CN101325972A

CN101325972A - A beta 1-42 specific monoclonal antibodies with therapeutic properties

Info

Publication number: CN101325972A
Application number: CNA2006800464669A
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: 2008-12-17
Anticipated expiration: 2026-12-08
Also published as: CN101325972B; UA102368C2; AR107352A2

Abstract

The present invention is related to methods and compositions for the therapeutic and diagnostic use in the treatment of diseases and disorders which are 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. The present invention provides novel methods and compositions comprising highly specific and highly effective antibodies having the ability to specifically recognize and bind to specific epitopes from a range of ss-amyloid proteins. The antibodies enabled by the teaching of the present invention are particularly useful for the treatment of diseases and disorders which are caused by or associated with amyloid or amyloid-like proteins including amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis including, but not limited to, neurological disorders such as Alzheimer's Disease (AD).

Description

A β 1-42 monoclonal antibody specific with therapeutic properties

The present invention relates to be used for the treatment of by amyloid or amyloid sample albumen method and composition that cause or relative disease and treatment of conditions and diagnosis, described disease and disease comprise amyloidosis, this is one group of disease relevant with amyloid and unusual, such as Alzheimer.

Amyloidosis is not single disease entity, but the multiple PD process that is deposited as feature of a group with waxy, amylaceous albumen (be referred to as amyloid, they are accumulated in one or more organ or the body system) ECT.Along with the amyloid sediment pile, they begin to disturb the normal function of organ or body system.At least there are 15 kinds of dissimilar amyloidosis.Principal mode is the primary amyloidosis that does not have known precedent, secondary amyloidosis and heritability shallow lake amyloidosis after other disease.

Secondary amyloidosis occurs in suffers from chronic infection or inflammatory diseases, in tuberculosis, the bacterial infection that is called familial Mediterranean fever, infection of bone (osteomyelitis), rheumatoid arthritis, small intestinal inflammation (granulomatous ileitis), Hodgkin and crowd leprous.

The amyloid deposit comprises three kinds of components usually.90% the amyloid protein fibril (fibril) that accounts for the amyloid material comprises a kind of in several dissimilar albumen.These albumen can be folded into so-called " beta sheet " lamellar fibril dimension, and the albumen configuration of this uniqueness demonstrates the binding site to Congo red (Congo red), has caused the coloring property of the uniqueness of amyloid protein.In addition, the amyloid deposit is closely related with amyloid P (pentagonal) component (AP) (with the relevant glycoprotein of normal serum amyloid P (SAP)), and closely related with sulfated glycosaminoglycans (GAG) (complex carbohydrates of connective tissue).

Many infirmitiess of age based on or relevant with amyloid sample albumen, and partly to promote disease to take place and the amyloid of disease process or the sedimentary accumulation in extracellular of amyloid sample material are feature.These diseases include but not limited to sacred disease, such as Alzheimer (AD), comprise that the loss with cognitive memory ability is the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body (Lewy body dementia), mongolism (Down ' ssyndrome), Hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam hirano disease.Other is progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease (Creutzfeld Jacob disease), parkinson, the relevant dementia of HIV, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors and other disease based on amyloid sample albumen or relative disease, comprises degeneration of macula.

Although the pathogeny of these diseases may be different, their distinctive deposits often comprise many common minute subconstiuent.On significance degree, thus these depositions (McGeer etc., 1994) when may cause activatory complement component, acute phase reactant, immunomodulator and other inflammation medium owing to the local activation that promotes inflammatory pathway.

Alzheimer (AD) is a kind of sacred disease, thinks that mainly by the amyloid speckle, promptly the accumulation of protein abnormal deposition is caused in brain.The type of the modal amyloid of finding in the infected individuals brain mainly comprises A β fibril.Scientific evidence shows that the generation of amyloid beta matter in speckle and cumulative increase cause nerve cell death, and this promotes development and the process of AD.The impairment that causes the minimizing and the memory of neurotransmitter in the loss of the neurocyte in crucial brain zone again.Mainly cause the accumulative protein of speckle to comprise amyloid precursor protein (APP) and two kinds of senilism albumen (senilism protein I and senilism protein I I).Cause the release of 39 to 43 amino acid whose A β peptides by enzyme β and gamma secretase continuous cracking amyloid precursor protein (APP) (they are constitutive expression and catabolism in most of cell).The degraded of APPs may increase their accumulative tendencies in speckle.Because two extremely hydrophobic amino acid residues of its C-terminal, A β (1-42) fragment especially have the high tendency that makes up aggregation.Therefore think that A β (1-42) fragment relates generally to and cause initial that neuritic plaques forms among the AD, and therefore have high pathology potentiality.Therefore needing can targeting and spread the specific antibody that the amyloid speckle forms.

The symptom of AD occurs slowly, and first symptom may only be slightly forgetful.In this stage, individuality may be forgotten the title of recently incident, activity, familiar people or thing and can not solve the simple mathematical problem.Along with progression of disease,, symptom cause the people or their household that suffer from AD to seek medical science help thereby more easily being noted and becoming enough serious.The symptom in mid-term of AD comprises forgets the simple task of how doing such as the arrangement cleaning, and the problem of speaking, understand, read or writing manifests gradually.The AD patient in later stage may become anxiety or aggressiveness may be wandered away and finally need total care from family.

At present, the method for unique AD that clarifies a diagnosis is speckle and the entanglement of differentiating in individuality obduction after death in the cerebral tissue.Therefore, when the people still lived, the doctor can only make the diagnosis that " possibility " or " probably " suffers from AD.Use present method, the internist utilizes the instrument of several diagnosis " probably " AD to diagnose AD at 90% temporally precise at the most.The inquisitorial general health of doctor physician, the medical care problem in past and this person finish the history of the hell and high water of daily routines.The performance testing of memory, problem solving, attention, calculating and language provides cognitive deterioration for information about, and the check of medical inspection such as blood, urine or spinal fluid and brain scans can provide some out of Memory.

The management of AD by based on medicine and do not form based on the treatment of medicine.To change the potential process of disease (delaying or the reverse process) is that the present major part of treatment of purpose is unsuccessful.Chemical messenger (neurotransmitter) not enough (defective) or the handicapped medicine, particularly cholinesterase inhibitor (ChEI) (such as tacrine and Rivastigmine (rivastigmine)) that recover neurocyte have demonstrated and can improve symptom.ChEI hinders the enzymatic degradation of neurotransmitter, therefore is increased in the amount of the chemical messenger that can be used for the transmission signal in the brain.

For some people in the disease early and middle portion stage, the medicine tacrine (

Morris Plains, NJ), donepezil (

Tokyo, JP), Rivastigmine (

East Hanover, NJ) or galantamine (

New Brunswick NJ) can help prevent some symptom to become more serious in the limited time.Another kind of medicine memantine (

New York NY) has been checked and approved the medium extremely serious AD of treatment.Also there is medicine to be used to tackle the psychiatry performance of AD.Some medicine also can help to control the behavior symptom of AD, such as insomnia, excited, in a trance, anxiety and depression.Treating these symptoms makes that usually the patient is more comfortable and makes easier treatment for the caretaker.Unfortunately, although significantly therapeutic advance to demonstrate this class reagent better than placebo reliably, disease still continues development, and only is limited to the mean effort of moral function.The medicine that much is used for the AD Drug therapy for example, such as ChEI, also has side effect, comprises gastrointestinal dysfunction, hepatotoxicity and loses weight.

Other is mild cognitive impairment, dementia with Lewy body (LBD), amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM) and degeneration of macula, particularly age-related macular degeneration (AMD) based on the proteic gathering of amyloid sample with deposition or the disease relevant with them.

Mild cognitive impairment (MCI) is a general term, the most often is defined as not obvious but measurable dysmnesia.People with MCI experiences more serious memory problems than normal with ageing is desired, but can not demonstrate other dementia symptom, such as judging or the reasoning infringement.MCI is the symptom that reflects the AD preclinical phase usually.

Beta amyloid albumen is considered to play the part of crucial role in the deposition of entorhinal cortex (EC) in the evolution of old people's mild cognitive impairment (MCI).In case this becomes clinical obvious with AD, then the promptly remarkable observed result that descends of CSF-A A β (1-42) level is consistent.Opposite with CSF-A β (1-42), the level of CSF-τ significantly increased in the MCI stage, and the continuation rising after this of these values, thereby the CSF-τ level that shows increase might help to detect the MCI person under inspection that prediction will develop into AD.

Dementia with Lewy body (LBD) is the neurodegenerative disease that can occur among the over-65s crowd, and it generally causes the symptom of cognition (thinking) damage and unusual behavior change.Symptom can comprise cognitive impairment, neural sign, sleep disorder and autonomic failure.Cognitive impairment is the feature that LBD presented in most of patients.The patient has confusional outbreak repeatedly, and serious day by day.The fluctuation of cognitive competence is relevant with the metastasis degree of attention and Vigilance usually.The fluctuation of cognitive impairment and thinking can with minute, hour or day and different.

The Louis body is formed by phosphorylation and unphosphorylated neural thread protein NTP, and they comprise the synapsin alpha-synapse nucleoprotein, and the ubiquitin that participates in damage or paraprotein elimination.Except that the body of Louis, also may there be Louis aixs cylinder (Lewy neurites), it is the inclusion body of neurocyte cell in prominent.The amyloid speckle can be formed in the patient's who suffers from DLB the brain, however they tend to quantitatively than suffer from seen in the patient of Alzheimer to lack.Another micropathology sign of AD, promptly neurofibrillary tangles is not the principal character of DLB, but also often has neurofibrillary tangles except that having the amyloid speckle.

Amyotrophic lateral sclerosis (ALS) is to be feature with upper and degeneration LM.In some ALS patient, may there be dementia or aphasia (ALS-D).Dementia is frontotemporal dementia (FTD) the most normally, and much these cases have the ubiquitin positive, the negative inclusion of τ in the neuron of dentate gyrus and volume and temporal lobe shallow-layer.

Inclusion body myositis (IBM) is to be typically found at the disabling disease among the crowd more than 50 years old, but wherein muscle fiber develops inflammation and begins atrophy---its deutocerebral region can not sustain damage and the patient keeps intelligence completely.Suffer from that this old people is the most general, among the patient myocyte of carrying out property muscle disease, finding to participate in two kinds of enzymes that amyloid-β albumen produces increases, wherein amyloid-β also increases.

Another is a degeneration of macula based on the proteic gathering of amyloid sample and deposition or relative disease.

Degeneration of macula is to cause that the central area of retina (at the equally thin tissue of the sensitive paper at eyes rear portion, transmitting visual signal to brain at this photoreceptor cell) is the common disease of macular area degeneration.Produce vision clear, distinct, " forward straight " by the macular area processing.The damage macular area causes the generation and the smudgy or metamorphopsia of blind spot.Age-related macular degeneration (AMD) is to cause VI main cause in the U.S., and for the people of over-65s, it is a legal blind main cause among the Caucasian.Nearly 1,800,000 40 years old and above American suffer from AMD in late period, and other 7,300,000 people who suffers from medium AMD has the real danger of visual loss.Government estimates the year two thousand twenty, will have 2,900,000 people and suffer from AMD in late period.The usually surprised and dejected discovery of AMD patient is appreciated that so few to the reason and the treatment of this Anton symptom.

The form that has two kinds of degeneration of macula: dryness degeneration of macula and moist degeneration of macula.Have 85 percent patients with macular degeneration to be diagnosed as the dryness form, wherein the cell of macular area begins to damage slowly.Other eyes keep uninfluenced although eye may DE, and two eyes all are subjected to the influence of dryness AMD usually.Subretinal yellow deposit, promptly druse is the early stage sign of dryness AMD.Along with the increase of the number of druse and size, develop into late period dryness AMD or the danger of moist AMD also increase.Dryness AMD may further develop and cause the forfeiture of vision under the situation of the moist form that does not change disease into; Yet early stage dryness AMD also might become moist form suddenly.

Although only account for 1 15 of case, it is blind 90 percent that moist form has caused, and be considered to the AMD moist AMD of mid-term stage (not early stage or) in late period.Moist AMD always is in after the dryness form of disease.When the dryness form becomes more serious, some begin that in the back of macular area unusual angiogenic growth is arranged.These blood vessels are highly brittle weak and meeting leakage liquid and blood (being " moist " degeneration of macula therefore), and this has caused the quick damage to macular area.

The AMD of dryness form begins usually to cause slight blurred vision.Particularly the center of vision may thicken unclearly, and this zone is along with advancing of disease becomes bigger.If have only eyes to be affected, then do not have symptom to be noted.In moist AMD, straight line may look like wavy, and the forfeiture of central vision may very fast appearance.

The diagnosis of degeneration of macula relates generally to expand eye examination, visual acuity test, and the rear portion of checking eyes with the method that is referred to as examination of ocular fundus is with assisted diagnosis AMD, and if the suspicion of moist AMD is arranged, then also may carry out fluoresecein angiography.If dryness AMD reaches late stage, do not prevent the existing treatment of visual loss.Yet the specific high dose prescription of antioxidant and zinc can delay or prevent mid-term AMD to be developed to late stage. (piperazine Jia Tani sodium injection), laser photocoagulation and photodynamic therapy may be able to be controlled the abnormal vascular growth in the macular area and bleed, and it is useful that this can suffer from the patient of moist AMD to some; Yet what vision had been lost can not recover by these technology.If vision is lost, existence can help the low vision aids of improving the quality of living.

The coindication the earliest of age-related macular degeneration (AMD) is the accumulation of extracellular deposit between retinal pigment epithelium (RPE) basal layer and Bruch's membrane (BM) that is referred to as druse.Recently the research of being undertaken by Anderson etc. has confirmed that druse comprises amyloid-beta (Experimental Eye Research 78 (2004) 243-256).

Ongoing research continues to explore environment, heredity and the dietary factor that may promote AMD.Also exploring new therapeutic strategy, comprising retina cell graft, the medicine that can prevent or slow down disease progression, X-ray therapy, gene therapy, be implanted to the amphiblestroid medicament that helps to stimulate the computer chip of vision and can prevent neovascularity growth under the macular area.

During the developing new drug thing, the key factor that needs to consider is the easiness of using concerning target patient.Because to patient's convenience, oral drugs are sent (particularly tablet, capsule and flexible glue agent) and are accounted for 70% of all consumption dosage forms.The drug development person admits the patient and prefers oral delivery, rather than acceptance is injected or other has more invasive drug administration mode.It also is preferred causing the preparation of short dosing interval (promptly once a day or continue to discharge).Caused the increase of patient compliance during the treatment with peroral dosage form administration antibiotic easiness.

