid residues, said residues being primarily involved in antibody binding, wherein said 4 distinct binding sites are located antigenically in close proximity to each other, separated by at least one amino acid residue which is not involved in antibody binding or which is significantly less involved in antibody binding than said one amino acid residue and said two consecutive amino acid residues of the 4 distinct binding sites, thereby forming an epitope which is conformationally discontinuous.

In particular, the first two consecutive amino acid residues predominantly involved in antibody binding are-Lys-Leu-inserted in the following core sequence and the second at least two consecutive amino acid residues are-Phe-Phe-inserted in the following core sequence; the first single amino acid residue is-His-and the second single amino acid residue is-Asp-inserted in the following core sequence:

-His-Xaa₂-Lys-Leu-Xaa₄-Phe-Phe-Xaa₅-Xaa₆-Asp-

wherein,

Xaa₂is an amino acid residue selected from Asn and Gln;

Xaa₅is an amino acid residue selected from Ala, Val, Leu, Ser and Ile;

Xaa₆is an amino acid residue selected from Glu and Asp;

and wherein said amino acid residue Xaa₂、Xaa₃、Xaa₄、Xaa₅、Xaa₆、Xaa₇Are not involved in antibody binding or are involved in antibody binding to a significantly lesser extent than-His-, -Asp-, -Lys-Leu and-Phe-Phe-binding sites.

In a particular embodiment of the invention, the recognition and binding sites as defined above form a conformationally discontinuous epitope within the region between amino acid residues 12 to 24, in particular between amino acid residues 14 to 23, more in particular between amino acid residues 14 to 20 of the β -amyloid protein, wherein three unique recognition and binding sites comprising 1 and 2 amino acid residues, respectively, are located at positions 16, 17 and 19 and 20, respectively, and 14, respectively, said residues being predominantly involved in the binding of the β -amyloid protein and wherein said three unique recognition and binding sites are separated by a single amino acid residue located at positions 15 and 18, respectively, which amino acid residue is not involved in the binding of the antibody, or at least to a substantially lesser extent.

In a particular embodiment, said consecutive amino acid residues predominantly involved in the binding of β -amyloid, in particular-Lys-Leu-at positions 16 and 17 and-Phe-at positions 19 and 20, are embedded within the following core sequence:

Val- His- His- Gln- Lys- Leu- Val- Phe- Phe- Ala- Glu- Asp

12 13 14 15 16 17 18 19 20 21 22 23

in a further particular embodiment, said consecutive amino acid residues predominantly involved in β -amyloid binding, in particular-Lys at position 16, -Leu at position 17-and-Phe at positions 19 and 20-and-His at position 14-are embedded within the following core sequence:

Val- His- His- Gln- Lys- Leu- Val- Phe- Phe- Ala- Glu- Asp-

12 13 14 15 16 17 18 19 20 21 22 23

in a particular embodiment of the invention, the antibody according to the invention is produced against an antigen fragment which does not comprise said unique binding site.

The variation in the epitope region may be caused at least in part by the use of a supramolecular antigenic construct comprising the peptide from β -amyloid, in particular β -amyloid peptide A β_1-16An antigenic peptide corresponding to the amino acid sequence, which antigenic peptide is modified with a hydrophilic moiety, such as for example polyethylene glycol (PEG), wherein said hydrophilic moiety is covalently linked to the end of each antigen by at least one, in particular one or two amino acids, such as for example lysine, glutamic acid and cysteine or any other suitable amino acid or amino acid analogue capable of being coupled as a linking means to a peptide fragment as a hydrophilic moiety, as described in the immunological methods hereinafter. As described herein, when PEG is utilized as the hydrophilic moiety, the free PEG terminus is covalently attached to phosphatidylethanolamine or any other compound suitable as an anchoring element, e.g., to embed the antigenic construct into the bilayer of the liposome.

The use of lipid a as part of an immunization protocol also promotes variation in the epitope region.

In a particular embodiment of the invention, there is provided a polypeptide comprising SEQ ID NO: 7 (LCVR).

In another particular embodiment, the invention relates to the nucleic acid sequence of SEQ ID NO: 7 Light Chain Variable Region (LCVR).

In another particular embodiment of the invention, there is provided a polypeptide comprising SEQ ID NO: 8 (HCVR).

In another particular embodiment, the invention relates to the nucleic acid sequence of SEQ ID NO: 8 (HCVR).

In one embodiment, the present invention relates to a polypeptide comprising SEQ ID NO: 8 and SEQ ID NO: 7 in the light chain variable region of the amino acid sequence of (1).

Also part of the invention are antibodies comprising a Light Chain Variable Region (LCVR) or a Heavy Chain Variable Region (HCVR) or both a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR) that are substantially identical to SEQ ID NO: 7 and 8.

In particular, the invention relates to an antibody or fragment thereof according to the invention and as described above, wherein the Light Chain Variable Region (LCVR) has an amino acid sequence as set forth in SEQ ID NO: 7, or a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

Furthermore, the invention relates to an antibody or fragment thereof according to the invention and as described above, wherein the Heavy Chain Variable Region (HCVR) has the amino acid sequence shown in SEQ ID NO: 8, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence given in seq id no.

Further, the invention relates to an antibody or fragment thereof according to the invention and as described above, wherein the Light Chain Variable Region (LCVR) and the Heavy Chain Variable Region (HCVR) together have an amino acid sequence identical to SEQ ID NO: 7 and 8, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

In another particular embodiment, the invention relates to a polypeptide having the amino acid sequence of SEQ ID NO: 7, or a light chain variable region of an amino acid sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence given in seq id no.

In another particular embodiment, the present invention relates to a polypeptide having the amino acid sequence of SEQ ID NO: 8, or a heavy chain variable region of an amino acid sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence given in seq id No. 8.

In another embodiment of the present invention, there is provided a polynucleotide comprising a nucleotide sequence encoding the antibody of the present invention described above.

In particular, the present invention relates to a polynucleotide comprising a nucleotide sequence encoding an antibody of the present invention, comprising:

a) at least SEQ ID NO: 9, the nucleotide sequence of the light chain variable region;

b) a nucleotide sequence that is unique in codon sequence from the nucleotide sequence of (a) due to the degeneracy of the genetic code;

c) the complement of (a) and (b); or

d) A fragment of the nucleotide sequence of (a), (b), or (c) comprising a contiguous stretch of nucleotides selected from: at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 35 contiguous nucleotides, at least 40 contiguous nucleotides, at least 45 contiguous nucleotides, and at least 50 contiguous nucleotides;

in another embodiment, the invention relates to a polynucleotide comprising a nucleotide sequence encoding an antibody according to the invention, comprising:

a) at least SEQ ID NO: 10, a nucleotide sequence of a light chain;

c) the complement of (a) and (b); or

in yet another embodiment, the invention relates to a polynucleotide comprising a nucleotide sequence encoding an antibody according to the invention, comprising:

a) at least SEQ ID NO: 11, or a heavy chain variable region of seq id no;

b) a nucleotide sequence that differs from the nucleotide sequence of (a) in codon sequence due to the degeneracy of the genetic code;

c) the complement of (a) and (b); or

a) at least SEQ ID NO: 12 or a sequence complementary thereto;

c) the complement of (a) and (b); or

the invention also encompasses a polynucleotide comprising:

a) SEQ ID NO: 9;

c) the complement of (a) and (b); or

the invention also encompasses a polynucleotide comprising:

a) SEQ ID NO: 10;

c) the complement of (a) and (b); or

the invention also encompasses a polynucleotide comprising:

a) SEQ ID NO: 11;

c) the complement of (a) and (b); or

the invention also encompasses a polynucleotide comprising:

a) SEQ ID NO: 12;

c) the complement of (a) and (b); or

in another embodiment, the invention relates to any nucleotide sequence that hybridizes to:

a. according to the invention and in SEQ ID NO: 9. 10, 11 and 12;

b. a nucleotide sequence that differs from the nucleotide sequence of (a) in codon sequence due to the degeneracy of the genetic code;

the complement of (a) and (b); or

A fragment of the nucleotide sequence of (a), (b), or (c) comprising a contiguous stretch of nucleotides selected from: at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 35 contiguous nucleotides, at least 40 contiguous nucleotides, at least 45 contiguous nucleotides, and at least 50 contiguous nucleotides.

In particular, the present invention relates to the hybridization of a polypeptide according to the invention and in particular in SEQ ID NO: 9. 10, 11 and 12, in particular any nucleotide sequence which hybridizes to its complementary strand.

In a further embodiment, the invention relates to the hybridization of a nucleic acid sequence according to the invention and respectively in SEQ ID NO: 9. 10, 11 and 12, in particular any nucleotide sequence to which the complementary strand thereof hybridizes. After hybridization, washing is preferably carried out firstly with 2XSSC, 1% SDS and subsequently with 0.2XSSC at a temperature between 35 ℃ and 70 ℃, preferably 65 ℃ (see Sambrook et al in the citation above for definitions of SSPE, SSC and Dermart solutions).

In particular, the present invention relates to the hybridization of a polypeptide according to the invention and in the respective sequence of seq id NO: 9. 10, 11 and 12, in particular any nucleotide sequence to which the complementary strand thereof hybridizes.

In a particular embodiment, the invention provides an antibody, particularly a bifunctional antibody, but especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, comprising any functionally equivalent antibody or functional parts thereof, said antibody having the characteristic properties of an antibody produced by a hybridoma cell line selected from the group consisting of FP12H3, FP12H 3-C2 and FP12H 3-G2 deposited at DSM ACC2752, DSM ACC2750 and DSMACC2751, respectively, at 12/1 and 12/9 of 2005, respectively.

More specifically, the invention relates to antibodies produced by the hybridoma cell line FP12H3 deposited with DSMACC2752 at 12/1 and 12/9 of 2005, respectively, including any functionally equivalent antibody or functional parts thereof.

More specifically, the present invention relates to monoclonal antibodies produced by the hybridoma cell line FP12H 3-C2 deposited with DSMACC2750 at 12/1/2005 and 12/9/2005, respectively, including any functionally equivalent antibody or functional portions thereof.

More particularly, the present invention relates to monoclonal antibodies produced by the hybridoma cell line FP12H 3-G2 deposited with DSMACC2751 at 12/1/2005 and 12/9/2005, respectively, including any functionally equivalent antibody or functional portions thereof.

In another particular embodiment, the invention provides an antibody, particularly a bifunctional antibody, but especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, comprising any functionally equivalent antibody or functional parts thereof, said antibody having the characteristic properties of the antibody produced by the hybridoma cell line ET7E3 deposited at day 8 of 2005 with DSM ACC 2755.

More specifically, the present invention relates to monoclonal antibodies produced by the hybridoma cell line ET7E3 deposited at day 8, 12/2005 with DSM ACC2755, including any functionally equivalent antibody or functional parts thereof.

In another particular embodiment, the invention provides an antibody, particularly a bifunctional antibody, but especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, comprising any functionally equivalent antibody or functional parts thereof, said antibody having the characteristic properties of the antibody produced by the hybridoma cell line EJ7H3 deposited at day 8 of 2005 with DSM ACC 2756.

More specifically, the present invention relates to monoclonal antibodies produced by the hybridoma cell line EJ7H3 deposited at day 8, 12/2005 with DSM ACC2756, including any functionally equivalent antibody or functional parts thereof.

It is a further object of the present invention to provide methods and compositions comprising an antibody according to the invention and as described above, for preventing and/or treating and/or alleviating the effects of diseases and disorders caused by or associated with amyloid or amyloid-like proteins, e.g. by passive immunization of a human or animal with an antibody according to the invention and as described above. Such diseases and disorders include amyloidosis, a group of diseases and disorders associated with the formation of amyloid plaques including secondary amyloidosis and age-related amyloidosis including, but not limited to, neurological diseases such as Alzheimer's Disease (AD), including diseases or disorders characterized by loss of cognitive memory capacity, e.g., diseases or disorders such as Mild Cognitive Impairment (MCI), lewy body dementia, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (dutch-type), guam parkinsonism-dementia syndrome; and other diseases based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis, creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases, including macular degeneration.

It is a further object of the present invention to provide methods for the use of the monoclonal antibodies according to the invention and/or functional parts thereof and compositions comprising the antibodies according to the invention and described hereinbefore for the diagnostic or therapeutic intervention of diseases and disorders caused by or associated with amyloid or amyloid-like proteins. Such diseases and disorders include amyloidosis, a group of diseases and disorders associated with the formation of amyloid plaques including secondary amyloidosis and age-related amyloidosis including, but not limited to, neurological diseases such as Alzheimer's Disease (AD), including diseases or disorders characterized by loss of cognitive memory capacity, e.g., such as Mild Cognitive Impairment (MCI), lewy body dementia, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (dutch-type), guam parkinsonism-dementia syndrome; and other diseases based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis, creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases, including macular degeneration.

In particular, it is an object of the present invention to provide methods for reducing and preventing the occurrence of neurological diseases, including but not limited to alzheimer's disease, using monoclonal antibodies according to the invention and/or functional parts thereof and compositions comprising antibodies, particularly but not exclusively bispecific or bi-effective antibodies, according to the invention and as described above.

The composition according to the invention comprises an antibody, in particular a bispecific or bimanual antibody, according to the invention and as described above, comprising any functionally equivalent antibody or functional parts thereof, in particular in a therapeutically effective amount, more particularly a monoclonal antibody, in particular a bispecific or bimanual monoclonal antibody, in particular in a therapeutically effective amount, according to the invention and as described above, comprising any functionally equivalent antibody or functional parts thereof; and optionally, a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In particular, the composition according to the invention comprises an antibody, preferably a monoclonal antibody, comprising any functionally equivalent antibody or functional parts thereof, which antibody is capable of inhibiting an amyloid monomeric peptide, in particular β -amyloid monomeric peptide, such as, for example, a β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially a β, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient_1-42The monomeric peptides aggregate into polymeric amyloid fibrils or filaments.

In addition toIn an embodiment, the present invention provides a composition comprising an antibody, preferably a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, in contact with an amyloid monomeric peptide, in particular β -amyloid monomeric peptide, e.g. such as a β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially a β, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient_1-42In particular, the inhibition reaches at least 50%, in particular at least 65%, more in particular at least 75%, even more in particular at least 80%, but especially at least 85% -90%, or more when compared to the respective amyloid peptide monomer incubated in buffer (control).

In particular, the co-incubation of the antibody according to the invention and the amyloid monomeric peptide is carried out at a temperature of 37 ℃ for 48 hours.

The aggregation inhibition potential of the antibodies according to the invention can be determined by density gradient ultracentrifugation followed by SDS-PAGE sedimentation analysis on preformed gradients and/or by thioflavin T (Th-T) fluorescence assay.

The invention also provides a composition comprising an antibody capable of disaggregating amyloid monomeric peptides, in particular β -amyloid monomeric peptides, such as, for example, a β monomeric peptides 1-39, 1-40, 1-41, 1-42 or 1-43, but especially a β, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient_1-42Polymeric amyloid fibrils or filaments formed by aggregation of monomeric peptides.

By disaggregating the amyloid-derived polymeric fibrils or filaments, the antibodies according to the invention are able to prevent or slow down the formation of amyloid plaques, thereby leading to the alleviation of disease-related symptoms and to delay or reverse its progression.

In another embodiment of the inventionThere is provided a composition comprising an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, together with an amyloid monomeric peptide, preferably β -amyloid monomeric peptide, e.g. such as the a β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially a β, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient_1-42Pre-formed high molecular polymeric amyloid fibrils or filaments formed by aggregation of monomeric peptides are capable of depolymerizing the pre-formed polymeric fibrils or filaments by at least 35%, particularly at least 40%, more particularly at least 50%, even more particularly at least 60%, but especially at least 70% or more after co-incubation at a molar concentration ratio of between 1: 30 and 1: 100, especially after co-incubation for 24 hours at a temperature of 37 ℃.

The disaggregation potential of the antibodies according to the invention can be determined by density gradient ultracentrifugation followed by SDS-PAGE sedimentation analysis on preformed gradients and/or by thioflavin T (Th-T) fluorescence assay.

The invention also provides a composition comprising a specifically effective amount, more specifically a therapeutically effective amount, of an antibody or a functional part thereof, which antibody is conformationally sensitive.

In a further embodiment of the invention, there is provided a composition comprising an antibody, preferably a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, in combination with an amyloid monomeric peptide, in particular β -amyloid monomeric peptide, e.g. such as a β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially a β, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient_1-42After co-incubation of preformed polymeric amyloid fibrils or filaments formed by aggregation of monomeric peptides, in particular after 24 hours at a temperature of 37 ℃, a transition of the β folded conformation to the α helical and/or random coil conformation, but in particular to the random coil conformation, even more particularly to the random coil conformation in the environment of a given region within the molecule, in particular Val12 of the a β protein, can be caused, which results in a random coil conformation at the expense of the β folded conformationSpecifically, the reduction in β fold conformation is up to at least 30%, particularly up to at least 35%, and more particularly up to at least 40% and more when compared to a respective preformed amyloid polymeric fibril or filament incubated in buffer (control).

The potential of the antibodies to induce secondary conformational transitions was determined by solid state 13C NMR spectroscopy, but specifically by measuring A β_1-42Integrated intensity of Val 12C β conformation in the peptide.

The invention also provides a composition comprising a particularly therapeutically effective amount of an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which is bifunctional in that it exhibits both aggregation inhibiting properties as defined above as well as disaggregating properties, preferably in combination with a high conformational sensitivity, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In another embodiment of the invention there is provided a composition comprising a bifunctional antibody, but especially a bifunctional monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient, said antibody being in association with an amyloid monomeric peptide, especially β -amyloid monomeric peptide, such as, for example, a β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially a β_1-42Aggregation of the A β monomer into polymeric fibrils of macromolecules upon co-incubation with monomeric peptides is inhibited, and furthermore, upon co-incubation with monomeric peptides derived from amyloid, particularly β -amyloid, monomeric peptides, such as, for example, A β monomeric peptides 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42Aggregation of monomeric peptides formed preformed polymeric high molecular amyloid fibrils or filaments after co-incubation are capable of disaggregating the preformed polymeric fibrils or filaments.

In a particular embodiment of the invention, the co-incubation of the bifunctional antibodies according to the invention, but especially of the bifunctional monoclonal antibodies according to the invention, with the amyloid monomeric peptides and the preformed polymeric high molecular amyloid fibrils or filaments is performed at a molar concentration ratio of up to 1: 100, in particular at a molar concentration ratio of between 1: 30 and 1: 100, and more in particular at a molar concentration ratio of 1: 100, respectively.

In a further particular embodiment of the invention, the co-incubation with the monomeric amyloid peptide and preformed polymeric amyloid fibrils or filaments is carried out at a temperature of 37 ℃ for 48 hours and 24 hours, respectively.

In another particular embodiment of the invention, there is provided a composition comprising a bifunctional antibody according to the invention, but especially a bifunctional monoclonal antibody according to the invention, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient, said antibody being capable of disaggregating preformed polymeric fibrils or filaments by at least 10%, especially by at least 25%, more especially by at least 35%, even more especially by at least 50%, but especially by at least 60-70% or more.

In another embodiment of the invention, there is provided a composition comprising a bifunctional antibody according to the invention, but especially a bifunctional monoclonal antibody according to the invention, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient that, in comparison with a respective amyloid peptide monomer (control) incubated in a buffer, provides an amyloid monomeric peptide, especially β -amyloid monomeric peptide, e.g. such as a β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, but especially a β_1-42Aggregation of the monomeric peptide is inhibited by at least 50%, particularly at least 65%, more particularly at least 75%, even more particularly at least 80%, but especially at least 85% -90%, or more.

In particular, the present invention provides compositions comprising an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which mediates inhibition of amyloid monomeric peptides, particularly β -amyloid monomeric peptides, such as, for example, a β monomeric peptides 1-39, 1-40, 1-41, 1-42 or 1-43, by causing a secondary conformational transition by specific and direct binding to a β fibers, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient,but especially A β_1-42Polymerisation of monomeric peptides, and/or induction of monomeric peptides from amyloid, especially β -amyloid, peptides, e.g. such as A β monomeric peptides 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42Dissolution of preformed high molecular polymeric amyloid fibrils or filaments formed by aggregation of monomeric peptides.

The invention also provides a composition comprising an antibody, in particular a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which binds specifically and targeted to the β -amyloid epitope region, in particular by amino acid residue aa, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient_n-aa_mRestricted β -amyloid epitope region, directly and specifically binds to β -amyloid fibers, such as fibers comprising the A β monomeric peptide 1-39, 1-40, 1-41, 1-42 or 1-43, for example, but especially to fibers comprising A β_1-42Fibres of monomeric peptides, and/or induced from monomeric amyloid peptides, particularly β -amyloid monomeric peptides, such as, for example, monomeric A β peptides 1-39, 1-40, 1-41, 1-42 or 1-43, but especially A β_1-42Solubilization of preformed high molecular polymeric amyloid fibrils or filaments formed by aggregation of monomeric peptides, wherein the amino acid residue aa_n-aa_mN in (1) 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 the same number and n must always be a number smaller than m, the difference between n and m being ≧ 2.

The binding of the antibody according to the invention is capable of inducing a transition in conformation in said protein, in particular a transition of the β folded conformation to the α helical and/or random coil conformation, but in particular to the random coil conformation, even more in particular to the random coil conformation in a given region within the molecule, in particular in the environment of Val12 of the a β protein.

In a further embodiment, the present invention provides a composition comprising an antibody, particularly a monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which antibody incorporates at least one of the above mentioned properties, i.e. inhibition of polymerization, disaggregation, induction of conformational transition, recognition and direct binding to 4-16 and/or 14 to 23, but particularly 14 to 20 epitope regions, but especially a combination of two or more of said properties, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In a particular embodiment, the present invention provides a composition comprising an antibody, particularly a bifunctional antibody, but especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which antibody pair is a β, optionally together with a pharmaceutically acceptable carrier and/or diluent and/or excipient_1-42Monomeric peptides showed high specificity, but for A β_1-38、Aβ_1-39、Aβ_1-40And/or A β_1-41The monomeric peptides show substantially no or only little cross-reactivity, in particular comprising antibodies, but especially monoclonal antibodies, including any functionally equivalent antibody or functional parts thereof, against the amyloid peptide a β_1-42Alignment A β_1-38、Aβ_1-39、Aβ_1-40、Aβ_1-41More sensitive up to 100 times, in particular 50 to 100 times, more in particular 80 to 100 times, but especially 100 times, and to the amyloid peptide A β_1-42Alignment A β_1-38More sensitive up to 1000-fold, in particular 500 to 1000-fold, more in particular 800 to 1000-fold, but especially 1000-fold, and thus capable of inhibiting in vitro and in vivo the formation of monomeric peptides of amyloid, but especially amyloid peptide A β_1-42To (3) is performed.

In another particular embodiment of the invention, there is provided a composition comprising an antibody, particularly a bifunctional antibody, but especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which is directed against the amyloid peptide a β, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient_1-42Has high binding sensitivity and can detect concentration as low as at least 0.001 μ gA β at a concentration range between 0.5 and 0.001. mu.g, more particularly between 0.1 and 0.001. mu.g, but especially at a concentration of 0.001. mu.g_1-42A fiber.

In a very particular embodiment of the invention, there is provided a composition comprising an antibody, particularly a bifunctional antibody, but especially a monoclonal antibody, particularly a bifunctional monoclonal antibody, including any functionally equivalent antibody or functional parts thereof, which is capable of detecting a minimum concentration of A β of fiber amounts as low as 0.001 μ g, optionally together with a pharmaceutically acceptable carrier and/or diluent and/or excipient_1-42Fibres and A β with a fibre weight down to a minimum concentration of 0.1. mu.g_1-40Fibres and A β at minimum concentration of as low as 1. mu.g_1-38A fiber.

In a particular embodiment, the invention relates to a composition comprising a monoclonal antibody including any functionally equivalent antibody or functional parts thereof, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient, said antibody having the characteristic properties of an antibody produced by a hybridoma cell line selected from the group consisting of FP12H3, FP12H 3-C2 and FP12H 3-G2 deposited at DSM ACC2752, DSM ACC2750 and DSMACC2751, respectively, at 12/1 and 12/9 of 2005.

More specifically, the present invention relates to a composition comprising a monoclonal antibody produced by the hybridoma cell line FP12H3 deposited with DSM ACC2752 at 1/12/2005 and 9/12/2005, respectively, said monoclonal antibody comprising any functionally equivalent antibody or functional parts thereof, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

More specifically, the present invention relates to a composition comprising a monoclonal antibody produced by the hybridoma cell line FP12H 3-C2 deposited with DSM ACC2750 at 1/12/2005 and 9/12/2005, respectively, said monoclonal antibody comprising any functionally equivalent antibody or functional parts thereof, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

The present invention also relates to a composition comprising a monoclonal antibody produced by the hybridoma cell line FP12H 3-G2 deposited with DSMACC2751 at 1/12/2005 and 9/12/2005, respectively, said monoclonal antibody comprising any functionally equivalent antibody or functional parts thereof, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In another embodiment, the invention relates to a composition comprising a monoclonal antibody having the characteristic properties of the antibody produced by the hybridoma cell line ET7E3 deposited as DSMACC2755 at 12/8.2005, including any functionally equivalent antibody or functional parts thereof, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

The present invention also relates to a composition comprising a monoclonal antibody produced by the hybridoma cell line ET7E3 deposited at day 8/12/2005 with DSM ACC2755, said monoclonal antibody comprising any functionally equivalent antibody or functional parts thereof, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In another particular embodiment, the invention relates to a composition comprising a monoclonal antibody having the characteristic properties of the antibody produced by the hybridoma cell line EJ7H3 deposited at 8/12/2005 with DSM ACC2756, including any functionally equivalent antibody or functional parts thereof, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

The present invention also relates to a composition comprising a monoclonal antibody produced by the hybridoma cell line EJ7H3 deposited at day 8/12/2005 with DSM ACC2756, said monoclonal antibody including any functionally equivalent antibody or functional parts thereof, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

The antibodies, especially the monoclonal antibodies according to the invention, including any functionally equivalent antibody or functional parts thereof, can be administered in combination with other biologically active substances and methods for the treatment of diseases. The other biologically active substance may be part of the same composition already comprising the antibody according to the invention in the form of a mixture, wherein the antibody and the other biologically active substance may be mixed into or with the same pharmaceutically acceptable solvent and/or carrier, or may be provided separately as part of separate compositions, which may be administered separately or together in the form of a kit of parts.

An antibody, in particular a monoclonal antibody, according to the invention, comprising any functionally equivalent antibody or functional parts thereof, may be administered simultaneously, intermittently or sequentially with one or more other biologically active substances. For example, an antibody according to the invention comprising any functionally equivalent antibody or functional parts thereof may be administered simultaneously with the first additional biologically active substance or sequentially after or before administration of the antibody. If an administration regimen is chosen in which more than one additional biologically active substance is administered together with at least one antibody according to the invention, the compounds or substances can be administered partly simultaneously and partly sequentially in different combinations.

It is another object of the invention to provide an antibody mixture comprising at least one antibody of the invention, and optionally one or more other biologically active substances, and to methods of use of the respective antibodies, or mixtures thereof or antibody mixtures comprising compositions comprising said antibodies, for the prevention and/or therapeutic treatment and/or alleviation of the effects of diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological diseases such as Alzheimer's Disease (AD), including diseases or disorders characterized by loss of cognitive memory capacity, e.g., such as Mild Cognitive Impairment (MCI), Dementia with lewy bodies, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (dutch type), parkinson-dementia complex of guam; and other diseases based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis, creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases, including macular degeneration.

The mixture of the invention may comprise, in addition to the antibody of the invention, a biologically active substance, such as, for example, compounds known 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 or amyloid-like proteins such as the a β protein involved in alzheimer's disease.

In another embodiment of the invention, the additional biologically active substance or compound may also be a therapeutic agent useful in the treatment of diseases and conditions caused by or associated with amyloid or amyloid-like proteins, including amyloidosis caused by amyloid β, or may be a therapeutic agent useful in the pharmacological treatment of other neurological diseases.

Other biologically active substances or compounds may exert their biological effect by the same or similar mechanisms as the antibodies according to the invention, or by unrelated mechanisms of action, or by a variety of related and/or unrelated mechanisms.

In general, other bioactive compounds may include neutron transmission enhancers, psychotherapeutic drugs, acetylcholinesterase inhibitors, calcium channel blockers, biogenic amines, benzodiazepines

A sedative, 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 an antibody according to the invention, at least one further biologically active compound selected from compounds against oxidative stress, anti-apoptotic compounds, metal chelators, DNA repair inhibitors such as pirenzepine and metabolites, 3-amino-1-propanesulfonic acid (3APS), 1, 3-propanedisulfonic acid (1, 3PDS), secretase activators, β -and γ -secretase inhibitors, tau protein, neurotransmitters, β fold disrupters, anti-inflammatory molecules, or cholinesterase inhibitors (ChEI) such as tacrine, rivastigmine, donepezil, and/or galantamine, as well as optionally pharmaceutically acceptable carriers and/or diluents and/or excipients.

In a further embodiment, the compound according to the invention may comprise niacin or memantine (memantine), the antibody according to the invention, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In another embodiment of the invention, a mixture is provided comprising an "atypical antipsychotic drug" such as, for example, clozapine, ziprasidone, risperidone, aripiprazole or olanzapine, for the treatment of behavioral and anhedonia, affective flattening, frigidity, and oddly positive and negative psychotic symptoms including hallucinations, delusions, thought disorders (manifested as marked disorganization, misthinking, misdirection) and bizarre or disorganized, and an antibody according to the invention, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In a particular embodiment of the invention, the compositions and mixtures according to the invention and as described above comprise a therapeutically effective amount of antibody and biologically active substance, respectively.

Other compounds suitable for use in combination with the antibodies according to the invention in the mixture, e.g. as described in WO2004/058258 (see especially pages 16 and 17), including therapeutic drug targets (pages 36-39), alkanesulfonic and alkyllsulfates (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 agents (pages 68-75), amyloid-inhibiting agents (pages 77-78), metal-chelating agents (pages 78-79), neuroleptic and antidepressant agents (pages 80-82), nutritional supplements (pages 83-89), 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.

Also provided are methods of making antibodies according to the invention, including any functionally equivalent antibody or functional parts thereof, in particularIs a method for the preparation of a monoclonal antibody, said method comprising the production of an antibody, but in particular a monoclonal antibody, against a supramolecular antigenic construct comprising the amino acid sequence of a supramolecular antigenic construct with β -amyloid peptide, in particular β -amyloid peptide A β_1-15，Aβ_1-16And A β_1-16(Δ14)An antigenic peptide corresponding to the amino acid sequence of (a), which antigenic peptide is modified with a hydrophobic moiety (e.g. such as palmitic acid) or a hydrophilic moiety (e.g. such as polyethylene glycol (PEG)) or a combination of both, wherein the hydrophobic and hydrophilic moieties are covalently linked to the end of each antigenic peptide by at least one, in particular one or two, amino acids, such as lysine or any other suitable amino acid or amino acid analogue, such as glutamic acid or cysteine, respectively, which can be used as a linking means for coupling the hydrophilic and hydrophobic moieties to the peptide fragment.

Also part of the invention is the use of a monoclonal antibody and/or a functional fragment thereof according to the invention and as described above, and/or a pharmaceutical composition or mixture comprising said antibody, for the manufacture of a medicament for the treatment or alleviation of the effects of diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, such as, but not limited to, neurological diseases such as Alzheimer's Disease (AD), and diseases or disorders characterized by loss of cognitive memory capacity, such as, for example, Mild Cognitive Impairment (MCI), lewy body dementia, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (dutch-type), Parkinson-dementia complex of guam; and other diseases based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis, creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases, including macular degeneration.

In another embodiment of the invention, there is provided a method of using an antibody and/or functional parts thereof, but especially a monoclonal antibody and/or functional parts or functionally equivalent antibodies, according to the invention for the preparation of a pharmaceutical composition for the treatment or alleviation of the effects of diseases and disorders, which method comprises formulating the antibody in a pharmaceutically acceptable form, wherein said diseases and disorders are caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, such as, but not limited to, neurological diseases such as Alzheimer's Disease (AD), and diseases or disorders characterized by loss of cognitive memory capacity, such as, for example, Mild Cognitive Impairment (MCI), Dementia with lewy bodies, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (dutch type), parkinson-dementia complex of guam; and other diseases based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis, creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases, including macular degeneration.

The antibodies and/or functional parts thereof, but especially the monoclonal antibodies and/or functional parts or functionally equivalent antibodies according to the invention as well as compositions and mixtures comprising said antibodies, may be used for the preparation of a medicament for preventing, treating or alleviating the effects of diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological diseases such as Alzheimer's Disease (AD), including diseases or disorders characterized by loss of cognitive memory capacity, such as, for example, Mild Cognitive Impairment (MCI), lewy body dementia, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (dutch-type), and compositions and mixtures comprising said antibodies, Parkinson-dementia complex of guam; and other diseases based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis, creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases, including macular degeneration.

In a further embodiment of the invention, there is provided a method for reducing the plaque load in the brain of an animal, particularly a mammal, but especially a human, suffering from a disease or condition which leads to an increased plaque load in the brain, comprising administering to an animal, particularly a mammal, more especially a human, in need of such treatment a therapeutically effective amount of an antibody and/or a functional part thereof, but especially a monoclonal antibody and/or a functional part or functional equivalent antibody thereof according to the invention and as described above, or a composition or mixture comprising said antibodies.