Required is the method and composition that is used to produce high specific and high efficiency antibody, if provide antibody with peroral dosage form, this is a prerequisite.Preferably, this antibody-like can be identified in the specificity epitope on the different antigen (such as amyloid).

Therefore, what is also needed is compositions useful and the method for handling complication, these complication with by amyloid or amyloid sample albumen is that cause or relative disease association, described disease comprises amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, include but not limited to sacred disease such as Alzheimer (AD), comprise that with cognitive memory ability forfeiture be the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.Particularly, what need is the physiology's performance that can eliminate a disease, such as the special and efficient antibody of the elimination speckle formation relevant with the gathering of the fiber of amyloid or amyloid sample peptide.

For human Alzheimer, proved complete or Freund is blended by A β with Fu Shi _1-42The anti-amyloid antibodies that causes can reduce the load (Schenk etc., 1999) of the amyloid of transgenic mice.

Four palmitoylation A β that will reconstruct in liposome _1-16Intraperitoneal is inoculated into the NORBA transgenic mice and causes significant anti-amyloid antibodies titre, and this antibody also can dissolve amyloid fiber and speckle (Nicolau etc., 2002) in vitro and in vivo.

Bard etc. (2000) have at first proposed the amyloid speckle and fiber decomposes the possibility mechanism that takes place, and they reach a conclusion based on their data, i.e. antibody conditioning speckle, and speckle is damaged by the macrophage of microgliacyte subsequently.(2001) such as De Mattos point out anti-amyloid beta division center territory Mab can in conjunction with and isolate the blood plasma amyloid fully.They think that the existence of these mAbs in the blood circulation has changed the A β balance in brain and the blood plasma, help the removing and the catabolism of periphery rather than deposit in brain.

The invention provides the new method and composition that comprises high specific and efficient antibody, described antibody has specific recognition and in conjunction with the ability of the antigenic specific epitopes of a series of amyloid betas.That the antibody that provides by the present invention instruction is caused by amyloid or amyloid sample albumen treatment especially or relative disease is useful, described disease comprises amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, include but not limited to sacred disease such as Alzheimer (AD), comprise that with cognitive memory ability forfeiture be the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula; These only are some examples wherein.

In addition, the invention provides the new method and the compositions that in the mammal that shows amyloid related diseases or disease, keep or improve cognitive memory ability, comprise animal to this treatment of needs, mammal particularly, more especially human administration treatment effective dose according to monoclonal antibody of the present invention.

The summary of accompanying drawing and sequence:

Fig. 1: derived from the peptide of A β sequence 1-15,1-16 and 1-16 (Δ 14), 22-35 and 29-40.

Fig. 2: mACI-01-Ab7 C2 monoclonal antibody and amyloid kind combines in western blotting (Western blot) and dot blotting;

Fig. 3: by the mACI-01-Ab7 C2 monoclonal antibody of transmission electron microscopy and combining of amyloid fiber;

Fig. 4: U- ¹³The Th-T fluoremetry of the amyloid beta 1-42 peptide of C Tyr10 and Val12 labelling and the parallel experimental result between the solid state NMR.

SEQ ID NO:1: antigenic peptides A β _1-15

SEQ ID NO:2: antigenic peptides A β _1-16

SEQ ID NO:3: antigenic peptides A β _{1-16 (Δ 14)}

SEQ ID NO:4: antigenic peptides A β _22-35

SEQ ID NO:5: antigenic peptides A β _29-40

SEQ ID NO:6: antigenic peptides A β _1-17

SEQ ID NO:7: the aminoacid sequence of mice C2 variable region of light chain

SEQ ID NO:8: the aminoacid sequence of mice C2 variable region of heavy chain

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

SEQ ID NO:10: the nucleotide sequence that comprises the mice C2 variable region of light chain of signal sequence

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

SEQ ID NO:12: the nucleotide sequence that comprises the mice C2 variable region of heavy chain of signal sequence

The aminoacid sequence variant of epitope regions on the SEQ ID NO:13-20:A β peptide

SEQ ID NO:21: the aminoacid sequence of mice C2 light chain

SEQ ID NO:22: the aminoacid sequence of mice C2 heavy chain

The present invention has utilized the exposure that causes increasing the preferred antigens conformation and stable, and finally causes having the antigen presentation of the antibody of peculiar property.

In one embodiment of the invention, the antibody that comprises any function equivalent antibody or its funtion part is provided, or more particularly, the monoclonal antibody that comprises any function equivalent antibody or its funtion part, the anti-supramolecular constructs of this antibody, described supramolecular constructs comprises corresponding to β-amyloid peptide, particularly beta-amyloid peptide A β_1-15、Aβ _1-16With A β_1-16(Δ14)The antigenic peptides of amino acid sequence, described antigenic peptides with hydrophobic part (for example, such as palmitic acid) or hydrophilic segment is (for example, such as polyethylene glycol (PEG)) or both combinations modify, wherein said hydrophobic and hydrophilic segment is respectively by at least one, one or two amino acid particularly, for example, such as lysine, glutamic acid and cysteine or any other each end that can covalently be connected to as the suitable amino acid of jockey that hydrophilic and hydrophobic part is coupled to fragments of peptides or amino acid analogue antigenic peptide. When using PEG as hydrophilic segment, free PEG end covalently is connected to phosphatidyl-ethanolamine or any other is suitable on the compound as anchoring element, for example, and so that the antigen construct is embedded in the bilayer of liposome.

In another embodiment of the invention, the antibody that comprises any function equivalent antibody or its funtion part is provided, monoclonal antibody particularly, the native conformation of its identification amyloid, because it is bonded to amyloid oligomer and fiber specifically, rather than be bonded to nonlinearized amyloid kind.

In another embodiment of the invention, provide according to the present invention and the antibody that comprises any function equivalent antibody or its funtion part, particularly monoclonal antibody described above, wherein antibody or fragment are with at least about 1 * 10 ^-6Extremely at least about 1 * 10 ^-8, especially at least about 1 * 10 ^-6Extremely at least about 1 * 10 ^-7, more particularly at least about 1 * 10 ^-7Extremely at least about 1 * 10 ^-8, even more particularly at least about 1 * 10 ^-7Extremely at least about 4 * 10 ^-7Binding affinity combine with the A beta monomers, but preferably do not demonstrate the cross reactivity of any significant and amyloid precursor protein (APP).

In another embodiment of the invention, provide according to the present invention and the antibody that comprises any function equivalent antibody or its funtion part, particularly monoclonal antibody described above, wherein antibody or fragment are with at least about 1 * 10 ^-7Extremely at least about 1 * 10 ^-9, especially at least about 1 * 10 ^-7Extremely at least about 1 * 10 ^-8, more particularly at least about 1 * 10 ^-8Extremely at least about 1 * 10 ^-9, even more particularly at least about 1 * 10 ^-8Extremely at least about 5 * 10 ^-8Binding affinity combine with A beta, fibril or fibril (filament), but preferably do not demonstrate the cross reactivity of any significant and amyloid precursor protein (APP).

In other embodiments, according to the present invention with the antibody that comprises any function equivalent antibody or its funtion part described above, particularly monoclonal antibody show than with at least 5 times of the binding affinity height of A beta monomers, at least 10 times especially, more particularly high at least 15 times and binding affinity A beta, fibril or fibril.

Can be in vivo form the monomeric peptide, particularly amyloid beta monomeric peptide of amyloid according to antibody of the present invention with vitro inhibition, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42Monomeric peptide is gathered into high molecular polymerization amyloid fibril or fibril.

In special embodiment of the present invention, the antibody that comprises any function equivalent antibody or its funtion part is provided, monoclonal antibody particularly, described antibody with the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After monomeric peptide is hatched altogether, suppressed the A beta monomers and be gathered into high molecular polymerization fibril.

In another embodiment of the invention, the antibody that comprises any function equivalent antibody or its funtion part is provided, monoclonal antibody particularly, described antibody with the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42Monomeric peptide is hatched altogether, especially with molar concentration rate up to 1: 100, more particularly with the molar concentration rate between 1: 30 to 1: 100 but after especially hatching altogether, suppressed the A beta monomers and be gathered into high molecular polymerization fibril with 1: 100 molar concentration rate.Particularly, when comparing with other amyloid peptide monomer (contrast) of branch of hatching with buffer, described inhibition reaches at least 50%, reach at least 65% especially, more particularly reach at least 75%, even more particularly reach at least 80%, but especially reach 85%-90% at least, perhaps more.

Particularly, according to being incubated in altogether between 28 ℃ to 40 ℃ of antibody of the present invention and amyloid monomeric peptide, between 32 ℃ and 38 ℃, more particularly carried out 24 hours to 60 hours especially 37 ℃ temperature, 30 hours to 50 hours especially, more particularly 48 hours.

In another embodiment, the invention provides the antibody that comprises any function equivalent antibody or its funtion part, monoclonal antibody particularly, described antibody is at 37 ℃ of molar concentration rate and amyloid monomeric peptides with 1: 100, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After monomeric peptide is hatched 48 hours altogether, when comparing with other amyloid peptide monomer (contrast) of branch of hatching with buffer, can at least 85%, at least 89% and more particularly at least 95% suppresses the amyloid monomer especially, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42Monomeric peptide is gathered into high molecular polymer fibril or fibril.

In special embodiment, the invention provides the antibody that comprises any function equivalent antibody or its funtion part, particularly monoclonal antibody, it is to A β _1-42Monomeric peptide shows high specific, but to A β _1-38, A β _1-39, β _1-40And/or A β _1-41Monomeric peptide demonstrates not to be had in fact or only very little cross reactivity; The antibody that particularly comprises any function equivalent antibody or its funtion part, especially monoclonal antibody, it is to amyloid peptide A β _1-42Comparison A β _1-38, A β _1-39, A β _1-40, A β _1-41More responsive to 100 times, 50 to 100 times especially, more particularly 80 to 100 times, but especially 100 times; And antibody is to amyloid peptide A β _1-42Comparison A β _1-38More responsive to 1000 times, 500 to 1000 times especially, more particularly 800 to 1000 times, but especially 1000 times; And therefore can suppress to form the monomeric peptide of amyloid in vitro and in vivo, but amyloid peptide A β especially _1-42Gathering.

In another embodiment of the invention, the antibody that comprises any function equivalent antibody or its funtion part, particularly monoclonal antibody are provided, it has the β to amyloid peptide A _1-42Height in conjunction with sensitivity, and can detect the concentration that is low to moderate at least 0.001 μ g, but especially between 0.5 μ g and 0.001 μ g, the concentration range between 0.1 μ g and 0.001 μ g more particularly, but the A β of 0.001 μ g concentration especially _1-42Fiber.

In the very special embodiment of the present invention, the antibody that comprises any function equivalent antibody or its funtion part, particularly monoclonal antibody are provided, antibody can the detection fibers amount be low to moderate the A β of the Cmin of 0.001 μ g _1-42Fiber and the A β that is low to moderate the Cmin of 0.1 μ g _1-40Fiber and the A β that is low to moderate the Cmin of 1 μ g _1-38Fiber.

Antibody according to the present invention and as described above and the monomeric peptide that forms amyloid, but particularly caused forming the inhibition that the amyloid monomeric peptide is gathered into macromolecule fibril or fibril with combining of amyloid form (1-42).By the accumulative inhibition of monomeric peptide to the formation amyloid, can prevent or slow down the amyloid speckle according to antibody of the present invention, the particularly formation of amyloid form (1-42), well-known amyloid form (1-42) becomes soluble by the change of secondary conformation, and is the major part of amyloid speckle in infected animal or the human brain.

Gathering inhibition potentiality according to antibody of the present invention can be measured by any suitable method known in the art, particularly by the density gradient ultracentrifugation method, subsequently by be pre-formed on the gradient the SDS-PAGE sedimentation analysis and/or by thio-flavin T (thioflavin, Th-T) fluorimetry is measured.

The present invention also provides antibody, its with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched altogether, amyloid fibril or fibril that can the described high molecular polymerization of depolymerization.

In another one embodiment of the present invention, provide antibody, the monoclonal antibody that particularly comprises any function equivalent antibody or its funtion part, wherein antibody with the molar concentration rate up to 1: 100, more particularly with the molar concentration rate between 1: 30 and 1: 100 but especially with 1: 100 molar concentration rate with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched altogether, can make the amyloid fibril or the fibril depolymerization at least 35% of preformed high molecular polymerization, especially at least 40%, more particularly at least 50%, even especially at least 60%, but especially at least 70% or higher.

Particularly, form high molecular polymerization amyloid fibril or fibril in advance between 28 ℃ to 40 ℃ according to antibody of the present invention and amyloid, especially between 32 ℃ to 38 ℃, more particularly hatched altogether 12 hours to 36 hours 37 ℃ temperature, 18 hours to 30 hours especially, more particularly 24 hours.

In special embodiment, the invention provides the antibody that comprises any function equivalent antibody or its funtion part, monoclonal antibody particularly, wherein antibody 37 ℃ with 1: 100 molar concentration rate with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched 24 hours altogether, when with and control vector (the having only amyloid) branch of hatching else form high molecular polymerization amyloid fibril or fibril (contrast) is compared in advance, can make described high molecular polymerization amyloid fibril or fibril depolymerization at least 35%, lack 40% especially, more particularly at least 50%, even more particularly at least 60%, but especially at least 70% or higher.

Depolymerization potentiality according to antibody of the present invention can be measured by any suitable method known in the art, particularly, measure by the SDS-PAGE sedimentation analysis on preformed gradient and/or by thio-flavin T (Th-T) fluorimetry by the density gradient ultracentrifugation method thereupon.

The present invention also provides antibody or its funtion part of conformation sensitization.

In other embodiments of the present invention, provide antibody, the monoclonal antibody that particularly comprises any function equivalent antibody or its funtion part, described antibody with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched altogether, can cause that the βZhe Die conformation is to α spiral and/or random-coil conformation, but random-coil conformation particularly, even the transformation of the random-coil conformation of (especially in the Val of a 12 environment) more particularly in the intramolecularly given area, this has caused with the βZhe Die conformation is the increase of random-coil conformation of cost and the improved dissolution that forms high molecular polymerization amyloid fibril or fibril in advance.Particularly, when comparing with other pre-formation amyloid polymerization fibril of the branch of hatching in buffer or fibril (contrast), the minimizing of βZhe Die conformation reaches at least 30%, reaches at least 35% especially, and more particularly reaches at least 40% and more.