In particular, the plaque load is reduced by at least 20%, in particular at least 25%, more in particular at least 30%, even more in particular more than 30%.

In a further embodiment of the invention, there is provided a method for reducing the amount of plaque in the brain of an animal, particularly a mammal, but especially a human, suffering from a disease or condition which leads to an increased plaque load in the brain, comprising administering to an animal, particularly a mammal, more especially a human, in need of such treatment a therapeutically effective amount of an antibody and/or a functional part thereof, but especially a monoclonal antibody and/or a functional part or a functionally equivalent antibody thereof according to the invention and as described above, or a composition or mixture comprising said antibodies.

In particular, the amount of plaques in the brain is reduced by at least 10%, particularly by at least 15%, more particularly by more than 15%.

In yet another embodiment of the present invention, there is provided a method for reducing the total amount of soluble a β in the brain of an animal, particularly a mammal, but especially a human, suffering from a disease or disorder that results in an increase in the concentration of soluble a β in the brain, comprising administering to an animal, particularly a mammal, more especially a human, in need of such treatment a therapeutically effective amount of an antibody and/or a functional part thereof, but especially a monoclonal antibody and/or a functional part or functionally equivalent antibody thereof, according to the invention and as described above, or a composition or mixture comprising said antibody.

It is an object of the present invention to provide a method for preventing, treating or alleviating the effects of diseases and disorders by administering an antibody according to the invention, but especially a monoclonal antibody or a composition or mixture comprising the same, to an animal or human suffering from such a condition, wherein the diseases and conditions are caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, this is a group of diseases and disorders associated with the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, including but not limited to neurological diseases such as Alzheimer's Disease (AD), including diseases or disorders characterized by loss of cognitive memory capacity, for example, such as Mild Cognitive Impairment (MCI), lewy body dementia, down's syndrome, 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, creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases including macular degeneration; said method comprising administering to an animal, in particular a mammal, more particularly a human, in need of such treatment a therapeutically effective amount of an antibody and/or a functional part thereof, but especially a monoclonal antibody and/or a functional part or functionally equivalent antibody thereof according to the invention and as described above, or a composition or mixture comprising said antibodies.

In a particular embodiment of the invention, a method is provided for maintaining or increasing the cognitive memory capacity of an animal, particularly a mammal or a human, suffering from a memory disorder (memomorphism) by administering to an animal, particularly a mammal or a human, in need of such treatment an antibody, but particularly a monoclonal antibody according to the invention, or a composition or mixture according to the invention and comprising such an antibody as described above.

In another embodiment of the invention, there is provided a method of using an antibody and/or functional parts thereof, but especially a monoclonal antibody and/or functional parts or functionally equivalent antibodies according to the invention for the preparation of a pharmaceutical composition for the prevention, treatment or alleviation of the effects of diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, a group of diseases and disorders associated with the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, such as, but not limited to, neurological diseases such as Alzheimer's Disease (AD), including diseases or disorders characterized by loss of cognitive memory capacity, e.g. such as Mild Cognitive Impairment (MCI), lewy body dementia, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (dutch-type), the treatment or alleviation of the effects of diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, such as, for example, Mild Cognitive Impairment (MCI), lewy body dementia, down, Parkinson-dementia complex of guam; and other diseases based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis, creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases, including macular degeneration.

In a particular embodiment of the invention, a method is provided for the preparation of a pharmaceutical composition using an antibody according to the invention and/or a functional part thereof, but especially a monoclonal antibody and/or a functional part or functionally equivalent antibody, for maintaining or increasing the cognitive memory capacity of an animal, especially a mammal or a human suffering from a memory disorder by administering to the animal, especially a mammal or a human an antibody, especially a monoclonal antibody or a composition or mixture comprising the antibody according to the invention and as described above.

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

The terms "polypeptide", "peptide" and "protein" as used herein are interchangeable and are defined as a biomolecule composed of amino acids linked by peptide bonds.

The terms "a", "an", and "the" as used herein are defined as "one or more" and include the plural unless the context does not otherwise qualify.

The term "detecting" as used herein is defined as detecting a biomolecule using known techniques, such as immunochemical or histological methods, and refers to qualitatively or quantitatively detecting the presence or concentration of a biomolecule under investigation.

The term "amyloid β, A β or β -amyloid" is art-recognized term and refers to amyloid β protein and peptide, amyloid β precursor protein (APP), as well as modifications, fragments, or any functional equivalent thereof specifically, amyloid β as used herein refers to any fragment produced by the proteolytic cleavage of APP, but especially those fragments involved in or associated with amyloidosis, including but not limited to A β_1-38、Aβ_1-39、Aβ_1-40、Aβ_1-41、Aβ_1-42And A β_1-43。

The structure and sequence of the above-mentioned amyloid β peptide are well known to those skilled in the art, and methods for producing the peptide or extracting them from brain and other tissues are described, for example, in Glenner and Wong, biochem Biophys Res Comm129, 885-890 (1984). furthermore, the amyloid β peptide is also commercially available in a variety of forms.

The "a β fibrils" or "a β fibrils" or "amyloid fibrils" are polymeric forms of monomeric proteins forming single or bundled fibers of constant fiber diameter, which are insoluble in aqueous media and contain a large number of intersecting β structures in their core, mostly with β strands 1.2, 3 perpendicular to the fibril axis.

"monomer a β" or "a β monomer" is an amyloid β that is completely soluble in aqueous media without aggregated complexes.

"polymeric soluble amyloid" and "oligomeric A β" and "A β oligomer" refer to aggregated monomers of amyloid peptides, or amyloid-like peptides, or modified or truncated amyloid peptides or other derivatives of amyloid peptides forming oligomers or polymeric structures, which are soluble in both aqueous media in vitro and in vivo in the body, particularly the brain, of a mammal or human, but specifically to aggregated monomers of amyloid β (A β), or modified or truncated amyloid β (A β) peptides or derivatives thereof, which are soluble in the body, more particularly the brain, of a mammal or human, respectively.

By "isolated" is meant that the biomolecule is free of components with which it is at least partially naturally found.

The term "antibody" as used herein is a art-recognized term which refers to a molecule or an active fragment of a molecule that binds to a known antigen, particularly an immunoglobulin molecule and immunologically active portions of immunoglobulin molecules, i.e., molecules that comprise a binding site that immunospecifically binds to an antigen. The immunoglobulins according to the present invention may be immunoglobulin molecules of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

"antibody" is intended to include within the scope of the present invention monoclonal, polyclonal, chimeric, single chain, bispecific or diabody, mimobody, 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 and 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 then subjected to HPLC gel filtration. The appropriate fraction containing the Fab fragments can then be collected and concentrated by membrane filtration or the like. Other commonly used techniques for the isolation of antigen-active fragments are described, for example, in Khaw, b.a. et al, j.nuclear.med.23: 1011-; rousseaux et al, Methods Enzymology, 121: 663-69, Academic Press, 1986.

"humanized antibodies" refers to a class of engineered antibodies in which the CDRs are derived from a non-human donor immunoglobulin and the other immunoglobulin-derived portions of the molecule are derived from one (or more) human immunoglobulins. In addition, framework support residues can be altered to maintain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art (see, e.g., Queen et al, Proc. Natl Acadsi 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/bioventures/ therapeutic.php)。

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

For the purposes of the present invention, it is understood that "monoclonal antibody" also encompasses antibodies produced by parent clones that have not yet reached complete monoclonality.

"functionally equivalent antibody" means within the scope of the present invention an antibody which shares substantially at least one of the main functional properties, including specific binding to β -amyloid protein, in particular A β, with the antibodies mentioned above and described herein_1-42Proteins, and more particularly A β_1-424-16 epitope region of protein, immunoreactivity in vitro, inhibition of A β_1-42Polymeric fibrils of monomers aggregated into macromolecules and/or depolymerizing preformed A β_1-42The destructive nature of the polymeric fibrils, and/or the β folds, and the effects of alleviating diseases and conditions caused by or associated with amyloid or amyloid-like proteins, including amyloidosis, when administered prophylactically or therapeuticallyDiseases and disorders associated with the formation of amyloid plaques, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, neurological diseases such as Alzheimer's Disease (AD), including diseases or disorders characterized by loss of cognitive memory capacity, e.g., such as Mild Cognitive Impairment (MCI), lewy body dementia, down's syndrome, 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, creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes, senile cardiac amyloidosis, endocrine tumors, and other diseases, including macular degeneration. The antibody may be of any class, such as IgG, IgM, IgA, or the like, or of any subclass, such as IgG1, IgG2a, and the like, as well as other subclasses mentioned hereinabove or known in the art, but particularly of the IgG2 class. 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 cell types or cell lines that produce antibodies (such as humanized antibodies) with desired properties. Antibodies can also be formed by combining Fab portions and Fc regions from different species.

The terms "bispecific" or "bifunctional" and "dual-potency" are used synonymously within the scope of this application to describe antibodies which simultaneously exhibit inhibitory properties against amyloid or amyloid-like fiber formation and disaggregation activity of amyloid or amyloid-like fibers.

The term "antigen" refers to an entity or fragment thereof that is capable of inducing 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.

The term "soluble" as used herein refers to partial or complete dissolution in an aqueous solution.

The term "immunogenic" as also used herein refers to a substance that causes or enhances the production of antibodies, T cells and other reactive immune cells to an immunogenic substance, and promotes an immune response in a human or animal.

When an individual produces sufficient antibodies, T cells, and other reactive immune cells against an administered immunogenic composition of the invention, an immune response occurs to alleviate or mitigate the condition being treated.

"polymeric soluble amyloid" refers to a plurality of aggregated monomers of amyloid peptides, or amyloid-like peptides, or modified or truncated amyloid peptides, or other derivatives of amyloid peptides, forming oligomers or polymeric structures, which are soluble in the mammalian or human body, particularly in the brain, and specifically refers to a plurality of aggregated monomers of amyloid β (a β) peptide, or modified or truncated amyloid β (a β) peptide, or derivatives thereof, which are soluble in the mammalian or human body, particularly in the brain.

The term "hybridoma" is art-recognized and is understood by one of ordinary skill in the art to refer to a cell resulting from the fusion of a cell that will produce an antibody and an immortalized cell (e.g., a multiple myeloma cell). Such hybrid cells are capable of producing a continuous supply of antibodies. See the definition of "monoclonal antibody" above and the examples describing the fusion method in more detail below.

The term "carrier" as used herein refers to a structure into which an antigenic peptide or supramolecular construct can be incorporated or to which it can be bound, thereby exposing or exposing portions of the antigenic peptide or peptide to a human or animal immune system. Any microparticle suitable for application in animal or human therapy, such as, for example, a liposome, microparticle or microsome, 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 the desired site through a delivery device. One example of such a delivery system includes the use of colloidal metals, such as colloidal gold.

Furthermore, the term "vector" also includes delivery mechanisms well known to those skilled in the art,including but not limited to a keyway

Hemocyanin (KLH), Bovine Serum Albumin (BSA), and other adjuvants.

In the supramolecular antigenic construct according to the present invention, the liposome may be bifunctional in that it can serve as a carrier comprising the supramolecular construct described above, and at the same time can function as an adjuvant to increase or stimulate an immune response in the target animal or human to be treated with the therapeutic vaccine according to the present invention

(polyoxyethylene-polyoxypropylene copolymer adjuvant from CytRx Corporation), modified lipid adjuvant from Chiron Corporation, saponin derivative adjuvant from Cambridge Biotech, killed Bordetella pertussis (Bordetella pertussis), Lipopolysaccharide (LPS) from gram-negative bacteria, large polymeric anions such as dextran sulfate, and inorganic gels such as alum, aluminum hydroxide, or aluminum phosphate.

Carrier proteins that can be used in the supramolecular antigenic construct compositions of the invention include, but are not limited to, maltose binding protein "MBP", bovine serum albumin "BSA", keyhole limpet

Form of hemocyanin 'KLH', ovalbumin, flagellin and formazanA globular gland globulin, serum albumin of any species, gamma globulin of any organism, a syngeneic cell with the Ia antigen, and a polymer of D-and/or L-amino acids.

Furthermore, the term "therapeutically effective amount" refers to an amount of an antibody that, when administered to a human or animal, elicits an immune response in the human or animal sufficient to produce a therapeutic effect. The therapeutically effective amount can be readily determined by one skilled in the art according to routine methods.

The "homology" between two sequences is determined by sequence identity. Sequence identity preferably refers to the percentage of nucleotide residues of a shorter sequence that are identical to the nucleotide residues of a longer sequence if the two sequences to be compared to each other differ in length. Sequence identity can be determined routinely using Computer programs such as the Bestfit program (Wisconsin sequence analysis package, version 8, Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wi 53711). Bestfit used the local homology algorithm of Smith and Waterman, Advances in applied Mathesics 2(1981), 482-489 to find the portion of the sequence with the highest sequence identity between two sequences. When using Bestfit or other sequence alignment programs to determine whether a particular sequence has, for example, 95% sequence identity with a reference sequence of the invention, the parameters are preferably set such that the percentage of sequence identity is calculated over the full length of the reference sequence and allows for gaps of homology of up to 5% of the total number of nucleotides in the reference sequence. When using Bestfit, the so-called optional parameters are preferably set at their predetermined ("default") values. Deviations that occur in the comparison between a given sequence and the above-described sequences of the invention may be caused, for example, by additions, deletions, substitutions, insertions or recombinations. Such sequence comparisons may also be preferably performed using the program "fasta 20u 66" (version 2.0u66, 9.1998, William R.Pearson and the University of Virginia; see also W.R.Pearson (1990), Methodsin Enzymology 183, 63-98, appended examples and http:// work bench. For this purpose, a "default" parameter setting may be used.

The term "hybridization" as used refers to conventional hybridization conditions, preferably to hybridization conditions using 5XSSPE, 1% SDS, 1 Xdenhardts (Denhardts) solution as a solution and/or a hybridization temperature between 35 ℃ and 70 ℃, preferably 65 ℃. After hybridization, washing is preferably carried out at a temperature of between 35 ℃ and 70 ℃, preferably 65 ℃, first with 2XSSC, 1% SDS and subsequently with 0.2XSSC (for the definition of SSPE, SSC and dengue solutions see Sambrook et al in the citation above). Stringent hybridization conditions, such as those described in Sambrook et al, supra, are particularly preferred. If the hybridization and washing are carried out at 65 ℃ as indicated above, for example, particularly preferred stringent hybridization conditions are given. Non-stringent hybridization conditions, such as hybridization and washing at 45 ℃ are less preferred, and at 35 ℃ even less preferred.

The invention may be understood more readily by reference to the following detailed description of specific embodiments included herein. While the present invention has been described with reference to specific details of certain embodiments thereof, these details are not intended to limit the scope of the invention.

The antibodies are useful for diagnostic and therapeutic intervention in diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including 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.

The antibodies are administered to individuals to passively immunize them against various diseases or disorders, including, but not limited to, amyloid-related diseases, such as alzheimer's disease.

The antibodies provided herein are monoclonal or polyclonal antibodies having binding specificity for an antigenic peptide representative of an amyloid-related condition (e.g., alzheimer's disease).

The antibodies according to the invention are prepared by immunizing an animal, such as a mouse, rat, rabbit or any other animal that can produce natural or human antibodies, with the supramolecular antigenic construct composition.

The supramolecular antigenic construct compositions disclosed herein generally comprise modified peptides to enhance antigenic effect, wherein such peptides are modified by pegylation (with polyethylene glycol or modified polyethylene glycol), or by other methods, such as modification by palmitic acid, polyamino acids (e.g., polyglycine, polyhistidine), polysaccharides (e.g., polygalacturonic acid, polylactic acid, polyglycolide, chitin, chitosan), synthetic polymers (polyamides, polyurethanes, polyesters), or copolymers (e.g., poly (methacrylic acid) and N- (2-hydroxy) propyl methacrylamide), and the like.

When the peptide is provided with an anchor in the liposomal bilayer, the modification by palmitic acid (palmitoylation) is due to C_16:0The relatively reduced length of the fatty acid moiety results in the peptide being located almost on the surface of the liposome. Thus, cells processing the antigen will have to take up the entire liposome with the peptide, which in most cases results in a relatively slower immune response.