Particularly, according to antibody of the present invention and amyloid preformed high molecular polymerization amyloid fibril or the temperature of fibril between 28 ℃ to 40 ℃, especially between 32 ℃ and 38 ℃, more particularly hatched altogether 12 hours to 36 hours at 37 ℃, 18 hours to 30 hours especially, especially 24 hours.

Particularly, the invention provides the antibody that comprises any function equivalent antibody or its funtion part, monoclonal antibody particularly, described antibody 37 ℃ with 1: 100 molar concentration rate with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched 24 hours altogether, can cause that the βZhe Die conformation is to α spiral and/or random-coil conformation, but random-coil conformation particularly, even the transformation of the random-coil conformation of (especially in the Val of a 12 environment) more particularly in the intramolecularly given area, this has caused with the βZhe Die is the increase of the random-coil conformation of cost.When other pre-forms amyloid polymerization fibril or fibril (contrast) is compared with the branch of hatching in buffer, the βZhe Die conformation has reduced at least 30%, especially at least 35%, and more particularly at least 40% reach more.

The potentiality of antibody in causing conformation transition can be measured by any suitable method known in the art, particularly by solid-state 13C NMR spectroscopy, but particularly, by measuring A β peptide, particularly A β peptide 1-39,1-40,1-41,1-42 or 1-43, but especially at A β _1-42The integrated intensity of the conformation of the Va1 12 C β in the monomeric peptide is measured.

The polymerization fibril by forming amyloid or the depolymerization of fibril, according to the formation that antibody of the present invention could prevent or slow down the amyloid speckle, this can cause and the delaying or reverse of the alleviation of disease related symptom and progress thereof.

Therefore, another embodiment of the invention provides the antibody that comprises any function equivalent antibody or its funtion part as described above, monoclonal antibody particularly, described antibody can reduce to suffer from and causes the disease that A β concentration in the brain increases or the animal of disease, mammal particularly, but the total amount of A β in the especially human brain.

In another embodiment of the invention, the antibody that comprises any function equivalent antibody or its funtion part as described above is provided, monoclonal antibody particularly, described antibody can decompose speckle, therefore can reduce and suffer from the disease that causes increasing speckle load in the brain or the animal of disease, mammal particularly, but the load of the speckle in the especially human brain.The antibody that comprises any function equivalent antibody or its funtion part according to the present invention makes that the speckle load has reduced at least 20% in the brain, and especially at least 25%, more particularly at least 30%, even more particularly more than 30%.

In another embodiment of the invention, the antibody that comprises any function equivalent antibody or its funtion part as described above is provided, monoclonal antibody particularly, described antibody can dissolve speckle, cause suffering from and cause the disease that the load of speckle in the brain increases or the animal of disease, mammal particularly, but the minimizing of plaque volumes in the especially human brain.The antibody that comprises any function equivalent antibody or its funtion part according to the present invention makes that plaque volumes has reduced at least 10% in the brain, and especially at least 15%, more particularly at least 20%.

Should be appreciated that, can show the various combination of a kind of, two or more character of above-described specific physique according to antibody of the present invention.

For example, in one embodiment, the invention provides antibody, the monoclonal antibody that particularly comprises any function equivalent antibody or its funtion part, described antibody shows difunctional, because they show as mentioned the gathering rejection characteristic of definition and depolymerization characteristic both, particularly be equipped with the height conformation sensitization.

In another one embodiment of the present invention, the bifunctional antibody that comprises any function equivalent antibody or its funtion part is provided, but the bifunctional monoclonal antibody that especially comprises any function equivalent antibody or its funtion part, described antibody and amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After monomeric peptide is hatched altogether, suppressed the A beta monomers and be gathered into high molecular polymerization fibril or fibril, and in addition, with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched altogether, can preformed polymerization fibril of depolymerization or fibril.

In special embodiment of the present invention, according to bifunctional antibody of the present invention, but hatching altogether respectively with the molar concentration rate up to 1: 100, more particularly with molar concentration rate between 1: 30 and 1: 100 and more particularly carry out with 1: 100 molar concentration rate according to bifunctional monoclonal antibody of the present invention and amyloid monomeric peptide and preformed high molecular polymerization amyloid fibril or fibril especially.

Particularly, according to being incubated in altogether between 28 ℃ to 40 ℃ of antibody of the present invention and amyloid monomeric peptide, between 32 ℃ to 38 ℃, more particularly carried out 24 hours to 60 hours especially 37 ℃ temperature, 30 hours to 50 hours especially, more particularly 48 hours; And and amyloid form being incubated in altogether between 28 ℃ to 40 ℃ of high molecular polymerization amyloid fibril or fibril in advance, especially between 32 ℃ and 38 ℃, more particularly carried out 12 hours to 36 hours 37 ℃ temperature, 18 hours to 30 hours especially, more particularly 24 hours.

In another embodiment of the invention, comprise any function equivalent antibody or its funtion part according to bifunctional antibody of the present invention, particularly according to bifunctional monoclonal antibody of the present invention, can make preformed polymerization fibril or fibril depolymerization at least 10%, especially at least 25%, more particularly at least 35%, even more particularly at least 50%, but 60-70% or more especially at least.

In another special embodiment of the present invention, comprise any function equivalent antibody or its funtion part according to bifunctional antibody of the present invention, particularly according to bifunctional monoclonal antibody of the present invention, make the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42The gathering of monomeric peptide has suppressed at least 50%, especially at least 65%, more particularly at least 75%, even more particularly at least 80%, but 85%-90% especially at least, perhaps more (comparing) with other amyloid peptide monomer (contrast) of the branch of in buffer, hatching.

Particularly, the invention provides the antibody that comprises any function equivalent antibody or its funtion part, bifunctional antibody particularly, but especially monoclonal antibody, particularly bifunctional monoclonal antibody, described antibody causes that the secondary conformation transition mediates and suppresses the amyloid monomeric peptide by and direct combination the special with the A beta, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42The polymerization of monomeric peptide, and/or induce, particularly amyloid beta monomeric peptide by the amyloid monomeric peptide, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42The pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or the dissolving of fibril.

The present invention also provides the antibody that comprises any function equivalent antibody or its funtion part, bifunctional antibody particularly, but especially monoclonal antibody, particularly bifunctional monoclonal antibody, described antibody is by targeting and be attached to amyloid beta epi-position district specifically particularly by amino acid residue aa _n-aa _mEpi-position in the amyloid beta epi-position district of restriction directly and specifically is attached to the amyloid beta fiber, and for example, the fiber such as comprising A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43 comprises A β but especially be attached to _1-42The fiber of monomeric peptide, and/or induce, particularly amyloid beta monomeric peptide by the amyloid monomeric peptide, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42The pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or the dissolving of fibril, wherein said amino acid residue aa _n-aa _mIn n be between 2 and 16, between special 5 and 16, the integer between 10 and 16 between 8 and 16 even more particularly more particularly, and m is between 3 and 25, between special 3 and 23, between special 3 and 20, between special 3 and 17, between special 6 and 17, the integer between 11 and 17 between 9 and 17 even more particularly more particularly, wherein n and m can not be that identical numeral and n must always little than m numerals, poor 〉=2 between n and the m.

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

Can induce the transformation of conformation in the described albumen according to the combination of antibody of the present invention, particularly the βZhe Die conformation is to α spiral and/or random-coil conformation, but particularly to random-coil conformation, even more particularly in the intramolecularly given area, the especially transformation of the random-coil conformation in the Val of a 12 environment.

In another embodiment, the invention provides the antibody that comprises any function equivalent antibody or its funtion part, special monoclonal antibody, described antibody has been incorporated at least a following characteristic of above having mentioned and be selected from into: assemble and suppress, depolymerization, induce conformation transition, identification and directly be attached to epi-position (particularly 14-23 zone especially the discontinuous epi-position of conformation in 14-20 zone), prevention or the formation that slows down the amyloid speckle, reduce soluble A β total amount in the brain, reduce speckle load in the brain, reduce plaque volumes in the brain, keep or improve cognitive memory ability, but two kinds or above combination of especially described characteristic.

In special embodiment, the present invention relates to comprise the antibody of any function equivalent antibody or its funtion part, special monoclonal antibody, described antibody has been incorporated at least 2 kinds, at least 3 kinds especially, more particularly at least 4 kinds even more particularly at least 5,6,7 or 8 kind into, but all characteristics above-mentioned especially.

In special embodiment, the invention provides the antibody that comprises any function equivalent antibody or its funtion part, particularly bifunctional antibody, but especially monoclonal antibody, particularly bifunctional monoclonal antibody, it is to A β _1-42Monomeric peptide shows high specific, but to A β _1-38, A β _1-39, A β _1-40And/or A β _1-41Demonstrating does not have in fact or only very little cross reactivity; The antibody that comprises any function equivalent antibody or its funtion part particularly is provided, but monoclonal antibody especially, it is to amyloid peptide A β _1-42Comparison A β _1-38, A β _1-39, A β _1-40, A β _1-41More responsive height to 100 times, 50 to 100 times especially, more particularly 80 to 100 times, but especially 100 times; And to amyloid peptide A β _1-42Comparison A β _1-38More responsive height to 1000 times, 500 to 1000 times especially, more particularly 800 to 1000 times, but 1000 times especially in particular; And therefore can suppress to form the monomeric peptide of amyloid in vitro and in vivo, but amyloid peptide A β especially _1-42Gathering.

In another embodiment of the invention, the antibody that comprises any function equivalent antibody or its funtion part, particularly bifunctional antibody are provided, but especially monoclonal antibody, particularly bifunctional monoclonal antibody, it has the β to amyloid peptide A _1-42High binding affinity, and can detect the concentration that is low to moderate at least 0.001 μ g but especially between 0.5 μ g and the 0.001 μ g, the more particularly concentration range between 0.1 μ g and 0.001 μ g but the A β of 0.001 μ g concentration especially _1-42Fiber.

In the very special embodiment of the present invention, the antibody that comprises any function equivalent antibody or its funtion part is provided, bifunctional antibody particularly, but monoclonal antibody especially, bifunctional monoclonal antibody particularly, described antibody can the detection fibers amount be low to moderate the A β of 0.001 μ g Cmin _1-42Fiber and the A β that is low to moderate 0.1 μ g Cmin _1-40Fiber and the A β that is low to moderate 1 μ g Cmin _1-38Fiber.

One special aspect, the present invention relates to antibody or its fragment, its identification and in conjunction with the binding site of at least one unique binding site, particularly at least two uniquenesses on the amyloid beta.

In special embodiment, the present invention relates to comprise the antibody of any function equivalent antibody or its funtion part, described antibody recognition and in conjunction with at least one the unique binding site on the amyloid beta, the binding site of at least two uniquenesses particularly, the binding site of wherein said at least one or described at least two uniquenesses comprises at least one amino acid residue and at least two successive amino acid residues of main participation antibodies respectively, wherein in the specific embodiment of the present invention, at least one residue that constitutes first unique combination position is the Leu that is inserted in the following core sequence, and at least two successive amino acid residues that constitute second unique combination position are inserted in the following core sequence-Phe-Phe-:

-Xaa ₁-Xaa ₂-Xaa ₃-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Xaa ₇-

Wherein

Xaa ₁It is the amino acid residue that is selected from His, Asn, Gln, Lys and Arg;

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₃It is the amino acid residue that is selected from Lys, His, Asn, Gln and Arg;

Xaa ₄It is the amino acid residue that is selected from Ala, Val, Leu, nor-leucine, Met, Phe and Ile;

Xaa ₅It is the amino acid residue that is selected from Ala, Val, Leu, Ser and Ile;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

Xaa ₇It is the amino acid residue that is selected from Glu and Asp.

Particularly, antibody or its fragment are provided, its identification and in conjunction with at least one the unique binding site on the amyloid beta, the binding site of at least two uniquenesses particularly, the binding site of wherein said at least one or described at least two uniquenesses comprises at least one amino acid residue and at least two successive amino acid residues of main participation antibodies respectively, wherein in the specific embodiment of the present invention, at least one residue that constitutes first unique combination position is the Leu that is inserted in the following core sequence, and at least two successive amino acid residues that constitute second unique combination position are inserted in the following core sequence-Phe-Phe-:

-Xaa ₁-Xaa ₂-Xaa ₃-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Xaa ₇-

Wherein

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

Xaa ₇It is the amino acid residue that is selected from Glu and Asp.

In another embodiment of the invention, antibody or its fragment are provided, wherein

Xaa ₁Be His or Arg, but His particularly;

Xaa ₂Be Gln or Asn, but Gln particularly;

Xaa ₃Be Lys or Arg, but Lys particularly;

Xaa ₄Be Val or Leu, but Val particularly;

Xaa ₅Be Ala or Val, but Ala particularly;

Xaa ₆Be Glu or Asp, but Glu particularly; And

Xaa ₇Be Asp or Glu, but Asp particularly.

On the other hand, the present invention relates to antibody or its fragment, its identification and in conjunction with at least one the unique binding site on the amyloid beta, the binding site of at least two uniquenesses especially, the binding site of at least three uniquenesses more particularly, the binding site of wherein said one or at least two or at least three uniquenesses each self-contained at least one, the amino acid residue of at least two successive main participation antibodies especially.

Particularly, according to antibody of the present invention or its fragment binding site in conjunction with at least two uniquenesses on the amyloid beta, the amino acid residue of each self-contained at least two successive main participation antibodies of the binding site of wherein said at least two uniquenesses, the binding site of wherein said at least two uniquenesses is closely closely located on antigen each other, do not participated in antibodies or compare the remarkable less amino acid residue of participation antibodies degree separating by at least one, therefore form the discontinuous epi-position of conformation with described at least two successive amino residue.

In another embodiment of the invention, provide according to antibody of the present invention or its fragment, its identification and in conjunction with at least one the unique binding site on the amyloid beta, the binding site of at least two uniquenesses especially, the binding site of at least three uniquenesses more particularly, the binding site of wherein said uniqueness comprises the amino acid residue of at least one and at least two successive main participation antibodies respectively, wherein do not participated in antibodies by at least one or compare with the amino acid residue of main participation antibodies participate in the antibodies degree significantly at least one and at least two the successive aminoacid that separate of less amino acid residue be inserted in respectively in the following core sequence-His-and-Lys-Leu-:

-His-Xaa ₂-Lys-Leu-Xaa ₃-Xaa ₄-Xaa ₅-Xaa ₆-Xaa ₇-Xaa ₈-

Wherein

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₃It is the amino acid residue that is selected from Ala, Val, Leu, nor-leucine, Met, Phe and Ile;

Xaa ₅It is the amino acid residue that is selected from Ala, Val, Leu, nor-leucine, Met, Phe and Ile;

Xaa ₆It is the amino acid residue that is selected from Ala, Val, Leu, Ser and Ile;

Xaa ₇It is the amino acid residue that is selected from Glu and Asp;

Xaa ₈It is the amino acid residue that is selected from Glu and Asp;

And wherein said amino acid residue Xaa ₂, Xaa ₃, Xaa ₆, Xaa ₇, Xaa ₈Do not participate in antibodies or compare with-Lys-Leu-binding site with-His-that to participate in the antibodies degree significantly less.

In another embodiment, antibody or its fragment are provided, its identification and in conjunction with at least one the unique binding site on the amyloid beta, the binding site of at least two uniquenesses especially, the binding site of at least three uniquenesses more particularly, the binding site of wherein said uniqueness comprises the amino acid residue of at least one and at least two successive main participation antibodies respectively, wherein represent at least two successive amino acid residues of first binding site to be inserted in the following core sequence-Phe-Phe-, and at least one amino acid residue is inserted in the following core sequence-His-:

-Xaa ₁-His-Xaa ₃-Xaa ₄-Xaa ₅-Xaa ₆-Phe-Phe-Xaa ₇-Xaa ₈-Xaa ₉-

Wherein,

Xaa ₃It is the amino acid residue that is selected from Asn and Gln;

Xaa ₄It is the amino acid residue that is selected from His, Asn, Gln, Lys and Arg;

Xaa ₆It is the amino acid residue that is selected from Ala, Val, Leu and Ile;

Xaa ₇It is the amino acid residue that is selected from Ala, Val, Leu and Ile;

Xaa ₈It is the amino acid residue that is selected from Glu and Asp;

Xaa ₉It is the amino acid residue that is selected from Glu and Asp;

And wherein said amino acid residue Xaa ₁, Xaa ₃, Xaa ₆, Xaa ₇, Xaa ₈And Xaa ₉Do not participate in antibodies or compare with-Phe-Phe-binding site with-His-that to participate in the antibodies degree significantly less.

In the specific embodiment of the present invention, first kind of main at least two successive amino acid residue that participate in antibodies comprise be inserted in the following core sequence-Lys-and-Leu-, and second kind at least two successive amino acid residues comprise and are inserted in the following core sequence-Phe-Phe-:

-Xaa ₁-Xaa ₂-Lys-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Xaa ₇-

Wherein

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

Xaa ₇It is the amino acid residue that is selected from Glu and Asp;

And wherein said amino acid residue Xaa ₂, Xaa ₃, Xaa ₄, Xaa ₅, Xaa ₆, Xaa ₇Do not participate in antibodies or compare with-Phe-Phe-binding site with Lys-Leu that to participate in the antibodies degree significantly less.

Xaa ₁Be His or Arg, but His particularly;

Xaa ₂Be Gln or Asn, but Gln particularly;

Xaa ₄Be Val or Leu, but Val particularly;

Xaa ₅Be Ala or Val, but Ala particularly;

Xaa ₆Be Glu or Asp, but Glu particularly; And

Xaa ₇Be Asp or Glu, but Asp particularly.

In other embodiments of the present invention, according to antibody of the present invention or its fragment binding site in conjunction with at least three uniquenesses on the amyloid beta, the binding site of wherein said at least three uniquenesses comprises at least one amino acid residue and at least two successive amino acid residues respectively, described residue mainly participates in the combination of antibody, the binding site of wherein said at least three uniquenesses is closely closely located on antigen each other, do not participated in antibodies by at least one respectively or compare the remarkable less amino acid residue of participation antibodies degree separating, therefore form the discontinuous epi-position of conformation with described at least one amino acid residue and described at least two successive amino acid residues.

In the specific embodiment of the present invention, the amino acid residue of first kind at least two successive main participation antibodies comprises and is inserted in the following core sequence-Lys-Leu-, and second kind at least two successive amino acid residues comprise and are inserted in the following core sequence-Phe-Phe-; And the third at least one amino acid residue comprises and is inserted in the following core sequence-His-:

-His-Xaa ₂-Lys-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Xaa ₇

Wherein,

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

Xaa ₇It is the amino acid residue that is selected from Glu and Asp;

And wherein said amino acid residue Xaa ₂, Xaa ₃, Xaa ₄, Xaa ₅, Xaa ₆And Xaa ₇Do not participate in antibodies or with-His-,-Lys-Leu compares with-Phe-Phe-binding site that to participate in the antibodies degree significantly less.

Xaa ₂Be Gln or Asn, but Gln particularly;

Xaa ₄Be Val or Leu, but Val particularly;

Xaa ₅Be Ala or Val, but Ala particularly;

Xaa ₆Be Glu or Asp, but Glu particularly; And

Xaa ₇Be Glu or Asp, but Asp particularly.

In the specific embodiment of the present invention, the amino acid residue of first kind at least two successive main participation antibodies comprises and is inserted in the following core sequence-Lys-Leu-, and second kind at least two successive amino acid residues comprise and are inserted in the following core sequence-Phe-Phe-; And the third at least one amino acid residue comprises and is inserted in the following core sequence-Asp-:

-Xaa ₁-Xaa ₂-Lys-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Asp.-

Wherein,

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

And wherein said amino acid residue Xaa ₂, Xaa ₃, Xaa ₄, Xaa ₅, Xaa ₆And Xaa ₇Do not participate in antibodies or with-Asp-,-Lys-Leu compares with-Phe-Phe-binding site that to participate in the antibodies degree significantly less.

Xaa ₁Be His or Arg, but His particularly;

Xaa ₂Be Gln or Asn, but Gln particularly;

Xaa ₄Be Val or Leu, but Val particularly;

Xaa ₅Be Ala or Val, but Ala particularly; And

Xaa ₆Be Glu or Asp, but Glu particularly;

In other embodiments of the present invention, provide according to antibody of the present invention or its fragment, it is in conjunction with the binding site of 4 uniquenesses on the amyloid beta, the binding site of wherein said 4 uniquenesses comprises an amino acid residue and two successive amino acid residues respectively, described residue mainly participates in antibodies, the binding site of wherein said 4 uniquenesses is closely closely located on antigen each other, do not participated in antibodies by at least one respectively or compare participating in the significantly less amino acid residue of antibodies degree and separating, therefore form the discontinuous epi-position of conformation with the described amino acid residue of the binding site of 4 uniquenesses and described two successive amino acid residues.

Particularly, the amino acid residue of first kind of two successive main participation antibodies is inserted in the following core sequence-Lys-Leu-, and second kind at least two successive amino acid residues are inserted in the following core sequence-Phe-Phe-; First kind of monamino acid residue be inserted in the following core sequence-and His-and second kind of monamino acid residue be inserted in the following core sequence-Asp-:

-His-Xaa ₂-Lys-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Asp-

Wherein,

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

And wherein said amino acid residue Xaa ₂, Xaa ₃, Xaa ₄, Xaa ₅, Xaa ₆, Xaa ₇Do not participate in antibodies or with-His-,-Asp-,-Lys-Leu compares with-Phe-Phe-binding site that to participate in the antibodies degree significantly less.

In the specific embodiment of the present invention, identification as hereinbefore defined and binding site have formed between 12 to 24 amino acids residues of amyloid beta, especially between 14 to the 23 amino acids residues, more particularly between 14 to the 20 amino acids residues zone in conformation on discontinuous epi-position, the identification and the binding site that wherein comprise three uniquenesses of 1 and 2 amino acid residue respectively lay respectively at position 16,17, and position 19 and 20, and position 14, described residue mainly participates in the combination of amyloid beta and the identification of wherein said three uniquenesses and the monamino acid residue that binding site is positioned at position 15 and 18 respectively and separates, described amino acid residue does not participate in the combination of antibody, or littler at least basically degree.

In special embodiment, the bonded continuous amino acid residue of described main participation amyloid beta, especially in the position 16 and 17-Lys-Leu-and in the position 19 and 20-Phe-Phe-, be embedded in 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 other special embodiment, the bonded continuous amino acid residue of described main participation amyloid beta, special in the position 16-Lys-, in the position 17-Leu-and in the position 19 and 20-Phe-Phe-and in the position 14-His-, be embedded in 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 the specific embodiment of the present invention, antibody according to the present invention is to produce by the antigen fragment that resists the binding site that does not comprise described uniqueness.

In the change of epitope regions may be that application by supramolecular constructs causes at least in part, this supramolecular constructs comprise with beta-amyloid peptide, especially with beta-amyloid peptide A β _1-16The corresponding antigenic peptides of aminoacid sequence, this antigenic peptides hydrophilic segment, for example, modify such as Polyethylene Glycol (PEG), wherein said hydrophilic segment passes through at least one, especially one or two aminoacid, for example, can be coupled to the suitable aminoacid or the amino acid analogue of the connecting device of fragments of peptides as hydrophilic segment such as lysine, glutamic acid and cysteine or any other, and covalently be connected to each antigenic end, as described in the immunization method hereinafter.As described herein, when utilizing PEG as hydrophilic segment, free PEG end covalently is connected to PHOSPHATIDYL ETHANOLAMINE or any other is suitable on the chemical compound as anchoring element, for example, and so that the antigen construct is embedded in the bilayer of liposome.

Lipid A also can be promoted the change of epitope regions as the part of immunization protocol.

In special embodiment of the present invention, provide the antibody of the variable region of light chain (LCVR) that comprises SEQ ID NO:7.

In another special embodiment, the present invention relates to the variable region of light chain (LCVR) of SEQ ID NO:7.

In another special embodiment of the present invention, provide the antibody of the variable region of heavy chain (HCVR) that comprises SEQ ID NO:8.

In another special embodiment, the present invention relates to the variable region of heavy chain (HCVR) of SEQ ID NO:8.

In one embodiment, the present invention relates to comprise the two the antibody of variable region of light chain of the variable region of heavy chain of SEQ ID NO:8 and SEQ ID NO:7.

Also be a part of the present invention be to comprise the two antibody of variable region of light chain (LCVR) or variable region of heavy chain (HCVR) or variable region of light chain (LCVR) and variable region of heavy chain (HCVR), variable region of light chain (LCVR) and variable region of heavy chain (HCVR) respectively with SEQ ID NO:7 and 8 in any peptide homology of providing.

Particularly, the present invention relates to according to the present invention and as described above antibody or its fragment, wherein variable region of light chain (LCVR) has the same aminoacid sequence of the sequence that provides with SEQ ID NO:7 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In addition, the present invention relates to according to the present invention and as described above antibody or its fragment, wherein variable region of heavy chain (HCVR) has the same aminoacid sequence of the sequence that provides with SEQ ID NO:8 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

Further, the present invention relates to according to the present invention and as described above antibody or its fragment, wherein variable region of light chain (LCVR) and variable region of heavy chain (HCVR) have the same aminoacid sequence of the sequence that provides with SEQ ID NO:7 and 8 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% together.

In another special embodiment, the present invention relates to have the variable region of light chain of the same aminoacid sequence of the sequence that provides with SEQ ID NO:7 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In other special embodiment, the present invention relates to have the variable region of heavy chain of the same aminoacid sequence of the sequence that provides with SEQ ID NO:8 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In another one embodiment of the present invention, polynucleotide are provided, it comprises the nucleotide sequence that code book is invented antibody as described above.

Particularly, the present invention relates to comprise the polynucleotide of nucleotide sequence of antibody of the present invention of encoding, it comprises:

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

B) since the degeneracy of genetic codon on the codon sequence with the nucleotide sequence of (a) nucleotide sequence uniqueness;

C) complementary series (a) and (b); Or

D) fragment of (a) and (b) or nucleotide sequence (c), it comprises and is selected from following continuous nucleotide section: at least 20 successive nucleotide, at least 25 successive nucleotide, at least 30 successive nucleotide, at least 35 successive nucleotide, at least 40 successive nucleotide, at least 45 successive nucleotide and at least 50 successive nucleotide;

In another embodiment, the present invention relates to comprise the polynucleotide of coding according to the nucleotide sequence of antibody of the present invention, it comprises:

A) nucleotide sequence of the light chain of SEQ ID NO:10 at least;

C) complementary series (a) and (b); Or

In another embodiment again, the present invention relates to comprise the polynucleotide of coding according to the nucleotide sequence of antibody of the present invention, it comprises:

A) nucleotide sequence of the variable region of heavy chain of SEQ ID NO:11 at least;

B) because the degeneracy of the genetic codon nucleotide sequence different on the codon sequence with the nucleotide sequence of (a);

C) complementary series (a) and (b); Or

In another one embodiment again, the present invention relates to comprise the polynucleotide of coding according to the nucleotide sequence of antibody of the present invention, it comprises:

A) nucleotide sequence of the heavy chain of SEQ ID NO:12 or sequence complementary at least with it;

C) complementary series (a) and (b); Or

The present invention has also comprised polynucleotide, and described polynucleotide comprise:

A) nucleotide sequence of the SEQ ID NO:9 of encoded light chain variable region;

C) complementary series (a) and (b); Or

A) nucleotide sequence of the SEQ ID NO:10 of coding light chain;

C) complementary series (a) and (b); Or

A) nucleotide sequence of the SEQ ID NO:11 of encoding heavy chain variable region;

C) complementary series (a) and (b); Or

A) nucleotide sequence of the SEQ ID NO:12 of encoding heavy chain;

C) complementary series (a) and (b); Or

In another embodiment, the present invention relates to any nucleotide sequence with following sequence hybridization:

A. according to the present invention and respectively at SEQ ID NO:9,10,11 and 12 nucleotide sequences that provide;

B. because the degeneracy of the genetic codon nucleotide sequence different on the codon sequence with the nucleotide sequence of (a);

C. (a) and complementary series (b); Or

D. the fragment of (a) and (b) or nucleotide sequence (c), it comprises and is selected from following continuous nucleotide section: at least 20 successive nucleotide, at least 25 successive nucleotide, at least 30 successive nucleotide, at least 35 successive nucleotide, at least 40 successive nucleotide, at least 45 successive nucleotide and at least 50 successive nucleotide.

Particularly, the present invention relates under the conventional hybridization condition, especially under stringent hybridization condition with according to the present invention and respectively SEQ ID NO:9,10,11 and 12 nucleotide sequences that provide, particularly with any nucleotide sequence of its complementary strand hybridization.

In other embodiments, the present invention relates to use 5xSSPE, 1%SDS, 1x step on Hart (Denhardts) solution as reaction solution and/or hybridization temperature between 35 ℃ and 70 ℃, under the preferred 65 ℃ conventional hybridization condition, and according to the present invention with respectively at SEQ ID NO:9,10,11 and 12 nucleotide sequences that provide, particularly any nucleotide sequence of its complementary strand hybridization.After the hybridization, preferably between 35 ℃ and 70 ℃, preferred 65 ℃ temperature, at first with 2xSSC, 1%SDS and wash (about the definition of SSPE, SSC and denhardt solution referring to Sambrook in the above-mentioned quoted passage etc.) with 0.2xSSC subsequently.