In one embodiment of the invention, a modified amyloid 1-15 peptide is used for the preparation of antibodies according to the invention, in particular monoclonal antibodies, may be synthesized according to the method reported in Nicolau et al 2002, the method reported by Nicolau et al comprises modification of the antigenic peptide by grafting a lipophilic or hydrophobic moiety onto a preformed peptide terminal amino acid residue on a resin, which results in a product of considerable purity_1-42To synthesize A β_1-15An antigenic peptide, thereby producing a polypeptide having the amino acid sequence of SEQ ID NO: 1 peptide fragments of the sequence given. In the final step, two other protected amino acids are coupled to the growing peptide fragment. The Mtt group can then be selectively cleaved and coupled to palmitic acid. In washingAfter the resin, the protecting group is removed and the resin is cleaved simultaneously, followed by side chain deprotection using standard methods. The final product can then be obtained in high purity and its identity confirmed by methods well known in the art, such as electrospray mass spectrometry, for example.

The lipophilic or hydrophobic moiety according to the present invention may be a fatty acid, a triglyceride or a phospholipid wherein 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 up to about 24 carbon atoms, and individual numbers of carbon atoms falling within this range are also part of the invention. More particularly, the lipophilic or hydrophobic portion has a carbon backbone of at least 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 lipophilic or hydrophobic moiety is palmitic acid.

To enhance the immune response, other anchors/spacers may be used to reconstitute the peptide in the liposome, such as polyethylene glycol (PEG).

The PEG is covalently linked to amino acid residues at both ends of the peptide, in particular Glu, Cys or Lys amino acid residues or any other amino acid residue that may be suitable for covalently binding the PEG to the peptide. At the other end of the chain, a hydrophobic moiety may be covalently attached to serve as an anchoring element in the liposome bilayer, such as Phosphatidylethanolamine (PEA). Thus, the liposome still functions as an adjuvant, and peptides sufficiently far from the bilayer can be processed separately, and thus increase its immunogenicity compared to palmitoylated antigen.

In certain embodiments, the supramolecular antigenic constructs used within the scope of the present invention comprise a peptide sequence covalently linked to pegylated lysines (one 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-20 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, and even more in particular n-10.

The supramolecular constructs described herein can be synthesized using automated peptide synthesis and well-known protection chemistries, particularly Fmoc/tBu chemistry and standard side chain protecting groups. Generally, pegylation of a peptide produces a mixture of regioisomers (regiooisomers).

For these peptide sequences containing internal Lys or His residues, orthogonally protected Lys (ivDde) is added to each terminus.

Resins, in particular 2-chlorotrityl resins, which are acid-sensitive and enable the isolation of protective peptides are advantageous.

In a particular embodiment of the invention, the coupling reaction is carried out in the solution phase. The resin is then selectively cleaved under mild conditions to release the internal protective peptide.

Successful completion of peptides derived from β -amyloid protein sequence (e.g., such as A β_1-16(SEQ ID NO: 2)) is coupled to a solution of PEG molecules modified with fatty acid-phosphatidylcholine (e.g., such as DSPE). Separation of the mono-and di-coupled products prior to final side chain deprotection can be accomplished by applying cation exchange chromatography. Subsequent deprotection of the peptide side chains results in isolation of the conjugate of interest with acceptable purity. Purification can be accomplished by methods well known in the art, such as HPLC and the like.

This method of synthesis of the N-and C-terminal lipid-PEG β -amyloid antigen using protective peptides can be applied to a variety of peptide sequences.

Liposomal antigens according to the invention can then be prepared using the methods as described in Nicolau et al, 2002 modified amyloid A β antigen peptides, in particular modified PEGylated and palmitoylated A β_1-15、Aβ_1-16、Aβ_1-16(Δ14)、Aβ_22-35And A β_29-40The antigenic peptide may be reconstituted in a construct consisting of liposomes, in particular made of dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylethanolamine (DMPEA), dimyristoyl phosphatidylglycerol (DMPG) and cholesterol, optionally liposomes comprising monophosphoryl lipid a.

In a particular embodiment of the invention, liposomes having lipid a are used as adjuvant to prepare a vaccine against amyloid.dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol and cholesterol are mixed, in particular in a molar ratio of 0.9: 1.0: 0.7.then a strong immunomodulator, e.g. monophosphoryl lipid a is added in a suitable concentration, in particular in a concentration of between 30mg/mmol and 50mg/mmol, more in particular 40mg/mmol of phospholipid.then the modified antigen a β peptide is added in a molar ratio of peptide to phospholipid of between 1: 30 and 1: 200, in particular in a molar ratio of between 1: 50 and 1: 120, more in particular in a molar ratio of peptide to phospholipid of 1: 100.

Liposomes can also be prepared by cross-flow injection (cross-flow injection) techniques such as those described in Wagner et al (2002) Journal of lipid research Vol 12(3), pp 259-270. During injection of the lipid solution into the aqueous buffer system, the lipids tend to form "precipitates" that subsequently align themselves into liposomes. The size of the resulting liposomes depends on factors such as lipid concentration, agitation rate, injection rate, and choice of lipid. The preparation system may consist of a cross-flow injection module, a container of a polar phase (e.g., PBS buffer), a container of an ethanol/lipid solution, and a pressurizing device, but in particular a nitrogen pressurizing device. The ethanol/lipid solution is injected into the polar phase with different applied pressures as the aqueous or polar solution is pumped through the cross-flow injection assembly.

The liposome still acts as an adjuvant and the peptide sufficiently far from the bilayer can be processed separately and thus increase its immunogenicity compared to the palmitoylated antigen.

The free PEG end is covalently attached to the phosphatidylethanolamine molecule (where the fatty acid can be: myristic acid, palmitic acid, stearic acid, oleic acid, etc., or combinations thereof) to serve as an anchoring element. This supramolecular structure can be anchored by reconstitution in liposomes composed of phospholipids and cholesterol (phosphatidylethanolamine, phosphatidylglycerol, cholesterol in various molar ratios). Other phospholipids may also be used. Lipid A was used at a concentration of approximately 40. mu.g/pmole phospholipid.

In certain embodiments, palmitoylated or pegylated supramolecular antigenic constructs comprise a peptide having the amino acid sequence of β -amyloid_1-16(SEQ ID NO：2)、Aβ_1-16(Δ14)(SEQ ID NO：3)、Aβ_1-15(SEQ ID NO: 1), and active fragments thereof.

To elicit and produce antibodies and to determine the immunogenicity of the modified A β antigen construct, a suitable animal selected from the group consisting of mouse, rat, rabbit, pig, bird, etc., but particularly mouse, especially C57BL/6 mouse, is immunized with the antigenic peptide the immunogenicity of the antigen construct is determined by probing serum samples with an immunoassay (e.g., such as an ELISA assay) at suitable intervals following immunization.

Immunogens (antigens) of interest may be administered (e.g., intraperitoneally injected) to wild-type or inbred mice (e.g., BALB/C or, in particular, C57BL/6 mice), rats, rabbits, or other animal species capable of producing natural or human antibodies or transgenic mice

Hemocyanin (KLH) and bovine serum albumin), against which an immune response is desired.In these cases, the peptide acts as a hapten with a carrier protein. After boosting the animal, e.g., two or more times, splenocytes are harvested from the immunized animal and hybridomas are produced by fusing the sensitized splenocytes with a myeloma cell line, such as murine SP2/O myeloma cells (ATCC, Manassas, VA), 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)).

In a particular embodiment of the invention, the antigenic construct of the invention, in particular the composition comprising said antigenic construct in a pharmaceutically acceptable form, is administered in repeated doses, in particular in 1 to 15 doses, more particularly in 2 to 10 doses, even more particularly in 3 to 7 doses but especially in 4 to 6 doses, at intervals of between 1 and 10 weeks, in particular at intervals of between 1 and 6 weeks, more particularly at intervals of between 1 and 4 weeks, and even more particularly at intervals of between 2 and 3 weeks. The immune response is monitored by taking a serum sample at a suitable time after the boost, particularly 3 to 10 days after the boost, more particularly 4 to 8 days after the boost and more particularly 5 to 6 days after the boost, and detecting the immunogenicity of the antigen construct by one of the well-known methods, particularly commonly used immunoassays (e.g., such as the ELISA assay).

Immunization with the antigenic construct according to the invention, in particular with a vaccine composition comprising the antigenic construct according to the invention in a pharmaceutically acceptable form, results in a significant immune response in the treated animals. Animals, particularly mice, with therapeutic titers are selected for fusion of antibody-producing cells, particularly B-lymphocytes, with continuously growing cells or an immortal cell line, such as a myeloma cell line. Cell fusion was induced by the addition of polyethylene glycol. Those for which a dilution with a therapeutic titer of between 1: 4000 and 1: 6000, in particular between 1: 4500 and 1: 5500, more particularly 1: 5000, gives a positive result in an ELISA assay.

The resulting hybrid cells are then cloned by conventional methods, for example, by limiting dilution, and the resulting clones producing the desired monoclonal antibodies are cultured.

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

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

Polyclonal antibodies are prepared by immunizing an animal, such as a mouse or rabbit, or other suitable animal, with the antigen construct compositions of the invention described above. Serum was then collected from the animals and screened for antibodies in the serum based on binding reactivity to amyloid antigen.

The antibodies according to the invention can be prepared in physiologically acceptable formulations using well known techniques and may comprise pharmaceutically acceptable carriers, diluents and/or excipients. For example, an antibody according to the present invention and described above comprising any functionally equivalent antibody or functional parts thereof, in particular a monoclonal antibody comprising any functionally equivalent antibody or functional parts thereof, is combined with a pharmaceutically acceptable carrier, diluent and/or excipient to form a pharmaceutical composition. Suitable pharmaceutical carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffered saline, water, emulsions (such as oil/water emulsions), various wetting agents, sterile solutions and the like.

The formulation of the pharmaceutical composition according to the invention can be accomplished according to standard methods well known to those skilled in the art.

The compositions of the present invention may be administered to a subject in the form of a solid, liquid or aerosol in a suitable, pharmaceutically effective dose. Examples of solid compositions include tablets, creams, and implantable dosage units. The tablets may be administered orally. The therapeutic cream may be administered topically. The implantable dosage unit may be administered locally, e.g., at the tumor site; or may be implanted for systemic release of the therapeutic composition, e.g., subcutaneously. Examples of liquid compositions include formulations suitable for intramuscular, subcutaneous, intravenous, intraarterial injection, and for topical or intraocular administration. Examples of aerosol formulations include inhalation formulations for administration to the lungs.

The compositions may be administered by standard routes of administration. In general, the compositions may be administered by topical, oral, rectal, nasal, intradermal, intraperitoneal or parenteral (e.g., intravenous, subcutaneous, intramuscular) routes. In addition, the compositions can also be incorporated into sustained release matrices, such as biodegradable polymers, which are implanted at the desired delivery site, e.g., near the tumor site. The method includes administration of a single dose, repeated administration of the dose at predetermined time intervals, and continued administration for a predetermined period of time.

As used herein, a sustained release matrix is a matrix made of materials such as degradable polymers that are generally hydrolyzed or broken down by enzymes or acids/bases. Once implanted in the body, the matrix is affected by enzymes and body fluids. The desired sustained release matrix is selected to be a biocompatible substance such as liposomes, polylactides (polylactide acids), polyglycolides (polymers of glycolic acid), polylactide co-glycolides (copolymers of lactic and glycolic acids), polyanhydrides, polyorthoesters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone, and polysiloxanes. A preferred biodegradable matrix is one of polylactide, polyglycolide, or polylactide co-glycolide (a copolymer of lactic acid and glycolic acid).

It is well known to those skilled in the art that the dosage of a composition will depend on a variety of factors such as, for example, the condition to be treated, the particular composition used, and other clinical factors such as the weight, size, sex, and general health of the patient, the body surface area, the particular compound or composition to be administered, other drugs being administered concurrently, and the route of administration.

The compositions may be administered in combination with other compositions comprising biologically active substances or chemicals, in particular at least one of the compounds selected from the group consisting of compounds against oxidative stress, anti-apoptotic compounds, metal chelators, DNA repair inhibitors such as pirenzepine and metabolites, 3-amino-1-propanesulfonic acid (3APS), 1, 3-propanedisulfonic acid (1, 3PDS), secretase activators, β -and gamma-secretase inhibitors, tau protein, neurotransmitters, β fold disruptors, anti-inflammatory molecules, "atypical antipsychotics" (e.g. such as clozapine, ziprasidone, risperidone, aripiprazole or olanzapine), or cholinesterase inhibitors (Ch) (such as tacrine, rivastigmine, donepezil, and/or galantamine) and other pharmaceutical and nutritional supplements such as vitamin B12, cysteine, acetylcholine, lecithin, choline, theophylline, acetyl-purine L, carnitine, L-arginine, or a derivative of ginkgetin, and the antibodies of the invention, and optionally pharmaceutically acceptable carriers and/or diluents and/or excipients.

The proteinaceous pharmaceutically active substance may be present in an amount between 1ng and 10mg per dose. In general, the administration regimen may be in the range between 0.1 μ g and 10mg of an antibody according to the invention, in particular in the range between 1.0 μ g and 1.0mg, more particularly in the range between 1.0 μ g and 100 μ g, all individual values falling within these ranges also being part of the invention. If administered by continuous infusion, more appropriate doses may range from 0.01 μ g to 10mg per kg body weight per hour, and all individual values falling within these ranges are also part of the invention.

Administration is typically parenteral, e.g., intravenous. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Non-aqueous solvents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). The aqueous solution may be selected from water, an alcohol/water solution, an emulsion or a suspension comprising a salt and a buffer medium. Parenteral carriers include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution or fixed oils. Intravenous carriers include fluid and nutrient supplements, electrolyte supplements (such as those based on ringer's dextrose), and other substances. Preservatives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

The pharmaceutical composition may further comprise a protein carrier, such as, for example, serum albumin or immunoglobulin, in particular of human origin. Other biologically active substances may also be present in the pharmaceutical composition of the invention, depending on its intended use.

In further embodiments of the invention, methods and kits are provided which may be used for detecting and diagnosing amyloid-related diseases or disorders, diagnosing predisposition to amyloid-related diseases or disorders, or monitoring minimal residual disease in a patient, or predicting patient response to treatment with an antibody or vaccine composition according to the invention and as described above. These methods include well-known immunological methods commonly used to detect or quantify substances in biological samples or under in situ conditions.

Diagnosis of an amyloid-related disease or disorder or a predisposition to an amyloid-related disease or disorder may be accomplished by detecting immunospecific binding of a monoclonal antibody or active fragment thereof to a sample or an in situ amyloid epitope, which comprises contacting a sample or a specific body part or body area suspected to contain amyloid antigen with an antibody that binds an epitope of amyloid, allowing the antibody to bind to the amyloid antigen to form an immune complex, detecting the formation of the immune complex, and correlating the presence or absence of immune complexes with the presence or absence of amyloid antigen in the sample or specific body part or area, optionally comparing the amount of said immune complexes with a normal control value, wherein an increase in the amount of said aggregates compared to a normal control value indicates that said patient has or is at risk of developing an amyloid-related disease or disorder.

Monitoring of minimal residual disease in a patient treated with an antibody or immunological composition according to the invention may be accomplished by detecting immunospecific binding of a monoclonal antibody or active fragment thereof to a sample or to an epitope of amyloid in situ, which comprises contacting a sample or a specific body part or body area suspected to contain amyloid antigen with an antibody that binds an epitope of amyloid, allowing the antibody to bind to the amyloid antigen to form an immune complex, detecting the formation of the immune complex, and correlating the presence or absence of immune complexes with the presence or absence of amyloid antigen in the sample or specific body part or area, optionally comparing the amount of said immune complexes with a normal control value, wherein an increase in the amount of said aggregates compared to a normal control value indicates that said patient may still suffer from minimal residual disease.

Predicting a patient's response to treatment with a vaccine composition according to the invention may be accomplished by detecting immunospecific binding of a monoclonal antibody or an active fragment thereof to amyloid epitopes in a sample or in situ, comprising contacting a sample or a specific body part or body area suspected to contain amyloid antigens with an antibody that binds amyloid epitopes, allowing the antibody to bind to amyloid antigens to form an immune complex, detecting the formation of the immune complex, and correlating the presence or absence of the immune complex with the presence or absence of amyloid antigens in the sample or specific body part or area, optionally comparing the amount of said immune complex before and after treatment, wherein a reduction in the amount of said aggregates indicates that said patient has a high potential to respond to treatment.

Biological samples that can be used for diagnosing amyloid-related diseases or disorders, for diagnosing predisposition to amyloid-related diseases or disorders, or for monitoring minimal residual disease in a patient, or for predicting a patient's response to treatment with an antibody or vaccine composition according to the present invention and as described above are, for example, fluids such as serum, plasma, saliva, gastric secretions, mucus, cerebrospinal fluid, lymph, and the like; or a tissue or cell sample obtained from a body, such as neural, brain, heart, or vascular tissue. To detect the presence or absence of amyloid in a sample, any immunoassay known to those of ordinary skill in the art (see Harlow and Lane, Antibodies: immune Manual, Cold spring harbor Laboratory, New York 1988555-612)) may be used, for example, assays utilizing indirect detection methods employing a second reagent for detection, ELISA and immunoprecipitation methods, and agglutination tests. A detailed description of these tests is given, for example, in WO96/13590, Zrein et al (1998) and WO96/29605 by Maertens and Stuyver.

For in situ diagnosis, the antibody or any active and functional part thereof may be administered to the organism to be diagnosed using methods well known in the art, e.g. intravenous, intranasal, intraperitoneal, intracerebral, intraarterial injection, whereby specific binding between the antibody according to the invention and the epitope region on the amyloid antigen may occur. The antibody/antigen complex may be detected by a label attached to the antibody or functional fragment thereof.

Immunoassays which can be used for diagnostic applications, or for diagnosing the predisposition to amyloid-related diseases or disorders, or for monitoring minimal residual disease in a patient, or for predicting the patient's response to treatment with an antibody or vaccine composition according to the invention and as described above, typically rely on a labeled antigen, antibody or second reagent for detection. Proteins or reagents may be labeled with compounds known to those skilled in the art, including enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold and latex microspheres. Among these methods, radiolabelling is applicable to almost all types of tests and has the most variation. Labeling of conjugating enzymes is particularly useful when radioactivity must be avoided or rapid results are required. Although fluorescent dyes require expensive equipment in use, fluorescent dyes provide a very sensitive detection method. Antibodies used for these assays include monoclonal antibodies, polyclonal antibodies, and affinity-purified polyclonal antibodies.

Alternatively, the antibody may be indirectly labeled by reaction with a labeled substance having affinity for immunoglobulin, such as protein a or protein G, or a second antibody. The antibody may be conjugated to a second substance and detected with a labeled third substance having affinity for the second substance conjugated to the antibody. For example, the antibody may be conjugated to biotin, and the antibody-biotin conjugate detected with labeled avidin or streptavidin. Similarly, the antibody may be conjugated to a hapten, and the antibody-hapten conjugate detected with a labeled anti-hapten antibody.

These and other suitable markers useful in the present invention are well known to those skilled in the art. Binding of these labels to the antibody or fragment thereof can be accomplished using standard techniques generally known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J.H., et al, 1976(Clin.Chim.acta 70: 1-31), and Schurs, A.H.W.M., et al, 1977(Clin.Chim Acta 81: 1-40). The latter coupling techniques are mentioned as the glutaraldehyde method, the periodate method, the bismaleimide method, and others, all of which are incorporated herein by reference.

Current immunoassays utilize a two-antibody approach to detect the presence of an analyte, wherein an antibody is indirectly labeled by reacting with a second antibody that has been labeled with a detectable label. The second antibody is preferably an antibody that binds to an antibody of an animal from which the monoclonal antibody is derived. In other words, if the monoclonal antibody is a mouse antibody, the labeled secondary antibody is an anti-mouse antibody. For the monoclonal antibodies used in the tests described below, such labels are preferably antibody-coated beads, in particular magnetic beads. For polyclonal antibodies to be used in immunoassays as will be described herein, preferred labels are detectable molecules, such as radioactive, fluorescent, or electrochemiluminescent substances.

Alternative diabody systems, often referred to as fast format systems (fast formats systems) because they are suitable for rapid determination of the presence of an analyte, may also be applied within the scope of the present invention. This system requires a high affinity between the antibody and the analyte. According to one embodiment of the invention, the presence of amyloid antigen is determined using a pair of antibodies each specific for amyloid. One of the pair of antigens is referred to herein as a "detection antibody" and the other of the pair of antigens is referred to herein as a "capture antibody". The monoclonal antibodies of the invention can be used as capture or detection antibodies. The monoclonal antibodies of the invention can also be used in a single assay as both capture and detection antibodies. One embodiment of the invention thus applies a double antibody sandwich method for the detection of amyloid antigens in a biological fluid sample. In this method, the analyte (amyloid antigen) is sandwiched between a detection antibody and a capture antibody, which is irreversibly immobilized on a solid support. The detection antibody comprises a detectable label to identify the presence of the antibody-analyte sandwich and thus the analyte.

Exemplary solid phase materials include, but are not limited to, microtiter plates, polystyrene test tubes, magnetic, plastic or glass beads and slides as are well known in the field of radioimmunoassays and enzyme immunoassays. Methods for coupling antibodies to solid phases are also well known to those skilled in the art. Recently, various porous materials such as nylon, nitrocellulose, cellulose acetate, glass fiber, and other porous polymers have also been used as solid supports.

The invention also relates to a diagnostic kit for the detection of amyloid antigens in a biological sample, comprising a composition as defined above. Furthermore, the present invention relates to the latter diagnostic kit comprising, in addition to the composition as defined above, the detection reagent as defined above. The term "diagnostic kit" generally relates to any diagnostic kit known in the art. More specifically, the latter term refers to a diagnostic kit as described in Zrein et al (1998).

It is another object of the present invention to provide novel immunoprobes and test kits comprising antibodies according to the present invention for the detection and diagnosis of amyloid-related diseases and disorders. For an immunoprobe, the antibody is linked, directly or indirectly, to a suitable reporter molecule (e.g., an enzyme or radionuclide). The test kit comprises a container holding one or more antibodies according to the invention, and instructions for using the antibodies for binding to amyloid antigen to form an immune complex and detecting the formation of the immune complex such that the presence or absence of the immune complex correlates with the presence or absence of amyloid antigen.

Examples

Antigens for the production of mouse monoclonal antibodies

Mouse mAb	Antigen/sequence	Connecting body	Anchor	Adjuvant
					mACI-01-Ab7	Aβ_1-16	PEG	DSPE	Lipid A
mACI-02-Ab6	Aβ_1-16(Δ14)	PEG	DSPE	Lipid A
					mACI-11-Ab9	Aβ_22-35	PEG	DSPE	Lipid A
mACI-12-Ab11	Aβ_29-40	PEG	DSPE	Lipid A
					mACI-24-Ab4	Aβ_1-15	-	Palm	Lipid A

Table 1: antibodies and antigenic constructs for producing said antibodies

Example 1 preparation of palmitoylated A β_1-15Methods for supramolecular antigenic constructs

Synthesis of tetra (palmitoyl lysine) -A β 1-15 peptide antigen

Palmitoylated amyloid 1-15 peptide was synthesized according to a modified previously reported method (Nicolau et al 2002). This new approach involves grafting palmitic acid onto the terminal Lys residue of a preformed peptide on a resin, rather than stepwise solid phase synthesis incorporating the modified amino acids 9-fluorenylmethoxycarbonyl (Fmoc) -Lys (pal) -OH. This new process improves coupling efficiency and provides a product of considerably higher purity. Thus, the orthogonally protected amino acids Fmoc-Lys (Mtt) -OH were attached to Wang resin (Wang resin) using [ 2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium hexafluorophosphate ] (HBTU) coupling chemistry. The Fmoc group was removed with 20% piperidine in DMF and a second Fmoc-Lys (Mtt) -OH residue was coupled. The following 15 amino acids were then coupled using standard automated peptide synthesis using Fmoc/tBu chemistry and standard side chain protecting groups to generate SEQ id no: 1, the peptide sequence given. Finally, the last two amino acids coupled are Fmoc-Lys (Mtt) -OH. The Mtt group was then selectively cleaved with 1% trifluoroacetic acid (TFA) in dichloromethane to release the peptide fragment and coupled to palmitic acid with HBTU. After resin washing, the Fmoc group was removed with 20% piperidine in Dimethylformamide (DMF), and finally resin cleavage and side chain deprotection were performed simultaneously with TFA under standard conditions. Trituration in cold diethyl ether yielded the product as a white solid. Electrospray mass spectrometry confirmed the identity of the product (M/z predicted: 1097.9([ M ]3 +); experimental: 1096.8([ M-3H ]3+), no other tri-, di-or mono-palmitoylated peptides were detected.

Example 2: method for preparing supramolecular antigenic constructs

Synthesis of Pegylated β -amyloid peptide antigen

To enhance the immune response, another anchor/spacer is used to reconstitute the peptide in the liposome, e.g., polyethylene glycol (PEG). PEG was covalently attached to lysine residues at both ends of the peptide. At the other end of the chain (PEGn ═ 70), phosphatidylethanolamine is covalently bound to serve as an anchoring element in the liposome bilayer. Thus, the liposome still functions as an adjuvant, and peptides sufficiently far from the bilayer can be processed separately and thus increase its immunogenicity compared to palmitoylated antigen.

The supramolecular constructs described herein were uniquely synthesized using standard Fmoc/tBu amino acid side chain protection in general, PEGylation of peptides produces a mixture of regioisomers Here, convenient methods for site-specific attachment of PEG-lipid conjugates to the C-and N-termini of A β are illustrated with partially protected peptides.

For those peptide sequences (1-16, 1-16. DELTA.14, 22-35) that contain internal Lys or His residues, orthogonally protected Lys (ivDde) was added to each end. Additional Gly was added to the C-terminus to facilitate synthesis. The Fmoc group was removed with 20% piperidine in DMF and N-acetylated with acetic anhydride. Selective cleavage of the ivDde group was accomplished with 3% hydrazine hydrate in DMF over one hour. The 2-chlorotrityl resin is preferred over the more widely used Wang resin, since the former has proven to be more resistant to hydrazinolysis. Furthermore, unlike Wang's resin, 2-chlorotrityl resin is extremely acid sensitive and therefore enables the isolation of protective peptides. In fact, it is desirable to perform the coupling reaction in solution phase, since the coupling of the resin bound peptide to the preactivated pegylated lipid reagent DSPE-PEG-SPA does not result in any coupling product. Thus, selective cleavage from the resin under mild conditions (acetic acid/trifluoroethanol/dichloromethane, 1: 8, 1 hour, room temperature) yielded internally protected peptides.

Successful completion in DMSO and excess base was derived from sequence A β_1-16(SEQ ID NO: 2) was coupled to a solution of DSPE-PEG-SPA. The reaction was then stopped by adding an excess of ethanolamine over 2 hours and the solution was lyophilized.

No special protection strategy is required for the sequences 29-40.

By HPLC (semi-preparative reverse phase C)₄Column) provided N-and C-terminal PEG-lipid conjugates of 50-70% purity, the identity of which was confirmed by MALDI (matrix assisted laser desorption ionization). Each sequence showed considerable differences in the ease of coupling reaction and the conditions (temperature, DSPE-PEG-SPA molar equivalents, time) were adjusted accordingly. To separate the excess DSPE-PEG-SPA from the desired product, HPLC purification was applied. Separation of the mono-and di-coupled products prior to final side chain deprotection can be accomplished by using cation exchange chromatography. Subsequent deprotection of the peptide side chains and isolation of the excess terminated DSPE-PEG-SPA results in isolation of the desired conjugate with acceptable purity.

Example 3 antibodies elicited by supramolecular antigenic constructs

_1-153.1 preparation of mAbs against palmitoylated A β supramolecular antigen constructs

Palmitoylation antigen (ACI-24, A β)_1-15) At 2 week intervals, the mice were immunized with C57 BL/6. 10-12 animals were immunized with each antigen (injection volume: 200. mu.l, containing 8nmol peptide). Animals were sacrificed 4 days after the last injection. After 5 boosts, mice with therapeutic titers (when 1: 5,000 dilutions of serum were positive in ELISA) were selected for fusion. Spleen cells were collected from immunized animals and hybridomas were produced by fusing sensitized spleen cells with a myeloma cell line. 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)ory, New York 1988)) was used to perform fusion of mouse B-lymphocytes from the spleen with cells of the myeloma cell line SP2-0(ATCC, Manassas, VA).

The resulting hybrid cells were then cultured for 10 + -14 days using conventional methods to allow clonal growth_1-42And culturing the resulting clone that produces the desired monoclonal antibody.

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

The hybridomas are then screened for the ability to produce monoclonal antibodies against a particular amyloid-related disease or disorder. Once the female parent clone was identified, it was subcloned four times to ensure monoclonality and to allow stabilization of the hybrid cells.

Hybridoma cells producing the antibody of interest are cloned, expanded and cryopreserved for further production.

Antibodies were typed by a commercially available mouse monoclonal antibody isotyping kit, and stable clones were adapted to serum-free medium and placed in a bioreactor to produce antibodies.

Preferred hybridoma cells produce monoclonal antibodies having the IgG isotype, more preferably the IgG1 isotype.

_1-16 _4-11 _22-35 _29-403.2 preparation of mAbs against the Ethanolated PEG-A β, A β, A β and A β supramolecular antigenic constructs

Liposomal antibodies were prepared as described in Nicolau et al, 2002, PNAS, 99, 2332-37 reconstitution of the sequence PEG-A β in the construct of liposomes made of dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylethanolamine (DMPEA), dimyristoyl phosphatidylglycerol (DMPG) and cholesterol (0.9: 0.1: 0.7 molar ratio) containing monophosphoryl lipid A (40mg/mM phospholipid)_1-16、Aβ_4-11、Aβ_22-35And A β_29-40(FIG. 1) pegylation of A β with these antigens_1-16C57BL/6 was immunized at two week intervals. 10-12 animals were immunized with each antigen. After 3 to 6 boosts, mice with therapeutic titers (when a 1: 5000 dilution of serum was positive in the ELISA) were selected for fusion. Spleen cells were collected from immunized animals and hybridomas were produced by fusing sensitized spleen cells with a myeloma cell line. Fusion of mouse B-lymphocytes from the spleen and myeloma cell line SP2-0(ATCC, Manassas, VA) cells 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)).

The resulting hybrid cells are then cloned by conventional methods, e.g., using limiting dilution methods, IgG-producing hybridoma cell clones are selected and tested by ELISA for A β_1-42Specific binding of the peptide, and culturing the resulting clone that produces the desired monoclonal antibody.

The hybridoma cells thus obtained were chemically selected by inoculating the cells into a selection medium containing hypoxanthine, aminopterin and thymidine (HAT).

The hybridoma cells are then screened for the ability to produce monoclonal antibodies against a particular amyloid-related disease or disorder. Hybridoma cells producing the antibody of interest are cloned, expanded and cryopreserved for further production. Preferred hybridoma cells produce monoclonal antibodies having the IgG isotype, more preferably the IgG1 isotype.

Example 4: specific determination of antibody mACI-24-Ab4

To analyze the specificity of the antibody mACI-24-Ab4, different concentrations of preformed amyloid 1-42, 1-40 and 1-38 fibrils were spotted onto 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 primary antibody for 2 hours at room temperature. After washing, the membrane was incubated with a sheep anti-mouse IgG antibody conjugated to horseradish peroxidase (Amersham Biosciences) for 1 hour at room temperature, washed and incubated with a chemiluminescent solution, followed by exposure of the membrane to X-ray film.

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 seven days and spotted on a membrane using 20 μ g/ml of antibody for measuring binding capacity, and bound antibody was detected by exposure to horseradish peroxidase-conjugated sheep anti-mouse IgG antibody for 20 minutes.

The antibody mACI-24-Ab4 bound to different pre-formed A β fibers with different sensitivities as demonstrated by dot blot analysis the antibody showed similarity to A β_1-40Or A β_1-38Comparative example A β_1-42Highest binding sensitivity of the fibers at least 0.001 μ g A β can be detected_1-42Fibres, however, of pair A β_1-40For fibers, the limit of detection of antibody is at least 0.1. mu.g vs. A β_1-38The fibres were 1 μ g, meaning that the sensitivity was 100-fold to less than 1000-fold for these types of amyloid fibres. These data indicate that antibody ACI-24-Ab4 is at least 100-fold more sensitive to the amyloid form (1-42), which is known to become insoluble through secondary conformational changes and is the major portion of amyloid plaques in the brains of AD patients.

Example 5: binding of the AC Immunomalonai antibody mACI-01-Ab7C2 to amyloid species in Western and dot blots

To test whether the binding of the mouse antibody mACI-01-Ab7C2 was dependent on the native conformation of A β, the binding to linearized amyloid was compared by Western blotting or native amyloid in dot blotting (FIGS. 2a and 2 b).

For preparing monomers, resuspend peptide film in DMSO to a concentration of 2.75 μ g/μ l and dilute to 1 μ g/μ l in PBS to prepare oligomers, resuspend dried peptide film in DMSO to 5mM, sonicate and add PBS to reach 400 μ M amyloid, then add SDS to 0.2% of 0.2% SDS toFinal concentration. After 6 hours of incubation at 37 ℃, the amyloid was diluted in water to a final concentration of 100 μ M and incubated for a further 18 hours at 37 ℃. The amyloid oligomers were precipitated with ice-cold 33% methanol, 4% acetic acid solution at 4 ℃ for 1 hour, rotated 16200g for 10 minutes and the precipitate resuspended in 5mM Na₂H₂PO₄35mM NaCl (pH7.4) to a final concentration of 1. mu.g/. mu.l. To prepare the fibers, the peptide films were diluted in Tris-HCl 50mM buffer to a concentration of 1mg/ml amyloid and incubated for 5 days at 37 ℃. The tube was spun at 10000g for 5 minutes and the pellet was resuspended in 0.1M carbonate buffer (pH 9.6) to 1. mu.g/. mu.l.