Particularly, the present invention relates to as the stringent hybridization condition of top descriptions such as Sambrook, more particularly as above point out under 65 ℃ of stringent hybridization conditions of hybridizing and washing, and according to the present invention and respectively at SEQ ID NO:9,10,11 and 12 nucleotide sequences that provide, particularly any nucleotide sequence of its complementary strand hybridization.

In special embodiment, the invention provides the antibody that comprises any function equivalent antibody or its funtion part, bifunctional antibody particularly, but monoclonal antibody especially, bifunctional monoclonal antibody particularly, described antibody have by the features characteristic that is selected from the antibody that produces with the hybridoma cell line of FP12H3, the FP 12H3-C2 of DSM ACC2752, DSM ACC 2750 and DSM ACC2751 preservation and FP 12H3-G2 respectively respectively at December in 2005 1 day and December in 2005 9 days.

In particular, the present invention relates to the antibody that produced with the hybridoma cell line FP 12H3 of DSM ACC2752 preservation in 9th by respectively at December in 2005 1 day and December in 2005, described antibody comprises any function equivalent antibody or its funtion part.

In particular, the present invention relates to the monoclonal antibody that produced with the hybridoma cell line FP 12H3-C2 of DSM ACC2750 preservation in 9th by respectively at December in 2005 1 day and December in 2005, described monoclonal antibody comprises any function equivalent antibody or its funtion part.

In particular, the present invention relates to the monoclonal antibody that produced with the hybridoma cell line FP 12H3-G2 of DSM ACC2751 preservation in 9th by respectively at December in 2005 1 day and December in 2005, described monoclonal antibody comprises any function equivalent antibody or its funtion part.

In another special embodiment, the invention provides the antibody that comprises any function equivalent antibody or its funtion part, bifunctional antibody particularly, but monoclonal antibody especially, bifunctional monoclonal antibody particularly, described antibody have the features characteristic of the antibody that is produced with the hybridoma cell line ET 7E3 of DSM ACC2755 preservation by December in 2005 8 days.

In particular, the present invention relates to by December in 2005 monoclonal antibody with the hybridoma cell line ET 7E3 generation of DSM ACC2755 preservation on the 8th, described monoclonal antibody comprises any function equivalent antibody or its funtion part.

In another special embodiment, the invention provides the antibody that comprises any function equivalent antibody or its funtion part, bifunctional antibody particularly, but monoclonal antibody especially, bifunctional monoclonal antibody particularly, described antibody have the features characteristic of the antibody that is produced with the hybridoma cell line EJ 7H3 of DSM ACC2756 preservation by December in 2005 8 days.

In particular, the present invention relates to by December in 2005 monoclonal antibody with the hybridoma cell line EJ 7H3 generation of DSM ACC2756 preservation on the 8th, and described monoclonal antibody comprises any function equivalent antibody or its funtion part.

Another object of the present invention provides and comprises according to the present invention and the method and composition of antibody described above, for example by using according to the present invention and antibody passive immunity human or animal described above, be used to prevent and/or treat and/or alleviate by amyloid or amyloid sample albumen effect that cause or relative disease and disease.Described disease and disease comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, include but not limited to sacred disease such as Alzheimer (AD), comprise that with cognitive memory ability forfeiture be the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.

Another purpose of the present invention provide application according to monoclonal antibody of the present invention and/or its funtion part and comprise according to the present invention and the compositions of the antibody described hereinbefore is used to diagnose or therapeutic intervention by amyloid or amyloid sample albumen method that cause or relative disease and disease.Described disease and disease comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, include but not limited to sacred disease such as Alzheimer (AD), comprise that with cognitive memory ability forfeiture be the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.

Particularly, an object of the present invention is to provide use according to monoclonal antibody of the present invention and/or its funtion part with comprise according to the present invention and antibody described above, bispecific or two effectiveness antibody but the compositions of bispecific or the two effectiveness monoclonal antibodies method that reduces and prevent the generation of sacred disease (including but not limited to Alzheimer) especially especially.

Compositions according to the present invention comprises according to the present invention and the antibody of any function equivalent antibody or its funtion part, particularly bispecific or the two effectiveness antibody of comprising described above, in particular with the treatment effective dose, more particularly according to the present invention with the monoclonal antibody of any function equivalent antibody or its funtion part, especially bispecific or the two effectiveness monoclonal antibody of comprising described above, in particular with the treatment effective dose; And optional, pharmaceutically suitable carrier and/or diluent and/or excipient.

Especially, compositions according to the present invention comprises the antibody that comprises any function equivalent antibody or its funtion part, monoclonal antibody preferably, and pharmaceutically suitable carrier and/or the diluent and/or the excipient of optional choosing, this antibody can suppress the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42Monomeric peptide is gathered into high molecular polymerization amyloid fibril or fibril.

In other embodiments, the invention provides compositions, it comprises the antibody that comprises any function equivalent antibody or its funtion part, monoclonal antibody preferably, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody with amyloid monomeric peptide, particularly amyloid beta monomeric peptide, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After monomeric peptide is hatched altogether, especially with molar concentration rate, more particularly with the molar concentration rate between 1: 30 to 1: 100 but after especially hatching altogether, suppressed the A beta monomers and be gathered into high molecular polymerization fibril or fibril with 1: 100 molar concentration rate up to 1: 100.Particularly, when comparing with other amyloid peptide monomer (contrast) of the branch of in buffer, hatching, described inhibitory action reaches at least 50%, reach at least 65% especially, more particularly reach at least 75%, even more particularly reach at least 80%, but especially reach 85%-90% at least, perhaps more.

Particularly, carried out 48 hours according to 37 ℃ the temperature of being incubated in altogether of antibody of the present invention and amyloid monomeric peptide.

Gathering inhibition potentiality according to antibody of the present invention can be passed through the density gradient ultracentrifugation method, then measure by the SDS-PAGE sedimentation analysis on preformed gradient and/or by thio-flavin T (Th-T) fluorometric investigation method.

The present invention also provides compositions, it comprises antibody and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody can depolymerization by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42High molecular polymerization amyloid fibril or fibril that the gathering of monomeric peptide forms.

Polymerization fibril or fibril by the depolymerization amyloid produces can prevent or slow down the formation of amyloid speckle according to antibody of the present invention, thereby cause the alleviation of disease related symptom and delay or reverse its progress.

In another embodiment of the invention, compositions is provided, it comprises the antibody that comprises any function equivalent antibody or its funtion part, monoclonal antibody especially, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody with by the amyloid monomeric peptide, preferred amyloid beta monomeric peptide, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42Pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are with between 1: 30 to 1: 100, after 1: 100 molar concentration rate is hatched altogether especially, especially after 37 ℃ temperature is hatched 24 hours altogether, can make preformed polymerization fibril or fibril depolymerization at least 35%, especially at least 40%, more particularly at least 50% even more particularly at least 60% but especially at least 70% or higher.

Depolymerization potentiality according to antibody of the present invention can be passed through the density gradient ultracentrifugation method, then measure by the SDS-PAGE sedimentation analysis on preformed gradient and/or by thio-flavin T (Th-T) fluorometric investigation method.

The present invention also provides compositions, its comprise effective dose especially, more particularly treat antibody or its funtion part of effective dose, described antibody is conformation sensitization.

In other embodiments of the present invention, compositions is provided, it comprises the antibody that comprises any function equivalent antibody or its funtion part, monoclonal antibody preferably, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched altogether, especially after 37 ℃ temperature is hatched 24 hours altogether, can cause that the βZhe Die conformation is to α spiral and/or random-coil conformation, but particularly to random-coil conformation, even more particularly in the intramolecularly given area, especially the transformation of the random-coil conformation in the Val of a 12 environment, this has caused with the βZhe Die conformation is the increase of random-coil conformation of cost and the improved dissolution that forms high molecular polymerization amyloid fibril or fibril in advance.Particularly, when comparing with other pre-formation amyloid polymerization fibril of the branch of in buffer, hatching or fibril (contrast), the minimizing of βZhe Die conformation reaches at least 30%, reaches at least 35% especially, and more particularly reaches at least 40% and more.

The potentiality of antibody induction secondary conformation transition are passed through solid-state 13C NMR spectroscopy determining, but particularly, by measuring A β _1-42The integrated intensity of Va1 12 C β conformations in the peptide and measuring.

The present invention also provides compositions, it comprises treats comprising of effective dose of any function equivalent antibody or antibody, the particularly monoclonal antibody of its funtion part especially, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody is bifunctional, because it show as hereinbefore defined the gathering rejection characteristic and the depolymerization characteristic both, preferably be equipped with the height conformation sensitization.

In another embodiment of the invention, compositions is provided, it comprises the bifunctional antibody that comprises any function equivalent antibody or its funtion part but bifunctional monoclonal antibody especially, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody and amyloid monomeric peptide, particularly amyloid beta monomeric peptide, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After monomeric peptide is hatched altogether, suppressed the A beta monomers and be gathered into high molecular polymerization fibril, and in addition, with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched altogether, can preformed polymerization fibril of depolymerization or fibril.

In the specific embodiment of the present invention, according to bifunctional antibody of the present invention, but especially according to bifunctional monoclonal antibody of the present invention and amyloid monomeric peptide with form the hatching altogether respectively with the molar concentration rate up to 1: 100, especially with the molar concentration rate between 1: 30 to 1: 100 and more particularly carry out with 1: 100 molar concentration rate of high molecular polymerization amyloid fibril or fibril in advance.

In the other special embodiment of the present invention, carried out respectively 48 hours and 24 hours with amyloid monomeric peptide and pre-37 ℃ the temperature of being incubated in altogether that forms high molecular polymerization amyloid fibril or fibril.

In another specific embodiment of the present invention, compositions is provided, it comprises according to bifunctional antibody of the present invention but especially according to bifunctional monoclonal antibody of the present invention, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody can make preformed polymerization fibril or fibril depolymerization at least 10%, and especially at least 25%, more particularly at least 35%, even more particularly at least 50%, but 60-70% or more especially at least.

In another embodiment of the invention, compositions is provided, it comprises according to bifunctional antibody of the present invention but especially according to bifunctional monoclonal antibody of the present invention, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, compare with other amyloid peptide monomer (contrast) of the branch of in buffer, hatching, described antibody makes the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42The gathering of monomeric peptide has suppressed at least 50%, and especially at least 65%, more particularly at least 75%, even more particularly at least 80%, but 85%-90% especially at least is perhaps more.

Particularly, the invention provides compositions, it comprises antibody, the particularly monoclonal antibody that comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody causes that by and direct combination the special with the A beta secondary conformation transition mediates inhibition amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42The polymerization of monomeric peptide, and/or induce, particularly amyloid beta monomeric peptide by the amyloid monomeric peptide, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42The pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or the dissolving of fibril.

The present invention also provides compositions, it comprises antibody, the particularly monoclonal antibody that comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody is by targeting and be attached to amyloid beta epi-position district specifically particularly by amino acid residue aa _n-aa _mEpi-position in the amyloid beta epi-position district of restriction directly and specifically is attached to the amyloid beta fiber, and for example, the fiber such as comprising A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43 comprises A β but especially be attached to _1-42The fiber of monomeric peptide, and/or induce, particularly amyloid beta monomeric peptide by the amyloid monomeric peptide, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42The pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or the dissolving of fibril, wherein said amino acid residue aa _n-aa _mIn n be between 2 and 15, between special 5 and 15, the integer between 10 and 15 between 8 and 15 even more particularly more particularly, and m is between 3 and 17, between special 6 and 17, the integer between 11 and 17 between 9 and 17 even more particularly more particularly, wherein n and m can not be that identical numeral and n must always little than m numerals, poor 〉=2 between n and the m.

Can induce the transformation of conformation in the described albumen according to the combination of antibody of the present invention, particularly the βZhe Die conformation is to α spiral and/or random-coil conformation, but in particular to random-coil conformation, even more particularly in the intramolecularly given area, the especially transformation of the random-coil conformation in the Val of a 12 environment.

In other embodiments, the invention provides compositions, it comprises antibody, the particularly monoclonal antibody that comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody has been incorporated at least a characteristic of above being mentioned into, promptly suppress polymerization, depolymerization, induce conformation transition, identification and directly be attached to 4-16 and/or 14 to 23, but particularly 14 to 20 epitope regions but two kinds or above combination of especially described characteristic.

In special embodiment, the invention provides compositions, it comprises antibody, the particularly bifunctional antibody that comprises any function equivalent antibody or its funtion part, but especially monoclonal antibody, particularly bifunctional monoclonal antibody, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody is to A β _1-42Monomeric peptide demonstrates high specific, but to A β _1-38, A β _1-39, A β _1-40And/or A β _1-41Monomeric peptide demonstrates not to be had in fact or only very little cross reactivity, particularly comprises the antibody that comprises any function equivalent antibody or its funtion part but monoclonal antibody especially, and described antibody is to amyloid peptide A β _1-42Comparison A β _1-38, A β _1-39, A β _1-40, A β _1-41More responsive to 100 times, 50 to 100 times especially, more particularly 80 to 100 times, but especially 100 times; And to amyloid peptide A β _1-42Comparison A β _1-38More responsive to 1000 times, 500 to 1000 times especially, more particularly 800 to 1000 times, but especially 1000 times; And therefore can suppress to form the monomeric peptide of amyloid in vitro and in vivo, but amyloid peptide A β especially _1-42Gathering.

In another special embodiment of the present invention, compositions is provided, it comprises antibody, the particularly bifunctional antibody that comprises any function equivalent antibody or its funtion part, but especially monoclonal antibody, particularly bifunctional monoclonal antibody, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody is to amyloid peptide A β _1-42Have highly, and can detect the concentration that is low to moderate at least 0.001 μ g but especially at the concentration range between 0.5 μ g to the 0.001 μ g, the A β of 0.001 μ g concentration more particularly between 0.1 μ g to 0.001 μ g but especially in conjunction with sensitivity _1-42Fiber.

In very special embodiment of the present invention, compositions is provided, it comprises antibody, the particularly bifunctional antibody that comprises any function equivalent antibody or its funtion part, but especially monoclonal antibody, particularly bifunctional monoclonal antibody, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody can the detection fibers amount be low to moderate the A β of 0.001 μ g Cmin _1-42Fiber and fibre weight are low to moderate the A β of 0.1 μ g Cmin _1-40Fiber and the A β that is low to moderate 1 μ g Cmin _1-38Fiber.

In special embodiment, the present invention relates to compositions, it comprises the monoclonal antibody that comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described antibody has by being selected from respectively at December in 2005 1 day and December in 2005 9 days respectively with the features characteristic of the antibody of the hybridoma cell line generation of FP12H3, the FP 12H3-C2 of DSM ACC2752, DSM ACC 2750 and DSM ACC2751 preservation and FP 12H3-G2.

In particular, the present invention relates to compositions, it comprises the monoclonal antibody that produced with the hybridoma cell line FP 12H3 of DSM ACC2752 preservation in 9th by respectively at December in 2005 1 day and December in 2005, described monoclonal antibody comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient.

In particular, the present invention relates to compositions, it comprises the monoclonal antibody that produced with the hybridoma cell line FP 12H3-C2 of DSM ACC2750 preservation in 9th by respectively at December in 2005 1 day and December in 2005, described monoclonal antibody comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient.

The invention still further relates to compositions, it comprises the monoclonal antibody that produced with the hybridoma cell line FP 12H3-G2 of DSM ACC2751 preservation in 9th by respectively at December in 2005 1 day and December in 2005, described monoclonal antibody comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient.

In another embodiment, the present invention relates to compositions, it comprises the features characteristic with the antibody that is produced with the hybridoma cell line ET 7E3 of DSM ACC2755 preservation by December in 2005 8 days, the monoclonal antibody that comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient.

The invention still further relates to compositions, it comprises by December in 2005 monoclonal antibody with the hybridoma cell line ET 7E3 generation of DSM ACC2755 preservation on the 8th, described monoclonal antibody comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient.

In another special embodiment kind, the present invention relates to compositions, it comprises the features characteristic with the antibody that is produced with the hybridoma cell line EJ 7H3 of DSM ACC2756 preservation by December in 2005 8 days, the monoclonal antibody that comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient.

The invention still further relates to compositions, it comprises by December in 2005 monoclonal antibody with the hybridoma cell line EJ 7H3 generation of DSM ACC2756 preservation on the 8th, described monoclonal antibody comprises any function equivalent antibody or its funtion part, and optional pharmaceutically suitable carrier and/or diluent and/or excipient.

The antibody of any function equivalent antibody or its funtion part, the particularly monoclonal of comprising according to the present invention can be co-administered with other bioactive substance and method that is used for disease treatment.Other bioactive substance can be that the form with mixture becomes a part that comprises according to the same combination of antibody of the present invention, wherein antibody and other bioactive substance can be mixed in identical acceptable solvent and/or the carrier or mix mutually with it, or a part that can be used as independent compositions provides separately, and it can be individually or gives together with the form of kits of parts.

According to of the present invention comprise the antibody of any function equivalent antibody or its funtion part, particularly monoclonal antibody can with one or more other bioactive substances side by side, grant off and on or one after the other.For example, the antibody of any function equivalent antibody or its funtion part that comprises according to the present invention can be granted simultaneously with first kind of extra bioactive substance, or after antibody is granted or before grant in succession.If the application program of having selected more than one extra bioactive substances to grant with at least a antibody according to the present invention, chemical compound or material can partly be granted simultaneously, partly grant in succession with different compound modes.

Another object of the present invention provides and comprises at least a antibody of the present invention, and the mixtures of antibodies of one or more optional other bioactive substances, and relate to and use to divide other antibody, or comprise that its mixture of the compositions that comprises described antibody or mixtures of antibodies are used to the treatment of preventing and/or treating property and/or alleviate method by amyloid or amyloid sample albumen effect that cause or relative disease and disease, described disease and disease comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, include but not limited to sacred disease such as Alzheimer (AD), comprise that with cognitive memory ability forfeiture be the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.

Mixture of the present invention is except comprising antibody of the present invention, can also comprise bioactive substance, for example, all being used for the treatment of as is known by amyloid or amyloid sample albumen chemical compound that cause or relative disease and disease, described disease and disease comprise amyloidosis, this be one group with amyloid or relevant disease and the disease of amyloid sample albumen such as a that relates to Alzheimer.

In another embodiment of the invention, other bioactive substance or chemical compound also can be therapeutic agents that can be used for being caused by amyloid or amyloid sample albumen or relative disease and disease (comprising the amyloidosis that is caused by amyloid-beta) treatment, or can be used for the therapeutic agent of other neurological disease drug treatment.

Other bioactive substance or chemical compound can by with the same or analogous mechanism of antibody according to the present invention, or by the incoherent mechanism of action, or by multiple relevant and/or incoherent its biology effect of mechanism performance.

Usually, other bioactive compound can comprise neutron transmission reinforcing agent, psychotherapeutic drugs, acetylcholinesteraseinhibitors inhibitors, calcium channel blocker, biogenic amine, benzodiazepine

Class tranquilizer, acetylcholine are synthetic, storage or release enhancers, acetylcholine postsynaptic receptor agonist, monoamine oxidase, MAO-A or-B inhibitor, N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal anti-inflammatory drug, antioxidant and 5-hydroxy tryptamine can receptor antagonists.

Particularly, can comprise according to antibody of the present invention according to mixture of the present invention, at least a other bioactive compound, and optional pharmaceutically suitable carrier and/or diluent and/or excipient, described other bioactive compound is selected from the chemical compound of anti-oxidation stress, the chemical compound of anti-apoptotic, metal-chelator, DNA repair inhibitors such as pirenzepine and metabolite, 3-amino-1-propane sulfonic acid (3APS), 1,3-third disulfonic acid (1,3PDS), the secretase activator, β-and inhibitors of gamma-secretase, tau protein, neurotransmitter, the βZhe Die disrupting agent, the antiinflammatory molecule, or cholinesterase inhibitor (ChEI) is (such as tacrine, Rivastigmine, donepezil, and/or galantamine) and other medicines and supplementary.

In other embodiments, according to chemical compound of the present invention can comprise nicotinic acid or Memantine hydrochloride (memantine), according to antibody of the present invention and optional pharmaceutically suitable carrier and/or diluent and/or excipient.

In another embodiment of the invention, mixture is provided, it comprises to be used for the treatment of and comprises hallucination, paranoea, thought disturbance (shows as significant irrationality, thinking is beside the point, never talk about anything serious) and eccentric or disorderly behavior and anhedonia, the emotion dullness, cold, " atypical antipsychotic drug " with the unsocial positive and negative psychotic symptoms, for example, such as clozapine, Ziprasidone, Risperidone, Aripiprazole or olanzapine, and according to antibody of the present invention, and optional pharmaceutically suitable carrier and/or diluent and/or excipient.

In special embodiment of the present invention, according to the present invention and compositions described above and mixture to comprise be respectively the antibody and the bioactive substance of treatment effective dose.

Be fit to according to antibody combined other chemical compound that is used for mixture of the present invention, for example, be described in WO 2004/058258 (especially referring to 16 and 17 pages), comprise curative drug target (36-39 page or leaf), alkanesulfonic acid and alkanol sulphuric acid (39-51 page or leaf), cholinesterase inhibitor (51-56 page or leaf), nmda receptor antagonist (56-58 page or leaf), estrogen (58-59 page or leaf), NSAID (non-steroidal anti-inflammatory drug) (60-61 page or leaf), antioxidant (61-62 page or leaf), peroxisome proliferator activated receptors (PPAR) agonist (63-67 page or leaf), depressant prescription under the cholesterol (68-75 page or leaf), amyloid inhibitors (77-78 page or leaf), metal-chelator (78-79 page or leaf), neuroleptics and antidepressant (80-82 page or leaf), nutritional supplement (83-89 page or leaf), increase the chemical compound (referring to the 89-93 page or leaf) and the prodrug (93 and 94 pages) of bioactive substance utilizability in the brain, the document is incorporated herein by reference herein.

Also provide preparation according to the method that comprises the antibody of any function equivalent antibody or its funtion part of the present invention, particularly prepare monoclonal antibody method, described method comprises the antibody that produces anti-supramolecular constructs but monoclonal antibody particularly, described supramolecular constructs comprises and beta-amyloid peptide, particularly beta-amyloid peptide A β _1-15, A β _1-16With A β _{1-16 (Δ 14)}The corresponding antigenic peptide of aminoacid sequence, this antigenic peptide with hydrophobic part (for example, such as Palmic acid) or hydrophilic segment is (for example, such as Polyethylene Glycol (PEG)) or both combinations modify, wherein said hydrophobic and hydrophilic segment is respectively by at least one, one or two aminoacid particularly, for example, maybe can be such as lysine as any other suitable aminoacid or amino acid analogue of hydrophilic and hydrophobic part being coupled to the connecting device of fragments of peptides, for example glutamic acid or cysteine, and covalently be connected to the end of each antigenic peptide.

Also be the invention a part be according to the present invention and monoclonal antibody described above and/or its function fragment, and/or comprise the pharmaceutical composition of described antibody or mixture the preparation treatment or palliate a disease and the medicine of the effect of disease in application, wherein said disease is caused by amyloid or amyloid sample albumen with disease or is relevant with it, comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, such as following disease, include but not limited to sacred disease such as Alzheimer (AD), and be the disease or the disease of feature with the forfeiture of cognitive memory ability, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.

In another embodiment of the invention, provide and used according to antibody of the present invention and/or its funtion part, but especially monoclonal antibody and/or its funtion part or function equivalent Antibody Preparation are used for the treatment of or palliate a disease and the method for the pharmaceutical composition of the effect of disease, this method comprises with pharmaceutically acceptable form preparation antibody, wherein said disease is caused by amyloid or amyloid sample albumen with disease or is relevant with it, comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, such as following disease, include but not limited to sacred disease such as Alzheimer (AD), and be the disease or the disease of feature with the forfeiture of cognitive memory ability, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.

According to antibody of the present invention and/or its funtion part, but especially monoclonal antibody and/or its funtion part or function equivalent antibody and comprise the compositions of described antibody and mixture can be used for preparation and is used for prevention, treatment or palliate a disease and the medicine of the effect of disease, wherein said disease is caused by amyloid or amyloid sample albumen with disease or is relevant with it, comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, include but not limited to sacred disease such as Alzheimer (AD), comprise that with cognitive memory ability forfeiture be the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.

In other embodiments of the present invention, provide to be used for reducing suffering from and caused the disease that brain speckle load increases or the animal of disease, mammal particularly, but the method for speckle load in the especially human brain, this method comprise to the animal of this treatment of needs, particularly mammal, more especially human grant the treatment effective dose according to the present invention and antibody described above and/or its funtion part but especially monoclonal antibody and/or its funtion part or function equivalent antibody, or comprise the compositions or the mixture of described antibody.

Particularly, speckle load has reduced at least 20%, and especially at least 25%, more particularly at least 30%, even more particularly more than 30%.

In other embodiments of the present invention, provide to be used for reducing suffering from and caused the disease that brain speckle load increases or the animal of disease, mammal particularly, but the method for plaque volumes in the especially human brain, this method comprise to the animal of this treatment of needs, particularly mammal, more especially human grant the treatment effective dose according to the present invention and antibody described above and/or its funtion part but especially monoclonal antibody and/or its funtion part or function equivalent antibody, or comprise the compositions or the mixture of described antibody.

Particularly, the plaque volumes in the brain has reduced at least 10%, and especially at least 15%, more particularly more than 15%.

Go back in another the embodiment in the present invention, provide to be used for reducing suffering from and caused the disease that brain solubilized A β concentration increases or the animal of disease, mammal particularly, but the method for solubilized A β total amount in the especially human brain, this method comprise to the animal of this treatment of needs, particularly mammal, more especially human grant the treatment effective dose according to the present invention and antibody described above and/or its funtion part but especially monoclonal antibody and/or its funtion part or function equivalent antibody, or comprise the compositions or the mixture of described antibody.

An object of the present invention is to provide by granting according to antibody of the present invention for animal or human's class of suffering from following symptom, but monoclonal antibody or comprise the compositions or the mixture of this antibody especially, thereby prevention, treatment or palliate a disease and the method for the effect of disease, wherein said disease is caused by amyloid or amyloid sample albumen with disease or is relevant with it, comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, include but not limited to sacred disease such as Alzheimer (AD), comprise that with cognitive memory ability forfeiture be the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula; Described method comprise to the animal of this treatment of needs, particularly mammal, more particularly human grant the treatment effective dose according to the present invention and antibody described above and/or its funtion part but especially monoclonal antibody and/or its funtion part or function equivalent antibody, or comprise the compositions or the mixture of described antibody.

In the specific embodiment of the present invention, provide and be used for keeping or increasing the animal that suffers from dysmnesia (memory impairment), the method of mammal or human cognitive memory ability particularly, need the animal of this treatment, particularly mammal or the mankind to grant but monoclonal antibody particularly by giving according to antibody of the present invention, or according to the present invention with compositions or the mixture that comprises this antibody described above.

In another embodiment of the invention, provide and used according to antibody of the present invention and/or its funtion part, but especially monoclonal antibody and/or its funtion part or function equivalent Antibody Preparation are used for prevention, treatment or palliate a disease and the method for the pharmaceutical composition of the effect of disease, wherein said disease is caused by amyloid or amyloid sample albumen with disease or is relevant with it, comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, such as following disease, include but not limited to sacred disease such as Alzheimer (AD), comprise that with cognitive memory ability forfeiture be the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.

In the specific embodiment of the present invention, provide and used according to antibody of the present invention and/or its funtion part but the method for monoclonal antibody and/or its funtion part or function equivalent Antibody Preparation pharmaceutical composition especially, by giving animal, particularly mammal or humanly granting according to the present invention and antibody described above, particularly monoclonal antibody or comprise the compositions of this antibody or mixture keeps or increases the cognitive memory ability of suffering from amnemonic animal, particularly mammal or the mankind.

After the detailed description and claims of the embodiment of having read following discloses, these and other objects of the present invention, feature and advantage can become apparent.

The term that uses in the literary composition " polypeptide ", " peptide " and " protein " can exchange, and are defined as the biomolecule of being made up of the aminoacid that connects by peptide bond.

Unless context is improper, otherwise the term that uses in the literary composition " ", " being somebody's turn to do " are defined as " one or more " and comprise plural number.

The term that uses in the literary composition " detection " is defined as with known technology (such as immunochemistry or Histological method) detection of biological molecule, and the existence or the concentration that refer to qualitative under study for action or detection by quantitative biomolecule.