Mu.g of monomers, oligomers or fibers were diluted in PBS and loading buffer and applied to 12% SDS-PAGE, and the gels were transferred to nitrocellulose membranes. Alternatively, 3 or 1 μ g or 100 and 10ng amyloid species were diluted in PBS and spotted directly onto nitrocellulose membranes, and the membranes were dried at room temperature for 1 hour. After 30 minutes of blocking with casein solution (vehicle), the membrane was incubated with mACI-01-Ab7C2 or 6E10(Chemicon) antibody diluted to 1. mu.g/ml in casein solution for 30 minutes. After 3 washes with casein solution, the membranes were incubated with HRP-labeled goat anti-mouse igg (Dako Cytomation) diluted in casein solution for 30 minutes at room temperature, washed 3 times and developed with DAB substrate (Dako Cytomation).

In dot blots, the monoclonal mouse antibody mACI-01-Ab7C2 specifically binds to monomers, oligomers, and fibers as does the positive control antibody 6E 10. In contrast, the machi-01-Ab 7C2 antibody did not recognize linearized amyloid species in western blots, whereas the 6E10 antibody clearly recognized all the linearized peptides. This result confirms that the binding of mACI-01-Ab7C2 to amyloid is dependent on the native conformation of amyloid.

Example 6 mACI-01Ab 7C 2-A β_1-42Interaction of

Study of main antibodies mACI-01-Ab7C 2(mC2) and amyloid peptide A β for AC immunization by surface plasmon resonance_1-42The mouse antibodies mACI-01-Ab7C2 and A β were determined_1-42A combination of monomers or fibers.

All SPR experiments were performed on a Biacore X instrument (Biacore AB.) the reagents for immobilization (EDC, NHS and ethanolamine), sensor chips CM5 and SA, and running and sample buffers HBS-EP were all purchased from Biacore AB. sodium acetate (10mM, pH 5.0) was used as coupling buffer to increase coupling yield_1-42Fibril A β was prepared by reaching a final concentration of 3mg/ml and placing the vial at 37 ℃ for 7 days_1-42(BAchem.) fibril A β_1-42Coupled to a CM5 sensor chip containing a surface-bound carboxymethyl dextran matrix biotinylated monomer A β_1-42(Bachem) coupled to a sensor chip SA comprising a carboxymethyl dextran matrix covalently bound to streptavidin, usually the mAb was analyzed at four to five concentrations by serial dilution with running buffer, injection was started at the lowest concentration and passed over fc 1 and fc 2 at a rate of 30 μ L/min for 3 minutes, flow cell 2 was not derivatized and the reaction was subtracted from fc 1 to correct for instrument noise and overall refractive index change_1-42The remaining bound antibody was removed from the fibrils and surface regeneration was performed by injecting 10mM NaOH pulses. Kinetic analysis was performed using BIAevaluation 3.0 with numerical integration and ensemble analysis algorithms. The curves obtained for injections of different concentrations of analyte were overlaid and the baseline was adjusted to zero. For curve fitting, all data were fit simultaneously to a 1: 1 homocomplex.

The binding of the mouse mACI-01-Ab7C2 antibody to amyloid was determined to be relatively strong. As shown in Table 2, the mouse antibody mACI-01-Ab7C2 was expressed at 3.8X 10^-4Average binding constant (ka) of M/s, 1.1X 10^-3s^-1And thus gives a dissociation constant (kd) of 3.5X 10^-8M's average KD to specifically bind to immobilized A β_1-42Fibers mACI-01-Ab7C2 bound to A β monomer similarly or slightly faster with 1.6X 10^-4Average ka of M/s, but faster dissociation, gives 2.5X 10^-7KD of M.

Table 2:

example 7: binding of mACI-01-Ab7C2 monoclonal antibody to amyloid fibers

To analyze the molecular binding sites of the antibodies on the preformed fibers, negative contrast Transmission Electron Microscopy (TEM) was performed (fig. 3a and 3 b).

According to 4, 5, the antibody mACI-01-Ab7C2 was coupled with 8nm colloidal gold for co-incubation of amyloid 1-42(a β 1-42) fibers, 6.65 μ M fibers were incubated with gold labeled antibody at a 1: 100 molar ratio for 24 hours at room temperature, then 5 μ l samples were incubated in fresh glow discharge Cu grid (200 mesh) covered with parylene/C film for 45 seconds, washed 3 times with water and 1 time with 2% fresh diluted and filtered uranyl acetate, the samples were stained in 2% uranyl acetate for 15-20 seconds, excess stain in the grid was aspirated and thus air dried 3 grids of each sample were prepared.

Monoclonal antibody mACI-01-Ab7C2 binds directly to A β_1-42The present invention relates to a method for inducing conformational transition in a fibrous web, and more particularly to a method for inducing conformational transition in a fibrous web, comprising the steps of contacting the fibrous web with an antibody, wherein the antibody binds to a particular region of the lateral branch of the fibrous web, and binding the amyloid fiber to the fibrous web.

Example 8: fractionation by density gradient ultracentrifugation

By density gradient ultracentrifugation (Rzepecki et al, 2004) based on the principle of distributing peptide fibers obtained after incubation with and different antibodies between different sizes, followed by pre-gradient (OptiPrep)^TM) SDS-PAGE precipitation on (S.sub.D.) study of monoclonal antibody inhibition of A β_1-42Fibre polymerisation and depolymerisation A β_1-42Simultaneous analysis of the Total amount of Pre-formed A β fibers, disaggregation of the Co-incubated antibodyAnd aggregation inhibition properties, as well as binding of antibodies to the fibers are significant advantages of this method.

All anti-A β were analyzed in the disaggregation assay_1-16(mACI-01-Ab7 C2)、Aβ_1-16(Δ14)(mACI-02-Ab6)、Aβ_1-15(mACI-24-Ab4)、Aβ_22-35(mACI-11-Ab9) and A β_29-40(mACI-12-Ab11), wherein the aggregation inhibiting properties were studied only for the monoclonal antibodies mACI-02-Ab6, mACI-24-Ab4 and mACI-01-Ab7C 2.

For A β_1-42Inhibition of aggregation, A β_1-42Monomer to mAb in two different molar ratios (monomer A β)_1-42Thirty or one hundred fold higher molar ratio than mAb) was incubated at 50 μ M final concentration of a β after 24 hours incubation at 37 ℃, in Optiprep^TMCovering the sample and rotating the tube at 259000 g for 3 hours at 4 ℃ 15 fractions were collected, fraction 1 being the least dense fraction from the top of the gradient and fraction 15 being the most dense fraction from the bottom of the gradient_1-42Concentration ratio of A β for depolymerization analysis_1-42Is five times lower, which reduces amyloid aggregation kinetics and ensures measurement in the linear phase range.

For cells obtained by co-incubation with mAb (mAb + monomer A β)_1-42Two different molar ratios of 1: 30 and 1: 100, final concentration of A β was 246. mu.M) to depolymerize preformed A β_1-42For fibrils, 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).

8.1 inhibition of A β_1-42Method of testing for aggregation

Successful and significant aggregation inhibition should lead to smaller fibers or polymeric soluble amyloid β (a β) protein, which should be present in the fractions with lower density (10-13). transfer of this band in the aggregation assay containing mACI-01-Ab7C2 can be confirmed with certainty, showing a β peptide distribution in fractions 11, 12 and 13.

This was confirmed in a second experiment in which mACC-01-Ab 7C2 again caused the transfer of most of the bands (the strongest band) from 14 to 13 and significant dissolution into fraction 14 to the precipitated band, meaning that mACC-01-Ab 7C2 exhibited strong ability to inhibit polymerization of A β peptide monomers into fibers and showed specific binding to A β fibers (at fraction 13).

The same observation was made when using antibodies mACC-24-Ab 4 and mACC-02-Ab 6, in the absence of mAb, the A β peptide aggregated after 24 hours incubation time and most of the protein appeared in fractions 13 to precipitate (little precipitate in 12), indicating complete polymerization of A β peptide monomers, successful and significant aggregation inhibition should result in smaller fibers or polymerized soluble amyloid β (A β) protein, which should appear in fractions with lower density, mACC-24-Ab 4 resulting in transfer of most of the bands (the strongest band) from 13 to 11 and 12 and significant dissolution into fraction 13 to precipitated bands, while mACC-02-Ab 6 results in transfer of bands from 13 to 10 but additional complete inhibition of larger fiber formation (fractionation in fraction 13 to precipitate), these data indicate that both mACC-24-Ab 4 and mACC-02-6 show stronger inhibition of the binding of A-24-Ab 4 and mACC-02-Ab 6 to fiber formation (aggregation) and the ability of the fiber A5811 and binding of the monomers to fiber in fraction 12 (fraction β).

In contrast, the aggregation assay comprising mACC-11-Ab 9 at a molar ratio of 1: 30 showed larger aggregates distributed between fractions 12-15 and in the precipitate, aggregates appeared in fractions 11-15 and precipitate, but with the strongest signal in fractions 11 and 12 when mACC-12-Ab 11 was present at a molar ratio of 1: 30, which means that mACC-01-Ab 7C2 and mACC-24-Ab 4 showed the strongest ability to inhibit the polymerization of A β peptide monomers into fibers, mACC-12-Ab 11 had significantly lower inhibitory properties than mACC-01-Ab 7C2, a molar ratio of more than 3 times was required to obtain this weak inhibitory activity, still a slight inhibition was observed when compared to mACC-11-Ab 9 which did not inhibit the aggregation of A β peptide fibers, all of which showed specific binding to A β fibers (for mACC-6301-Ab 2, in fractions 11-Ab 6312; and for mACC 8912 in fractions 11-Ab-12, and 11-Ab 8912; and for fractions 8512; and 11-Ab 12).

In all inhibition assays, peptides were detected in the bottom fraction. Unbound mAbs (37kDa, 95kDa and greater than 120kDa) appear in the upper half of the gradient (fractions 3-9 and 4-8, respectively).

_1-428.2 depolymerization test method for fibers of A β

Due to incomplete polymerization of the fibers, A β alone_1-42The distribution of fibrils is shown in a broader range of fractions (11-15). Thus, demonstration of the successful and significant disaggregation properties of the antibody upon co-incubation with preformed fibers is more difficult than in aggregation assays. Only migration of most fibers to lower density but still within the individual amyloid fractions could indicate aggregation activity; for amyloid alone, the major band is fraction 12. Addition of mACI-01-Ab7C2 at a molar ratio of 1: 100 did not show migration of amyloid fibers to the lower density fraction (still in fractions 11-15) when compared to amyloid alone, but the strongest signal shifted from 12 to 11 across the fraction. While not an optimal environment for incomplete fiber formation, mACI-01-Ab7C2 showed low but detectable depolymerization activity.

In contrast, A β was preformed when incubated at the same amyloid peptide to antibody molar ratio similar to mACI-01-Ab7C2_1-42Incubation of fibrils with mACI-02-Ab6 showed no migration of the bands to the lower density fraction. Only when a 3-fold higher molar ratio of 1: 30 was used did amyloid fibres migrate from 12-15 (amyloid alone without antibody co-incubation) to fractions 11-15. Thus, it appears that mACC-01-Ab 7C2 has slightly higher depolymerization properties than mACC-02-Ab 6.

Detection of the corresponding bands of mAb in the lower half of the gradient demonstrates mACC-02-Ab 6 and mACC-01-Ab 7C2 vs A β_1-42Binding of fibrils (fractions 11 to 15 for both mabs).

The disaggregation properties of mACI-01-Ab7C2 can be demonstrated in additional experiments, in whichBy A β in the absence of antibody_1-42The distribution of fibrils in fractions 13 to P (precipitate) demonstrates complete fiber aggregation. Here, migration of the fibers to the lower density fraction indicates disaggregation activity of the antibody upon co-incubation with the preformed fibers. Addition of mACI-01-Ab7C2 at a molar ratio of 1: 100 showed that most of the amyloid fibers migrated from 13 to 12, and furthermore the lowest density band migrated from 13 to 11. Thus, mACI-01-Ab7C2 also showed strong depolymerization activity.

Detection of the corresponding bands of mAb in the lower half of the gradient demonstrates mACI-01-Ab7C2 vs A β_1-42Fibril binding (fractions 11 to P) whereas the corresponding bands of antibody in fractions 4 to 7 show no antibody binding.

Summarizing these results, it could be successfully demonstrated that monoclonal antibody mACI-01-Ab7C2 targeting amyloid a β peptide binds to preformed fibers and is able to inhibit aggregation from monomeric a β peptide to fibers and disaggregate the preformed fibers in vitro.

Similar observations were made when mACI-24-Ab4 was used similar to aggregation analysis, a β alone was used_1-42The distribution of fibrils in fractions 12 to P (precipitate) confirms complete fiber polymerization. Here, migration of the fibers to the lower density fraction indicates disaggregation activity of the antibody upon co-incubation with the preformed fibers. Addition of mACI-24-Ab4 at a molar ratio of 1: 100 showed that most of the amyloid fibers migrated from 12 to 11. Thus, mACI-24-Ab4 also showed strong depolymerization activity.

Example 9 evaluation of inhibition of A β by incubation with mAb_1-42Filament aggregation and depolymerization preformed A β_1-42Fluorescence assay of fibrils

BIS-ANS fluorescence assay

To evaluate the inhibitory properties of the mAbs, detection of monomeric or non-fibrous A β was used_1-42BIS-ANS (LeVine, 2002) fluorescence assay for filament populations A β was tested before fluorescence measurement_1-42Monomers with buffer (as control) or mAb (mAb with A β)_1-42Molar ratio of peptides 1: 100) was preincubated at 37 ℃ for 14 hours. Relative fluorescence units were automatically recorded and results expressed as percent change from control.

mACI-02-Ab6 showed slight inhibitory capacity (125.8 + -28.5% vs. 100 + -29.5%) compared to the control. mACC-01-Ab 7C2 appeared to have a weaker activity (108.0 + -30.0%), and no increase was observed for mAbs mACC-11-Ab 9 and mACC-12-Ab 11 (93.5 + -21.9% and 73.2 + -47.7%) compared to the control. This result confirms the ultracentrifugation data in which mACI-01-Ab7C2 showed greater inhibitory capacity than mACI-11-Ab9 and mACI-12-Ab 11.

Example 10: thioflavin T (Th-T) fluorescence assay

To measure aggregation inhibition and disaggregation properties of the mAbs, a thioflavin T (Th-T) fluorescence assay was used which specifically binds to fibril A β_1-42Molecular and subsequent fluorescence emission intensity vs. A β present in solution_1-42The amount of fibrils is correlated.

Before fluorescence measurement, A β was added_1-42Monomers with buffer (as control) or mAb (mAb with A β)_1-42Molar ratio of peptides 1: 100) was preincubated at 37 ℃ for 48 hours. Relative fluorescence units were automatically recorded and results expressed as percent change from control.

mACI-01-Ab7C2 showed significant inhibitory capacity (11.03. + -. 20.7% versus 100. + -. 40.5%) compared to controls. This result confirms the ultracentrifugation data, in which mACI-01-Ab7C2 showed inhibitory capacity.

To measure the disaggregation properties of the mAb, a thioflavin T (Th-T) fluorescence assay was used which specifically binds to fibril A β_1-42Molecular and subsequent fluorescence emission intensity vs. A β present in solution_1-42Fibril mass correlation prior to measurement, a β fibers were preformed over 7 days (37 ℃, in PBS, pH 7.1) and then mixed with mAb or buffer (negative control) at a molar ratio of 1: 100 (mAb to a β)_1-42) Co-incubation was performed at 37 ℃ for 24 hours. Relative fluorescence units were automatically recorded by an ELISA microtiter plate reader and results were expressed as percent change from control.

Consistent with the ultracentrifugation data, mACC-01-Ab 7C2 also showed the best properties in the Th-T disaggregation assay in two independent experiments, with disaggregation capacities compared to the control of 35. + -. 11% and 64.57. + -. 13.58%, respectively (compared to 100.0. + -. 15.37%).

mACI-24-Ab4 also showed significant depolymerization properties in the Th-T assay (62.99 + -10.34% vs 100.0 + -10.03%; p < 0.0001).

mACI-11-Ab9 was slightly less active (28. + -. 14%), whereas ACI-02-Ab6 and ACI-12-Ab11 did not show significant depolymerization properties (17. + -. 12% and 13. + -. 11%, respectively).