" amyloid-beta, A β or amyloid beta " is the term of this area approval, and refers to amyloid-beta albumen and peptide, amyloid-beta precursor protein (APP), with and trim, fragment or any functional equivalent.Particularly, employed amyloid-beta is meant any fragment by the proteolytic cleavage generation of APP in the literary composition, but especially those participate in the of science or associated fragments of amyloidosis, include but not limited to A β _1-38, A β _1-39, A β _1-40, A β _1-41, A β _1-42With A β _1-43

The structure of the top amyloid-beta peptide of mentioning and sequence are well known to a person skilled in the art, and produce described peptide or from brain and other tissue their method of extraction be described in, for example Glenner and Wong, Biochem Biophys Res Comm129,885-890 (1984).In addition, amyloid-beta peptide also in a variety of forms commerce can obtain.

" A β fibril " or " A β fibril " or " amyloid fibril " are the polymerized forms that forms the monomeric protein of the single or bunched fiber with constant fibre diameter, it is insoluble to the aqueous medium and comprises a large amount of intersection beta structures in their core, great majority have the β thigh 1.2,3 perpendicular to the fibril axle.

" monomer A β " or " A beta monomers " is dissolving and do not assemble the amyloid-beta of complex fully in aqueous medium.

" polymeric soluble starch shape albumen " and " oligomerization A β " and " A beta oligomers " refer to amyloid peptide or amyloid sample peptide or amyloid peptide that modify or truncate or form a plurality of aggregated monomers of other derivant of the amyloid peptide of oligomer or paradigmatic structure, and it all is soluble in external aqueous medium neutralizes mammal in vivo or people's health, particularly brain; But be meant a plurality of aggregated monomers of amyloid-beta (A β) or amyloid-beta (A β) peptide or derivatives thereof that modify or truncate especially, its respectively at mammal or people's health, more especially be soluble in the brain.

" isolating " meaning is meant that biomolecule is not to the naturally occurring with it component of small part.

The term of using in the literary composition " antibody " is the term of this area approval, it refers to be attached to the molecule of known antigens or the active fragment of molecule, particularly the immunologic competence part of immunoglobulin molecules and immunoglobulin molecules promptly comprises the molecule of immune specific bond to antigenic binding site.According to immunoglobulin of the present invention can be any type (IgG, IgM, IgD, IgE, IgA and IgY) or the immunoglobulin molecules of class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

" antibody " is intended to comprise monoclonal antibody, polyclonal antibody, chimeric antibody, single-chain antibody, bi-specific antibody or two effectiveness antibody, analog antibody, people's antibody and humanized antibody within the scope of the invention, with and active fragment.Example in conjunction with the active fragment of the molecule of known antigens comprises Fab and F (ab ') ₂Fragment comprises product and any antibody above-mentioned and the segmental epi-position binding fragment in Fab immunoglobulin expression library.

These active fragments can be derived from antibody of the present invention by multiple technologies.For example, can use enzyme,, pass through the HPLC gel filtration then such as the monoclonal antibody of pepsin cutting purification.Can and concentrate the suitable segmental fraction of Fab that contains by collection such as membrane filter method then.Isolating other the conventional description with technology of antigen active fragments can referring to, Khaw for example, B.A. etc., J.Nucl.Med.23:1011-1019 (1982); Rousseaux etc., Methods Enzymology, 121:663-69, Academic Press, 1986.

" humanized antibody " refers to a class engineered antibody, and its CDRs is derived from inhuman donor immunity globulin, and the immunoglobulin derivative moiety of other of molecule is planted the human normal immunoglobulin derived from one (or many).In addition, can change framework supports residue to keep binding affinity.The method that obtains " humanized antibody " is to well known to a person skilled in the art (for example, referring to, Queen etc., Proc.Natl AcadSci USA, 86:10029-10032 (1989), Hodgson etc., Bio/Technoloy, 9:421 (1991)).

" humanized antibody " also can obtain by new gene engineering method, and it can be larger animal, such as the class people's polyclonal antibody that produces affinity maturation in the rabbit ( Http:// www.rctech.com/bioventures/therapeutic.php).

Term " monoclonal antibody " also is art-recognized, and refers in laboratory mass-produced and only discern an antigenic antibody from single clone.Monoclonal antibody generally cell such as the cancerous cell (being sometimes referred to as " immortality " cell) of the B cell by the generation antibody that will normally die young and growth fast merges and produces.Hybrid cell that is produced or hybridoma fast breeding, the clone of generation production lot of antibodies.

For purpose of the present invention, should understand " monoclonal antibody " and also comprise the antibody of cloning production by the mother who does not also reach complete monoclonicity.

" function equivalent antibody " is meant within the scope of the invention and the antibody of the total in fact at least a main functional characteristic of antibody above-mentioned and described here that described functional characteristic comprises: specific bond amyloid beta, particularly A β _1-42Albumen, and A β more particularly _1-42Proteic 4-16 epitope regions, external immunoreactivity, suppress A β _1-42Monomer aggregation becomes high molecular polymerization fibril and/or the preformed A β of depolymerization _1-42The polymerization fibril, and/or the destruction characteristic of βZhe Die, with when prophylactically or therapeutic palliate a disease when granting and the effect of disease, wherein said disease is caused by amyloid or amyloid sample albumen with disease or is relevant with it, comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, include but not limited to sacred disease such as Alzheimer (AD), comprise that with cognitive memory ability forfeiture be the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.Antibody can be any class, such as IgG, IgM or IgA etc., or any subclass, above mentioning or other subclass well known in the art such as IgG1, IgG2a etc. and other, but IgG2 class particularly.In addition, antibody can be produced by any method, such as phage display, or in any biology or cell line production, comprises that antibacterial, insecticide, mammal or other production have the cell type or the cell line of the antibody (such as humanized antibody) of desired characteristic.Antibody also can form by Fab part and the Fc district of combination from different plant species.

Term " bispecific " or " difunctional " and " two effectiveness " use of synonym in the scope of this application are to describe the active antibody of depolymerization that shows simultaneously amyloid or the fibroplastic rejection characteristic of amyloid sample and amyloid or amyloid sample fiber.

Term " antigen " is meant can be organism, particularly animal, more particularly comprise entity or its fragment of induction of immunity reaction in the human mammal.This term comprises the zone of immunogen and responsible antigenicity or antigenic determinant.

Refer to partially or completely dissolving in aqueous solution as the term " soluble " that uses in this article.

Also the term " immunogenic " that uses has in this article guided or has strengthened the generation of antibody, T cell and other reactive immunocyte at immunogenic substance, and promotes immunoreactive material in the mankind or the animal.

When individuality produces the enough antibody that resists the immunogenic composition of the present invention of being granted, T cell and other reactive immunocyte, immunoreation takes place to alleviate or to alleviate the disease that will treat.

" polymeric soluble starch shape albumen " finger-type becomes a plurality of aggregated monomers of other derivant of the amyloid peptide of oligomer or paradigmatic structure or amyloid sample peptide or amyloid peptide that modify or truncate or amyloid peptide, its in mammal or human body, particularly be soluble in the brain, be meant especially amyloid-beta (A β) peptide or modify or amyloid-beta (A β) peptide of truncate, a plurality of aggregated monomers of or derivatives thereof, it is soluble in the brain particularly in mammal or human body.

Term " hybridoma " is this area approval, and is interpreted as that by those of ordinary skills finger merges the cell that produces by the cell that will produce antibody and immortality cell (for example multiple myeloma cells).This hybrid cell can be produced successive antibody supply.Referring to the definition of top " monoclonal antibody " and the embodiment of following more detailed description fusion method.

Therefore term " carrier " refers to that antigenic peptides or supermolecule construct can mix wherein or combination with it as used herein, the part of antigenic peptides or peptide is represented or is exposed to the structure that the mankind or animal are exempted from system.Any microgranule that is fit to be applied to the treatment of animal or human's class for example, all can be used as carrier in the context of the present invention such as liposome, microgranule or microsome.

Term " carrier " also comprises delivering method, wherein the supramolecular constructs compositions that comprises antigenic peptides can be delivered to the site of expectation by delivery apparatus.An example of this type of delivery system comprises and utilizes colloidal metal, such as gold colloidal.

In addition, term " carrier " also comprises and well known to a person skilled in the art delivery mechanism, includes but not limited to the key hole

Shape keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and other adjuvant.

In supramolecular constructs according to the present invention, liposome can have difunctional, because it can be as the carrier that comprises above the supermolecule construct of describing, and the work that can play adjuvant simultaneously will used according to the target animal of therapeutic vaccine treatment of the present invention or the immunoreation among the mankind in order to increase or to stimulate.Should be appreciated that; supramolecular constructs compositions of the present invention can further comprise other adjuvant; for example; lipid A, Alumen, calcium phosphate, interleukin-11 and/or polysaccharide and proteic microcapsule, but particularly detoxification lipid A (such as single phosphinylidyne or two phosphinylidyne lipid A) or Alumen, comprise antiseptic, diluent, emulsifying agent, stabilizing agent and other known and component of using in the vaccine of prior art in addition.In addition, any adjuvant system well known in the art also can be applicable in the compositions of the present invention.These adjuvants include but not limited to acemannan (" Acemannan ") that incomplete Freund, Freund's complete adjuvant, polydispersion β-(1,4) connect,

The lipopolysaccharide (LPS) of the modification lipid adjuvant of (polyethylene glycol oxide of CytRx Corporation-polyoxypropylene copolymer adjuvant), Chiron Corporation, the saponin derivative adjuvant of Cambridge Biotech, the bordetella pertussis (Bordetella pertussis) that kills, gram negative bacteria, big polymerization anion (such as dextran sulfate) and inorganic gel (such as Alumen, aluminium hydroxide or aluminum phosphate).

The carrier protein that can be applied to supramolecular constructs compositions of the present invention includes but not limited to maltose-binding protein " MBP ", bovine serum albumin " BSA ", key hole The gamma Globulin of shape keyhole limpet hemocyanin " KLH ", ovalbumin, flagellin, Elityran, the serum albumin of any species, any biology, homology cell, have the antigenic homology cell of Ia and D-and/or L-polymer of amino acid.

In addition, term " treatment effective dose " refers to the amount of antibody, when it grants to the mankind or animal, causes the immunoreation that enough produces therapeutic effect in the described mankind or animal.Those skilled in the art can measure the treatment effective dose easily according to conventional method.

" homology " between two sequences determined by sequence homogeneity.Have any different on length if two treat the sequence that compares mutually, sequence homogeneity preferably refers to the nucleotide residue percent of the shorter sequence identical with longer consecutive nucleotides residue.Sequence homogeneity can be by using such as Bestfit program (Wisconsin sequence analysis bag, version 8, Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, WI 53711) computer program measure as usual.Bestfit utilizes Smith and Waterman, Advances in AppliedMathematics 2 (1981), and the local clustalw algorithm of 482-489, purpose is to find out the part that has highest serial homogeneity between two sequences.When using Bestfit or other sequence alignment program when determining that whether specific sequence for example has 95% sequence homogeneity with reference sequences of the present invention, preferably so parameter is set so that sequence homogeneity percentage ratio is to calculate and allow 5% homology breach up to nucleotide sum in the reference sequences for the total length of reference sequences.When using Bestfit, what so-called optional parametric optimization ground was located at them is scheduled to (" default ") value.The deviation that appears in the comparison between given sequence and the sequence of the present invention described above may be by for example adding, lack, replace, insert or the institute that recombinates causing.This class sequence more also can preferably be used program " fasta20u66 " (2.0u66 version, in JIUYUE, 1998, William R.Pearson and the University of Virginia; Also referring to W.R.Pearson (1990), Methods in Enzymology 183,63-98, appended example and http://workbench.sdsc.edu/).For this purpose, can use the setting of " default " parameter.

Employed term " hybridization " refers to conventional hybridization conditions, preferably refer to use 5xSSPE, 1%SDS, 1x step on Hart (Denhardts) solution as solution and/or hybridization temperature between 35 ℃ to 70 ℃, preferred 65 ℃ hybridization conditions.After the hybridization, washing preferably between 35 ℃ and 70 ℃, preferred 65 ℃ temperature, at first with 2xSSC, 1%SDS with carry out (about the definition of SSPE, SSC and denhardt solution referring to the Sambrook in the above-mentioned quoted passage etc.) with 0.2xSSC subsequently.The stringent hybridization condition that for example is described among top Sambrook etc. is particularly preferred.If as above hybridize and wash at 65 ℃ pointing out, then for example provided particularly preferred stringent hybridization condition.The low stringency hybridization condition is for example hybridized and washing is more not preferred at 45 ℃, and at 35 ℃ even more not preferred.

The detailed description of the special embodiment by being contained in this below the reference can be more readily understood the present invention.Although the present invention is described according to the specific detail of its specific embodiment, and is not intended to these details are used as and limits the scope of the present invention.

The invention provides antibody and funtion part thereof, described antibody is the antibody of conformation sensitization.Specific epitopes on the multiple amyloid proteantigen of these antibody recognition.Antibody capable is used for that diagnostic and therapeutic intervention are caused by amyloid or amyloid sample albumen or relative disease and disease, described disease and disease comprise amyloidosis, this be one group with amyloid or relevant disease and the disease of amyloid sample albumen (such as a that relates to Alzheimer).

Antibody is granted individuality, and they resist various diseases or disease with passive immunity, include but not limited to, the disease relevant with amyloid is such as Alzheimer.

At this antibody that provides is monoclonal or polyclonal antibody, and it has binding specificity to the antigenic peptides of representing amyloid related symptoms (for example Alzheimer).

, prepare by with supramolecular constructs compositions immune animal according to antibody of the present invention such as mice, rat, rabbit or any animal that other can produce natural or human antibodies.

Supramolecular constructs compositions disclosed herein comprises the peptide of modification usually, with the enhancement antigen effect, wherein this class peptide is modified by Pegylation (with the Polyethylene Glycol of Polyethylene Glycol or modification), or by other method, such as passing through Palmic acid, polyamino acid (polyglycine for example, polyhistidyl), polysaccharide (polygalacturonic acid for example, polylactic acid, poly-Acetic acid, hydroxy-, bimol. cyclic ester, chitin, chitosan), synthetic polymer (polyamide, polyurethane, polyester) or modification such as copolymer (for example poly-(methacrylic acid) and N-(2-hydroxyl) propyl methyl amide).

When in the liposome bilayer, providing anchor for peptide, by the modification (palmitoylation) of Palmic acid because C _16:0The length that fatty acid part reduces relatively causes peptide almost to be positioned on the surface of liposome.Therefore, the cell of process antigen will absorb the whole liposome with peptide, and in most of the cases, it has caused slower comparatively speaking immunoreation.