However, in summary of the ultracentrifugation and fluorescence assays, mACC-01-Ab 7C2, mACC-01-Ab 6 and mACC-24-Ab 4 showed inhibition of fiber aggregation and reduction of preformed A β in centrifugation experiments _1-42The dual function capability of the filaments, which can be confirmed in fluorescence assays. In addition, centrifugation experiments confirmed the specific binding of mAb to amyloid.

mACI-11-Ab9 showed significantly lower inhibitory capacity in ultracentrifugation experiments at mACI-11-Ab9, which could be demonstrated in BIS-ANS, when compared to mACI-01-Ab7C2, even at 3-fold higher concentrations, as compared to mACI-01-Ab7C2 for disaggregation analysis, mACI-01-Ab7C2 showed a shortening preformed A β in both centrifugation analysis and ThT test_1-42The nature of the filaments. Three times higher concentrations of mACI-02-Ab6 in the centrifugation experiment were positive in both tests, but stronger in the centrifugation experiment.

From the above results, it is apparent that mACC-01-Ab 7C2 and mACC-02-Ab 6 are shown to be similar to A β_1-42The only antibodies that interact with the fibrils, inhibit aggregation and disaggregation of the bifunctional activity of the preformed fibers.

Example 11: mACI-01-Ab7C2 monoclonal antibody and¹³NMR and fluorescence Properties of C-labeled β -amyloid 1-42 peptide interactions

To assess the possible mechanism of mAb dissolution of preformed fibers or inhibition of fiber formation, U-¹³Parallel experiments between the Th-T fluorescence assay and solid-state NMR of the C Tyr10 and Val12 labeled β amyloid 1-42 peptides (FIG. 4) therefore, the objective of this study was to track the β -fold transition in β amyloid and in the presence of monoclonal antibodies by solid-state NMR spectroscopy and directly compare this with the depolymerization ability measured by the Th-T fluorescence assay.

Solid state NMR spectroscopy not only detects the transition in secondary structure, but also locates A β which dominates the structural transition_1-42Solid state NMR has demonstrated its applicability to this problem because of its suitability for A β_1-42A contribution is made to the determination of the structure of the fibers (Petkova et al, 2004, Petkova et al, 2002). In particular, the method of manufacturing a semiconductor device,¹³C_αand¹³C_βthe correlation between chemical shifts and secondary structure of (Cornilescu et al, 1999, Luca et al, 2001, Iway et al, 1999) is a valuable tool for testing for changes in secondary structure within peptides.

Inclusion at position 12 by Fmoc synthetic protocol¹³C Pre-labeled valine (C)¹²Val) and in position 10¹³C pre-labeled tyrosine: (¹⁰Synthesis of labelled peptides of Tyr) identity and purity of the peptides was confirmed by MALDI mass spectrometry analysis using labelled β amyloid peptide (1-42) to generate fibres by incubating peptide solutions in PBS buffer at 37 ℃ for 1 week the main problem, i.e. poor solubility of amyloid β peptide in PBS buffer, was solved by temporarily raising the pH of PBS buffer by trace ammonia to dissolve amyloid β peptide, exploiting the property of ammonia evaporation to recover the initial pH of PBS buffer by incubating samples in the presence of a larger bath of PBS buffer.

To measure the effect of β fold-disrupting antibodies, the fiber solution was incubated with the antibodies at 37 ℃ for 24 hours for NMR and Th-T assays.

In the first pass and pre-formation¹³After co-incubation of C-labeled amyloid β fibers and determination of the disaggregation capacity of mACI-01-Ab7C2 using the Th-T fluorescence assay, it was shown that mAb disaggregates the fibers by 38%.

To study the differences between PBS (control) and mAb incubations, each spectrum was deconvoluted with PeakFit (http:// www.systat.com/products/PeakFit) the lines were matched well by applying the mixed Lorentzian/Gaussian fitting procedure, the results of which are shown in fig. 4 the results are summarized in table 3, but the most significant difference is the integrated intensity of the two populations required to fit the double peak around 30-33ppm, the peak at C33ppm corresponds to β folding of the fiber, while the peak at 30ppm is the result of the random coil conformation, the sample incubated in PBS shows the majority of the label in the β folded conformation (81.7%) (top of fig. 2), when the sample was incubated with mACI-01-7C 2, the label in the β folded conformation is reduced (53.5%) (bottom of fig. 38964% of the total amount of the coil folding in the random conformation is reduced as determined by the Val 12C 5 study, and thus approximately 35% of the total amount of the fluorescence is measured.

Table 3 comparison of fitting parameters for the two conformations Val 12C β the fitted chemical shifts for the two conformations are very similar but the integrated intensities are very different, reflecting that the original β folded conformation is reduced by about 35% (1- (53.5/81.7)). this is in good agreement with the values obtained from fluorescence measurements.

Summarizing these results, it could be successfully demonstrated that the monoclonal antibody mACI-01-Ab7C2, which targets the N-terminal 1-16 region of the amyloid A β peptide, binds to preformed fibers and is able to inhibit aggregation of the monomeric A β peptide into and de-aggregate the preformed fibers in vitro, as demonstrated by density gradient ultracentrifugation experiments and by Th-T fluorescence assays, this antibody, in addition to binding to preformed fibers, also induced a shift of the β folded majority conformation of Val12 to a random coil secondary conformation environment, this could be a possible mechanism of being able to dissolve fibers by binding the monoclonal mACI-01-Ab7C2 antibody, also because detailed analysis of the Val 12C β peak showed a 35% reduction in the β folded component, which is closely consistent with the fluorescence data (38%).

Example 12: functionality of mACI-01-Ab7C2 on amyloid fibers

12.1 initiation of conformational Change and depolymerization of A β 1-42 fibers upon binding of mACI-01-Ab7C2 antibody

To assess the ability of the antibodies to disaggregate preformed β amyloid (A β)_1-42) Mechanism of fiber, measurement of depolymerized thioflavin T (T)h-T) fluorescence test method and U-plus for analyzing secondary structure¹³A parallel comparison of solid state Nuclear Magnetic Resonance (NMR) of C tyrosine 10 and valine 12 labeled A β -42 peptides the antibody solubilized 35.4% of preformed A β 1-42 fibers and simultaneously induced a transition in secondary conformation from β fold to random coil in the case of random coil, the total amount of β fold conformation was reduced by about 35% and thus closely matched to the total amount of reduction measured by the fluorescent Th-T assay.

12.2 conformation-dependent binding affinity of mACI-01-Ab7C2 antibody

Since it is well known in the scientific literature that partial antibody-antigen binding energies can be used for energy-dependent changes in antigen conformation⁶Thus, mACI-01-Ab7C2 antibody binding to the entire A β was performed_1-42For this comparison, biotinylated peptides covering the entire amino acid sequence of the epitope of mACI-01-Ab7C 2(A β) were used by ELISA_1-42Amino acids 13-21 of the sequence, manufactured by Mimotopes, available from ANAWA tracing SA) and biotinylated intact A β 1-42 peptide (Bachem) the affinity of the antibody mACI-01-Ab7C2 was analyzed according to the manufacturer's instructions (Mimotopes.) the antibody and the epitope comprising it (A β)_1-42Amino acids 13 to 21) of the sequence as compared to the binding affinity of the peptide to the entire A β_1-42The binding affinity of the protein is 38.40% higher. It is therefore suggested that the difference in binding affinity capacity is used for energy-consuming transitions in the secondary conformation of amyloid to present the antigen in a more acceptable position for antibody interaction. This may explain why the affinity of the antibody for the native antigen (whole amyloid peptide) is lower than for the isolated subunit.

Example 13: conformational specific binding of mACI-01-Ab7C2 to different classes of amyloid

To evaluate the availability of mACI-01-Ab7C2 to amyloid polymerized at different stages, i.e.to monomer and to polymerizationSpecificity of amylolytic proteins, in particular the amyloid β (A β) protein and fibrillar amyloid, an ELISA was carried out coating β amyloid protein polymerized in these different stages⁷A process is disclosed for preparing a monomer according to⁸Polymeric soluble amyloid, in particular amyloid β (a β) was prepared and fibers were prepared by incubating amyloid (Bachem, switzerland) at a final concentration of 1 μ g/μ l in Tris/HCl (pH 7.4) for 5 days at 37 ℃ followed by centrifugation (10,000rpm, 5 minutes.) the amyloid polymer was then coated on ELISA plates at a final concentration of 55 μ g/ml and subjected to a binding affinity ELISA using an alkaline phosphate labelled anti-mouse IgG monoclonal antibody (Jackson research Laboratories, Inc.).

The binding affinity of the antibody to polymeric soluble amyloid β (a β) protein (IC50 ═ 2.53nM) was higher than to fiber (IC50 ═ 5.27nM) and the binding affinity to monomer was lowest (IC50 ═ 8.3 nM).

Example 14: epitope mapping of monoclonal antibody mACI-01-Ab7C2

Epitope mapping of monoclonal antibodies mACI-01-Ab7C2 was performed by ELISA using three different peptide libraries, one library containing a total of 33 biotinylated peptides covering the entire amino acid (aa) sequence of a β 1-42 (manufactured by Mimotopes, purchased from ANAWA tracing SA), the second library containing biotinylated peptides using peptide 12 from the first peptide library (aa 12-20 of a β) and replacing each amino acid in the sequence with alanine (see table 4 below), and the third library containing biotinylated peptides 13, 14 or 15 (aa 13-21, 14-22 or 15-23 of a β) and replacing in each case the last amino acid with alanine or replacing aa 21 which is already alanine with glycine (see table 5 below), a third library containing biotinylated peptide 13, 14-15 (a) and replacing the last amino acid with alanine (see table 5 below), a total a β -42 peptide as positive control (Bachem) was used after incubation of the last amino acid with PBS (see table 5), mapping the epitope mapping with simple antibody mACI-01-Ab in PBS as positive control (PBS) after incubation with alkaline phosphatase in a PBS, 1-1% PBS, after washing with 0% PBS in a final PBS, washing with PBS in a final buffer with 0% alkaline buffer, 0% PBS, a buffer, a final buffer after incubation of the mouse antibody in a 1-10% buffer at room temperature, a1 h, a buffer, and a buffer.

Monoclonal antibodies mACI-01-Ab7C2 were shown to specifically bind to peptides 12, 13, 14 and 15 of the first peptide library these 4 peptides comprised aa12-20 (VHHQKLVFF), 13-21(HHQKLVFFA), 14-22(HQKLVFFAE) and 15-23(QKLVFFAED) of a β 1-42, suggesting that the epitope was located in region 12-23 of a β the second library substituted with alanine was used to determine the critical aa for binding to peptides 12-20(VHHQKLVFF) after aa16, 17, 19 or 20 was substituted with alanine, the binding of the mACI-01-Ab7C2 antibody was completely lost, indicating that these aa are absolutely critical for binding of the antibody to a β, when aa15 and 18 were substituted, the binding of the mACI-01-Ab7C2 antibody was partially lost.

When aa14 was replaced with alanine, binding was also almost completely lost, indicating that aa14 is also very important for binding.

Finally, a third library was used to determine whether amino acids 21, 21 or 23 were critical for epitope binding. When aa23 was replaced by alanine, the binding of the antibody to aa 15-23 was reduced, indicating that aa23 is also important for binding. Binding was partially lost when aa 21 was replaced by glycine, and slightly lost when aa 22 was replaced by alanine.

Table 4: overview of peptides used in the second library

Aa important for binding is marked in italics and underlining, and aa absolutely critical for binding is marked in italics and bold and underlining.

Table 5: overview of peptides used in the third library

Example 15: effect of Passive Vaccination with mACI-01-Ab7C2 on amyloid burden in brain of Single transgenic hAPP mice

To assess the ability of the mAb I-01-Ab7C2 monoclonal antibody to bind to soluble amyloid and clear it out of the brain in vivo, sex and age matched single hAPP mice at 6 months of age were used⁹At the end of the study, soluble amyloid burden was analyzed by collecting the animals' brains and performing a A β 1-40 and A β 1-42 specific ELISA (TGC, Germany).

Each group of 8-13 animals received two injections of 100, 300 and 1000 μ g of monoclonal antibody in 200 μ l PBS at weekly intervals, while PBS was used only as a control, after one day of the second injection, the animals were sacrificed for biochemical analysis of the soluble amyloid fraction, in order to quantify the amount of human a β 1-40 and human a β 1-42 in the soluble fraction of the brain homogenate and/or in the cerebrospinal fluid (CSF), enzyme linked immunosorbent assay (ELISA) kits (human amyloid β or β ELISA, high sensitivity, TGC, switzerland) were used (synthesized a β 1-40 or dilutions of a2 1-42) and samples were prepared according to the manufacturer's protocol briefly ELISA in 96-well polypropylene plates (Greiner, germany) without protein binding capacity, final concentrations of 1000, 500, 250, 125, 62.5, 31.3 and 15.6pg/ml of standard and dilutions of a 387 are prepared in sample diluents for linear dilution of the samples, the final concentrations of the samples were evaluated in the linear dilutions of the standard preparations, no increase in the sample concentration of the standard dilutions of the sample was used in the assay of the sample dilutions of the mouse, no linear dilutions of the standard dilutions of the sample was used for the analysis of the linear dilutions of the mouse, and no increase of the sample was evaluated in the sample concentration of the sample was used in the age of the standard dilutions of the sample, no age of the sample, no.

Samples, standards and blanks (50 μ l) were added to anti-a β coated polystyrene plates (capture antibody selectively recognizing the C-terminus of the antigen), with the addition of a selective anti-a β antibody conjugate (biotinylated detection antibody) and incubated overnight at 4 ℃ to allow formation of antibody-amyloid-antibody-complexes the next day, streptavidin-peroxidase-conjugate was added, after 30 minutes TMB/peroxide mixture was added, resulting in conversion of the substrate to colored product and color intensity was measured photometrically with an ELISA reader with a 450nm filter, quantification of the a β content in the samples was obtained by comparing absorbance to a standard curve made with synthetic a β 1-40 or a β 1-42, results are expressed as a respective change in relative average control values (as a percentage relative to control).

When single happp mice were passively immunized with the monoclonal antibody ACI-01-Ab7C2 injected intraperitoneally twice at a dose of 300 μ g, the total amount of a β in the brain homogenate was significantly reduced and the reduction in the total amount of a β was substantially insignificant (a β 040: -27.3 ± 13.9%, p < 0.05; a β: -8.6 ± 22.4, p ═ 0.56; unpaired Student T test), whereas 100 and 1000 μ g did not achieve significance the immunization with 100 μ g resulted in an increase in a β and a β in the brain homogenate (a β: 32.3 ± 36.8%; a β: 38.3 ± 51.4%), whereas treatment with 1000 μ g elicited the correct trend of a reduction in amyloid burden and was potentially effective when the number of animals per group increased (a β: -2.2 ± 26.0%; a β: -9.3 ± 15.9%) and the data for these animals appeared to be more relevant in the acute immunization schedule but the acute AD-5392-soluble mouse AD data was more interesting in these groups.

Example 16: effect of chronic passive administration of mACI-01-Ab7C2 on plaque burden in double transgenic hAPXPS 1 mice

To assess the ability of the mAbI-01-Ab 7C2 monoclonal antibody to bind and reduce amyloid plaques in brain in vivo, sex and age matched 3.5 month old double transgenic hAPPSS 1 mice were used¹⁰Used for a 4-month long chronic passive immunization study. At the end of the study, amyloid plaques were analyzed by histochemistry of the animal brain by binding of thioflavin S.

15 transgenic animals received 16 weekly injections of 500. mu.g of monoclonal antibody. Only 15 animals injected with PBS served as controls. All injections were given intraperitoneally. At sacrifice, mice were anesthetized and heart-flushed with physiological serum at 4 ℃ to remove blood from cerebral vessels. Subsequently, the brain is removed from the cranium and the forebrain and hindbrain are separated by cutting at the coronal/frontal plane. The forebrain was evenly divided into left and right hemispheres by using a midline sagittal incision. One hemisphere was fixed post-fixed in 4% paraformaldehyde used histologically. Sagittal vibrating microtome sections (40 μm) were cut for free floating incubation and stored at 4 ℃ until stained with 1% sodium azide in PBS. For dense plaques, five different levels of sections were stained with thioflavin S. Sections of all animals were randomly stained and quantified blindly. Images were obtained with a Leica DMR microscope equipped with a SonyDXC-9100P camera and analyzed in silico using Leica Q-Win software. The light intensity and condenser settings of the microscope are kept constant during image acquisition. All acquired images were processed through the same computer subroutine to minimize the bias of the investigator. The density split threshold is applied consistently throughout the analysis. The brain underfoot area was selected for automated quantification of amyloid burden in thioflavin S staining.

Total plaque load and number of plaques in the subbrain foot region could be significantly reduced upon passive immunization of double hAPP/PS1 mice for 4 months as described above. In plaque burden, a significant reduction of 31% can be achieved (mACI-01-Ab7C 2: 1.11 ± 0.21%, and control: 1.61 ± 0.35%; p ═ 0.003, Mann-Whitney U-Test), while chronic passive immunization significantly reduced plaque volume by 19% (mACI-01-Ab7C 2: 8.73 ± 1.36 and control: 10.78 ± 1.36; p ═ 0.006, Mann-Whitney U-Test), indicating that plaque lysis occurs to a slightly lesser extent than plaque lysis.

Example 17: effect of Passive Vaccination with mACI-01-Ab7C2 on memory Capacity of Single transgenic hAPP mice

To analyze the ability of the machi-01-Ab 7C2 antibody to alter or increase cognitive function in vivo, passive immunization studies were performed using sex and age-matched 9-month-old single hAPP mice. At the end of the immune phase, non-spatial cognition is measured by a new Object Recognition Task (ORT).

Each group of 12 animals received two intraperitoneal injections of 400 μ g monoclonal antibody in 200 μ l PBS, while PBS alone was used as a control. One day after the second injection, on a new Object Recognition Task (ORT)^12，13To study cognitive ability. For ORT enrollment, mice were placed in the behavioral arena for 10 minutes and faced with new unknown objects. The exploration time was recorded. Three hours later, the same mice were replaced in the same arena for a second period, but faced with previously explored objects and additional new objects. The times of exploration of the two objects are again recorded and calculated as the ratio of the time of exploration of the new object to the total exploration time and the change in proportion to the control represents the cognition factor.

Passive vaccination with mACI-01-Ab7C2 resulted in a significant increase in cognitive memory capacity in single transgenic AD mice (mACI-01-Ab7C 2: 131.6 ± 9.1%, and control: 100.0 ± 9.2%, p < 0.05; unpaired Student T-test, with each group n ═ 12).

And (4) preservation:

the following hybridoma cell lines were deposited under the Budapest treaty at the "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ)" located in Wenge, Brunan, 38124, and Olderwig, Masschel 1B.

Hybridoma strain Name (R)	Name of antibody	Date of storage	Accession number
				FP 12H3	mACI-01-Ab7	12/1/2005	DSM ACC2752
FP 12H3-C2	mACI-01-Ab7C2	12/1/2005	DSM ACC2750
				FP 12H3-G2	mACI-01-Ab7G2	12/1/2005	DSM ACC2751
ET 7E3	mACI-02-Ab6	12/8/2005	DSM ACC2755
				EJ 7H3	mACI-24-Ab4	12/8/2005	DSM ACC2756

Reference to the literature

Bard F，Cannon C，Barbour R，Burke RL，Games D，Grajeda H，Guido T，Hu K，Huang J，Johnson-Wood K，Khan K，Kholodenko D，LeeM，Lieberburg I，Motter R，NguyenM，Soriano F，Vasquez N，Weiss K，Welch B，Seubert P，Schenk D，Yednock T.(2000).

Nature Med.6，916-919.

Barghorn S, Nimmrich V, Streibinger A, Krantz C, Keller P, Janson B, Bahr M, Schmidt M, Bitner RS, Harlan J, Barlow E, Ebert U, Hillen H (2005), globular amyloid β peptide oligomer-a neuropathic protein that is homogeneous and stable in Alzheimer' S disease, J Neurochem 95: 834-.

Baschong W, Wrigley NG (1990), small colloidal gold linked to Fab fragments or immunoglobulin G as a high resolution marker for electron microscopy: technical review, J electronic microsystem Tech 14: 313-323.

Blond and Goldberg, 1987, PNAS, 3/1/1987, vol 84, stage 5, page 1147-1151.

Cornilescu G，Delaglio F，Bax A.(1999)J.Biomol.NMR；13：289-302.

Burdick.D. et al, the assembly and aggregation properties of synthetic amyloid peptide analogs of Alzheimer's A4/β, J.biol.chem.267, 546-554(1992).

DeMattos，Bales，KR，Cummins，DJ，Dodart，JC，Paul，SM，Holtzmann，D.M(2001).Proc Natl Acad Sci U S A 98，8850-8855.

Dewachter I, Van DJ, Smeijers L, GiNs M, Kuiperi C, Laenen I, caluwaters N, Moechars D, cherer F, vanderstrile H, Van LF (2000), amyloid peptides increasing with age in the brain of old APP/V717I transgenic mice and amyloid plaques caused by a mechanism other than mutated presenilin 1, J Neurosci 20: 6452-6458.

Dewalter I, Reverse D, caluwaters N, Ris L, Kuiperi C, Van den hc, Spittaels K, Umans L, Serneels L, third E, moechar D, Mercken M, Godaux E, VanLeuven F (2002), neuronal defects of presenilin 1 inhibit amyloid precursor protein [ V717I ] amyloid plaque formation in transgenic mice and correct long-term dystrophies of the hippocampus rather than cognitive defects, J Neurosci 22: 3445-3453.

Glenner and Wong, Biochem Biophys Res Comm129, 885-890(1984)

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

Heneka MT, Sastre M, dumitrecu-Ozimek L, Dewachter I, Walter J, Klockgether T, Van LF (2005), central glial activity consistent with increased BACE1 activity in APP [ V717I ] transgenic mice and precipitated prior to amyloid plaques, jneuroinflumation 2: 22.

hodgson et al, Bio/Technoloy, 9: 421(1991)

Iwadate M，Asakura T，Williamson MP.(1999)J.Biomol.NMR；13：199-211.

Kirschner.da, Abraham, C, & selkoe.d.j., X-ray diffraction from intra-neuronal paired helical filaments and extra-neuronal amyloid fibers in alzheimer's disease showed a crossed β conformation, proc.natl.acad.sci.u.s.a 83, 503-.

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

Kennedy, j.h. et al, 1976(clin. chim. acta 70: 1-31)

Klein WL (2002), A β toxicity in Alzheimer's disease globular oligomers (ADDLs) as novel vaccine and drug targets, Neurochem lnt 41 (5): 345-.

Kohler and Milstein (Nature 256: 495-497(1975))

LeVine，H.Ill，(2002).Arch Biochem Biophys 404，106-115.

Luca et al 2001

McGeer et al, 1994

Moechars D, Dewachter I, Lorent K, Reverse D, baekeladt V, naidusa, tesseur I, Spittaels K, Haute CV, cheler F, Godaux E, corell B, VanLF (1999), early phenotypic changes in transgenic mice overexpressing different mutants of amyloid precursor protein in the brain, J Biol Chem 274: 6483-6492.

Nelson, R. & eisenberg.d., recent atomic model of amyloid fibril structure, curr.opin.struct.biol. (2006).

Nicolau, C, Greferath, R., Balaban, T.S., Lazarte, J.E., and Hopkins, R.J, (2002) Proc Natl Acad Sci U S A99, 2332-.

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

Pearson W.R.(1990)，Methods in Enzymology 183，63-98

Petkova AT，Buntkowsky G，Dyda F，Leapman RD，Yau WM，TyckoR.J.Mol.Biol.2004；335：247-260.

Petkova AT，lshii Y，Balbach JJ，Antzutkin ON，Leapman RD，Delaglio F，Tycko R.(2002)Proc.Nat..Acad.Sci.U.S.A；99：16742-16747.

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

Rzepecki, p., Nagel-Steger, l., Feuerstein, s., Linne, U., Molt, o., Zadmard, R., Aschermann, K., Wehner, M., Schrader.T., and Riesner, D. (2004) J Biol Chem 279, 47497. 47505-.

Sambrook et al, supra.

Schenk D, Barbour R, Dunn W, Gordon G, Graj eda H, Guido T, HuK, Huang J, Smith, S.O., and Bormann, B.J, (1995). Proc Natl Acad Sci U SA 92, 488-491.

Schenk et al, 1999

Schurs, A.H.W.M., et al, 1977(Clin.Chim Acta 81: 1-40)

Slot JW, Geuze HJ (1985), a novel method for preparing gold probes for multi-labeled cell chemistry, Eur JCell Biol 38: 87-93.

Smith and Waterman, Advances in Applied Mathemitics 2(1981), 482. ang. 489

Van dA, I, Wera S, Van LF, Henderson ST (2005), ketogenic diet reduced amyloid β 40 and 42 in a mouse model of Alzheimer' S disease, Nutr Metab (Lond) 2: 28.

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

Ye, j., Dave, u.p., Grishin, n.v., Goldstein, j.l., and Brown, m.s. (2000) ProcNatl Acad Sci U S a 97, 5123-.

Zrein et al, (1998), clinical and Diagnostic Laboratory Immunology, 5 (1): 45-49.

Experimental Eye Research 78(2004)243-256

WO 2004/058258

WO96/1359

WO96/29605

Statement regarding deposited microorganisms or other biological materials

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	Deposit unit address (including postal code and country) Germany, 38124 Braun-Sawig, Australian Oldeweger 1b
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Statement regarding deposited microorganisms or other biological materials

(PCT rule No. 13 of 2)

A. The following instructions relate to the deposited microorganisms or other organisms indicated on page 39, line 7 of the description Material
	B. Deposit identification other deposits are identified on another page □
Name of depository organization DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
	Deposit unit address (including postal code and country) Germany, 38124 Braun-Sawig, Australian Oldeweger 1b
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Statement regarding deposited microorganisms or other biological materials

(PCT rule No. 13 of 2)

A. The following instructions relate to the deposited microorganisms or other biological materials referred to on page 39, line 7 of the description Material
	B. Deposit identification other deposits are identified on another page □
Name of depository organization DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
	Deposit unit address (including postal code and country) Germany, 38124 Braun-Sawig, Australian Oldeweger 1b
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Statement regarding deposited microorganisms or other biological materials

(PCT rule No. 13 of 2)

A. The following instructions relate to the deposited microorganisms or other organisms indicated on page 39, line 23 of the description Material
	B. Deposit identification other deposits are identified on another page □
Name of depository organization DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
	Deposit unit address (including postal code and country) Germany, 38124 Braun-Sawig, Australian Oldeweger 1b
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(PCT rule No. 13 of 2)

A. The following instructions relate to the deposited microorganisms or other biological materials referred to in the specification at page 40, line 3 Material
	B. Deposit identification other deposits are identified on another page □
Name of depository organization DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
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Claims

1. A monoclonal antibody or a functional part thereof produced by the hybridoma cell line FP 12H3-C2 deposited at 12.1.2005 with DSM ACC 2750.

2. An antibody or functional part thereof, wherein said antibody or functional part thereof comprises the heavy chain variable region of SEQ ID NO 8 and the light chain variable region of SEQ ID NO 7, wherein said antibody or functional part thereof recognizes and binds β -amyloid.

3. An antibody or functional part thereof, which antibody or functional part thereof is capable of specifically binding β -amyloid and comprises all the CDRs of the light chain variable region of SEQ ID NO. 7 and all the CDRs of the heavy chain variable region of SEQ ID NO. 8.

4. An antibody or functional part thereof, wherein said antibody or functional part thereof recognizes and binds an epitope of β -amyloid and comprises all the light chain variable region CDRs and all the heavy chain variable region CDRs of an antibody produced by the hybridoma cell line FP 12H3-C2 deposited at 12.1.2005 with DSM ACC 2750.

5. An antibody or functional part thereof, wherein said antibody or functional part thereof recognizes and binds an epitope of β -amyloid and comprises all the CDRs of the light chain variable region of SEQ ID NO:7 and all the CDRs of the heavy chain variable region of SEQ ID NO: 8.

6. An antibody or functional part thereof that recognizes and binds β -amyloid in its native conformation, wherein the antibody or functional part thereof comprises a light chain variable region and a heavy chain variable region having the sequences of the light chain variable region and the heavy chain variable region, respectively, of an antibody produced by hybridoma cell line FP 12H3-C2 deposited at 1.12.2005 with DSM ACC 2750.

7. The antibody of any one of claims 2-6, wherein the antibody is a monoclonal antibody.

8. The antibody or functional part thereof of any one of claims 3 to 5, wherein the antibody or functional part thereof is a humanized antibody or a chimeric antibody.

9. A humanized antibody comprising a humanized form of the light chain variable region of SEQ ID NO:7 and the heavy chain variable region of SEQ ID NO:8, wherein the antibody binds β -amyloid.

10. A humanized antibody comprising a humanized form of both the light chain variable region and the heavy chain variable region of an antibody produced by hybridoma cell line FP 12H3-C2 deposited at DSM ACC2750 at 12/1 of 2005, wherein the antibody binds β -amyloid.

11. According to claim 1-6 or a functional part thereof, which antibody is raised against a supramolecular antigenic construct comprising the amino acid sequence of β -amyloid peptide A β_1-16The antigenic peptide of (1), which corresponds to the amino acid sequence of (1), said antigenic peptide being modified with polyethylene glycol, wherein said polyethylene glycol is covalently linked to the terminus through an amino acid capable of serving as a linking molecule.

12. The antibody or functional part thereof of any one of claims 1-6 and 9 and 10, wherein the antibody or functional part thereof is isolated.

13. A polynucleotide comprising a nucleotide sequence encoding an antibody or functional part thereof according to any one of claims 1-12.

14. The polynucleotide of claim 13, comprising a nucleotide sequence encoding an antibody or functional portion thereof, comprising:

a) the nucleotide sequence of the heavy chain variable region of SEQ ID NO. 11; or

b) A nucleotide sequence which differs from the nucleotide sequence of (a) in codon sequence due to the degeneracy of the genetic code, wherein said nucleotide sequence encodes the polypeptide encoded by SEQ ID NO: 11; or

c) A nucleotide sequence having a complementary sequence of (a) or (b).

15. The polynucleotide of claim 13, comprising a nucleotide sequence encoding an antibody or functional portion thereof, comprising:

a) 12, the nucleotide sequence of the heavy chain variable region of SEQ ID NO; or

b) A nucleotide sequence which differs from the nucleotide sequence of (a) in codon sequence due to the degeneracy of the genetic code, wherein said nucleotide sequence encodes the polypeptide encoded by SEQ ID NO 12; or

c) A nucleotide sequence having a complementary sequence of (a) or (b).

16. A composition comprising an antibody or functional part thereof according to any one of claims 1 to 12.

17. The composition according to claim 16, further comprising a pharmaceutically acceptable carrier.

18. The composition according to claim 17, comprising a therapeutically effective amount of an antibody or functional part thereof.

19. Use of a composition according to any one of claims 16-18 in the manufacture of a medicament for the treatment of alzheimer's disease.

20. A method of using an antibody or functional part thereof according to any one of claims 1 to 12 for the preparation of a composition according to any one of claims 16-18, said method comprising formulating said antibody or functional part thereof in a pharmaceutically acceptable form.

21. Use of a composition according to any one of claims 16-18 in the manufacture of a medicament for treating or reducing the effects of alzheimer's disease.

22. Use of a composition according to any one of claims 16-18 in the manufacture of a medicament for reducing plaque load or reducing the amount of plaque in the brain of a mammal suffering from alzheimer's disease.

23. Use according to claim 22, wherein the plaque load in the brain is reduced by 20-31%.

24. Use according to claim 22, wherein the amount of plaques in the brain is reduced by 10-20%.

25. Use of a composition according to any one of claims 16-18 in the manufacture of a medicament for reducing the total amount of soluble β -amyloid in the brain of a mammal with alzheimer's disease.

26. Use of an antibody or functional part thereof according to any one of claims 1-12 for the manufacture of a medicament for preventing, treating or reducing the effects of alzheimer's disease.

27. Use of a composition according to any one of claims 16-18 in the manufacture of a medicament for maintaining or increasing cognitive memory capacity in a mammal having alzheimer's disease.

28. A hybridoma cell line that produces the antibody of any one of claims 1-12.

29. Hybridoma cell line FP 12H3-C2, deposited at 1.12.2005 with DSM ACC 2750.

30. Use of an antibody or functional part thereof according to any one of claims 1 to 12 for the manufacture of a medicament for the treatment of alzheimer's disease.

31. Use of an antibody or functional part thereof according to any one of claims 1 to 12 for the manufacture of a medicament for treating or reducing the effects of alzheimer's disease.

32. Use of an antibody or a functional part thereof according to any one of claims 1 to 12 for the preparation of a medicament for reducing the plaque load or reducing the plaque amount in the brain of an animal suffering from alzheimer's disease.

33. Use of an antibody or functional part thereof according to any one of claims 1 to 12 for the preparation of a medicament for reducing the total amount of soluble β -amyloid in the brain of an animal with alzheimer's disease.

34. Use of an antibody or functional part thereof according to any one of claims 1 to 12 for the preparation of a medicament for maintaining or increasing cognitive memory capacity in a mammal suffering from alzheimer's disease.

35. A cell line producing an antibody or functional part thereof according to any one of claims 1-12.

36. A cell line comprising the polynucleotide of any one of claims 13-15.

37. A method of producing an antibody or functional part thereof comprising the steps of culturing the cell line of claim 35 or 36 and producing the antibody or functional part thereof.

38. A method of making an antibody, or functional portion thereof, comprising expressing the polynucleotide of any one of claims 13-15.