In one embodiment of the invention, use the amyloid 1-15 peptide of modifying to be used for preparation according to antibody of the present invention, particularly monoclonal antibody.Can be according to amyloid 1-15 peptide at reported method synthetic modifications in 2002 such as Nicolau.Reported method such as Nicolau comprise by on resin lipophilic or hydrophobic part being grafted to preformed peptide terminal amino acid residue with the modified antigen peptide, and this has produced quite highly purified product.Particularly, with known coupling chemistry, will be through the aminoacid of protection, particularly the aminoacid of Fmoc protection is connected on the resin.Remove blocking group, and second amino acid residue through protection of coupling.Use standard automated peptide synthetic (it has used known protection chemistry, particularly Fmoc/tBu chemistry and standard side chain protected group) to pass through then at amyloid A β _1-42Amino acid/11 to 15 on coupling synthesize A β _1-15Antigenic peptides has the fragments of peptides that SEQ ID NO:1 provides sequence thereby produce.In last step, two other protection aminoacid is coupled to the fragments of peptides in the growth.Then can selective ablation Mtt group and be coupled to Palmic acid.Behind washing resin, remove blocking group and the resin of cracking simultaneously, then carry out the side chain deprotection with standard method.Can obtain high-purity end-product then, and can for example, confirm its identity by means commonly known in the art such as electron spray mass spectrometry.

According to lipophilic of the present invention or hydrophobic part can be fatty acid, triglyceride or phospholipid, and wherein the carbon backbone chain of fatty acid has at least 10 carbon atoms.Especially, lipophilic or hydrophobic part are the fatty acids that carbon backbone chain has about at least 14 carbon atoms and about at the most 24 carbon atoms, and the single numerical value that drops on the carbon atom in this scope also is a part of the present invention.More particularly, lipophilic or hydrophobic part have the carbon backbone chain of at least 14 carbon atoms.The example of hydrophobic part includes but not limited to Palmic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, linolenic acid and cholesterol or DSPE.In the specific embodiment of the present invention, lipophilic or hydrophobic part are Palmic acids.

In order to improve immunoreation, the anchor/interval that can use other is basic to rebuild the peptide in the liposome, for example Polyethylene Glycol (PEG).

PEG covalently is connected on the amino acid residue at peptide two ends, particularly Glu, Cys or Lys amino acid residue or any other can be suitable for PEG is covalently bound to the amino acid residue of peptide.At another end of chain, can covalently bound hydrophobic part with as the anchoring element in the liposome bilayer, PHOSPHATIDYL ETHANOLAMINE (PEA) for example.Therefore, liposome still works as adjuvant, and can be processed separately from bilayer peptide enough far away, and therefore compares the immunogenicity that has increased it with palmitoylation antigen.

In certain embodiments, the supramolecular constructs that uses within the scope of the present invention comprises covalently bound peptide sequence to Pegylation lysine (each terminal one).The length of PEG (Polyethylene Glycol) chain can be from n=8 to n=150, and 000 or more, particularly from n=10 to n=80,000, more particularly from n=20 to n=10,000 changes.In the specific embodiment of the present invention, the length of PEG chain is no more than n=45, particularly between n=5 to n=40, and more particularly between n=10 to n=30, and even n=10 more particularly.

Supermolecule construct described here can be synthetic with the synthetic and known protection chemistry of automated peptide (particularly Fmoc/tBu chemistry with standard side chain protected group).Usually, the Pegylation of peptide produces the mixture of regional isomer (regioisomer).

In order to realize that the PEG-lipid conjugates is connected with the C of A β and the locus specificity of N-terminal, can utilize the peptide of part protection.Comprise the peptide sequence of inner Lys or His residue for these, add the Lys (ivDde) of orthogonally protect to each end.Extra Gly can be added to C end is terminal to be synthesized promoting.Remove blocking group and carry out the N-acetylation, optionally excise the ivDde group subsequently with acetic anhydride.

Resin, particularly 2-chlorine trityl resin is favourable, its be acid-sensitive sense and make it possible to separate the protection peptide.

In the specific embodiment of the present invention, carry out coupling reaction in mutually at solution.Cracking resin and discharge inner protection peptide optionally under the condition of gentleness then.

Success finish peptide (for example, such as A β derived from the amyloid beta protein sequence _1-16(SEQID NO:2)) with the solution phase coupling of the PEG molecule of modifying with fatty acid-phosphatidylcholine (for example, such as DSPE).Can finish separating of single coupled product and double couple crosslinking product before final side chain goes to protect by using cation exchange chromatography.Peptide side chain subsequently goes to protect to have caused separating has the purpose conjugate that can accept purity.Can be by means commonly known in the art, for example HPLC etc. finishes purification.

The N-of this application protection peptide and the terminal lipid of C--antigenic synthetic method of PEG amyloid beta can be applied in the multiple peptide sequence.

Then can with as be described in Nicolau etc., 2002 method preparation is according to liposome antigen of the present invention.Amyloid A the beta antigen peptide of modifying, the particularly PEGization of Xiu Shiing and the A β of palmitoylation _1-15, A β _1-16, A β _{1-16 (Δ 14)}, A β _22-35With A β _29-40Antigenic peptides can be made up of liposome, particularly make, reconstituted in the construct that the optional liposome that comprises monophosphoryl lipid A is formed by two myristoyl phosphatidylcholines (DMPC), two myristoyl phosphatidyl cholamine (DMPEA), two myristoyl phosphatidyl glycerol (DMPG) and cholesterol.

In the specific embodiment of the present invention, the liposome with lipid A is used as the vaccine of adjuvant with the preparation anti-amyloid.Mix two myristoyl phosphatidylcholines, two myristoyl phosphatidyl glycerol and cholesterol, especially with 0.9: 1.0: 0.7 mixed in molar ratio.With strong immunomodulator, for example monophosphoryl lipid A adds with suitable concentration then, and especially with between the 30mg/mmol to 50mg/mmol, more particularly the concentration of 40mg/mmol phospholipid adds.Then with the peptide between 1: 30 to 1: 200 and the mol ratio of phospholipid, with between 1: 50 to 1: 120, more particularly add the antigen A β peptide of modifying especially with 1: 100 the peptide and the mol ratio of phospholipid.For example remove solvent by evaporation, and with sterile buffer (for example, such as the PBS) thin film that hydration produced.

Also can be by for example being described in (2002) Journal of Liposome ResearchVol 12 (3) such as Wagner, the cross flow one of pp 259-270 is injected (crossflow injection) technology and is prepared liposome.During lipid soln was injected into the aqueous buffer solution system, lipid was tending towards forming " precipitate ", and self is arranged as liposome subsequently.The size of resulting liposome depends on the factors such as selection such as lipid concentration, stir speed (S.S.), charge velocity and lipid.Preparation system can be by cross flow one fill assembly, polar phase (for example, the PBS buffer) container, ethanol/lipid soln container and pressue device, but particularly the nitrogen pressue device is formed.When by cross flow one fill assembly suction aqueous or polar solvent, ethanol/lipid soln is injected in the polar phase with different exerting pressure.

Liposome still works as adjuvant, and can be processed separately from bilayer peptide enough far away, and therefore compares the immunogenicity that has increased it with palmitoylation antigen.

Free PEG end covalency be connected to the PHOSPHATIDYL ETHANOLAMINE molecule (wherein fatty acid can be: myristic acid, Palmic acid, stearic acid, oleic acid etc., or its in conjunction with), with as anchoring element.This supramolecular structure can be anchored by reconstruct in the liposome of being made up of phospholipid and cholesterol (with PHOSPHATIDYL ETHANOLAMINE, phosphatidyl glycerol, the cholesterol of various mol ratios).Also can use other phospholipid.Concentration with about 40 μ g/pmole phospholipid is used lipid A.

In certain embodiments, the supramolecular constructs of palmitoylation or Pegylation comprises the peptide of the aminoacid sequence with amyloid beta.Peptide can comprise or corresponding to whole amyloid-beta peptide and its active fragment.In addition, be applicable to that peptide of the present invention also comprises A β _1-16(SEQ ID NO:2), A β _{1-16 (Δ 14)}(SEQ ID NO:3), A β _1-15(SEQ ID NO:1) and active fragment thereof.

In order to bring out and to prepare antibody and the immunogenicity of A beta antigen construct that measure to modify, be selected from suitable animals such as mice, rat, rabbit, pig, birds with the antigenic peptides immunity, but particularly mice, especially C57BL/6 mice.The immunogenicity of antigen construct is measured by survey serum sample with suitable interval with immunoassay (for example, such as elisa assay) after immunity.

Can prepare monoclonal antibody of the present invention with the clone and the cell-fusion techniques of standard well known in the art.Usually interested immunogen (antigen) is granted (for example intraperitoneal injection) wild type or inbreeding mice (for example BALB/c or especially C57BL/6 mice), rat, rabbit or other can produce the animal species or the transgenic mice of natural or human antibodies.Immunogen can be granted separately or mix with adjuvant, or from carrier (VEE replicon carrier, cowpox) expression or as DNA or as fusion rotein, reacts with induction of immunity.Fusion rotein comprises and is coupled to carrier protein (for example, such as beta galactosidase, glutathione s-transferase, key hole

Shape keyhole limpet hemocyanin (KLH) and bovine serum albumin) peptide, wherein the immunoreation at described peptide is desired.In these cases, peptide is as the hapten with carrier protein.After making animal for example strengthen secondary or more times, from the animal of immunity, gather in the crops splenocyte, and pass through with well-known Kohler and Milstein (Nature 256:495-497 (1975)) and Harlow and Lane (Antibodies:A Laboratory Manual (Cold SpringHarbor Laboratory, the splenocyte of method fusion sensitization New York 1988)) and myeloma cell strain are (such as Mus SP2/O myeloma cell (ATCC, Manassas, VA)) to produce hybridoma.

In the specific embodiment of the present invention, with antigen construct of the present invention, the compositions that comprises described antigen construct of pharmaceutically acceptable form particularly, with the interval between 1 to 10 week, the particularly interval between 1 to 6 week, more especially the interval between 1 to 4 week and even more particularly the interval repeated doses between 2 to 3 weeks grant particularly 1 to 15 dosage, more particularly 2 to 10 dosage even more particularly 3 to 7 dosage but 4 to 6 dosage especially.By the time suitable after reinforcement, particularly strengthen back 3 to 10 days, more particularly strengthen back 4 to 8 days and more particularly strengthen back 5 to 6 days, obtain serum sample and monitor immunoreation with the immunogenicity of a kind of (for example, such as the ELISA method of testing) in known method, particularly normally used immunity test detection antigen construct.

Cause significant immunoreation in treated animal with antigen construct according to the present invention, particularly the immunity that the vaccine combination according to antigen construct of the present invention carries out that comprises with pharmaceutically acceptable form.Animal, particularly mice that selection has the treatment titre are used for and will produce the cell or immortal cell line (such as the myeloma cell line) fusion of cell (especially B-lymphocyte) with the continuous growth of antibody.Merge by adding the Polyethylene Glycol inducing cell.The treatment titre is between 1: 4000 to 1: 6000, particularly between 1: 4500 to 1: 5500, more particularly 1: 5000 dilution produces those of positive findings in ELISA measures.

Use conventional method then, the clone of the production expectation monoclonal antibody that for example hybrid cell that is produced with the limiting dilution assay clone, and cultivation is produced.

By in the selection culture medium that comprises hypoxanthine, aminopterin, thymidine (HAT), coming chemistry to select the hybridoma that obtains like this cell inoculation.

Come the examination hybridoma according to the ability of monoclonal antibody of create antagonism specific amyloid related diseases or disease subsequently.Clone, expansion produce the hybridoma of interested antibody, and freezing is used for further production.Preferred hybridoma production has the IgG isotype, the monoclonal antibody of preferred IgG2 isotype.

By with above-described antigen construct compositions immune animal of the present invention, such as mice or rabbit or other suitable animal and prepare polyclonal antibody.From animal, collect serum subsequently, and foundation is to the antibody in the antigenic binding reactive examination of the amyloid serum.

Can be prepared as the physiology with technique known according to antibody of the present invention and go up acceptable preparation, and it can comprise pharmaceutically suitable carrier, diluent and/or excipient.For example, the antibody of describing according to the present invention and hereinbefore that comprises any function equivalent antibody or its funtion part, the monoclonal anti that particularly comprises any function equivalent antibody or its funtion part combine the formation pharmaceutical composition with pharmaceutically suitable carrier, diluent and/or excipient.Suitable pharmaceutical carriers, diluent and/or excipient are well known in the art and comprise for example phosphate buffered saline(PBS), water, emulsion (such as oil/aqueous emulsion), various wetting agent, sterile solution etc.

Can be according to the preparation of pharmaceutical composition of the present invention according to well known to a person skilled in the art that standard method finishes.

Compositions of the present invention can be granted object with suitable, pharmacy effective dose with solid, liquid or aerocolloidal form.The example of solid composite comprises tablet, emulsifiable paste and implantable dosage unit.Tablet can orally be granted.The treatment emulsifiable paste can be granted the part.Implantable dosage unit can for example be granted at tumor locus partly; Or can be used for discharging capapie therapeutic combination through implantation, for example be implanted subcutaneously.The example of fluid composition comprises the preparation of suitable intramuscular, subcutaneous, intravenous, intra-arterial injection, and is used for the preparation that part or ophthalmic are granted.The example of aerosol preparations comprises the suction preparation that is used to grant to pulmonary.

Compositions can be granted by the approach of granting of standard.In general, compositions can be granted by (for example intravenous, subcutaneous, intramuscular) approach of local, oral, rectum, nose, intradermal, intraperitoneal or parenteral.In addition, also compositions can be incorporated into and continue in the release matrix,, polymer be implanted the expectation delivery location for example near the tumor locus such as Biodegradable polymeric.Method comprise the granting of single dose, with preset time at interval repeated doses grant and continue to grant predetermined a period of time.

Lasting release matrix used herein is the substrate that can be made by the such material of the polymer of the degraded of enzyme or acid/basic hydrolysis or decomposition by usually.In case be implanted in the body, substrate is subjected to the influence of enzyme and body fluid.The lasting release matrix of expectation is chosen as biocompatible substance, such as liposome, polylactide (polylactide acid), poly-Acetic acid, hydroxy-, bimol. cyclic ester (polymer of glycolic), polylactide Acetic acid, hydroxy-, bimol. cyclic ester (copolymer of lactic acid and glycolic), polyanhydrides, poe, polypeptide, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, fatty acid, phospholipid, polysaccharide, nucleic acid, polyamino acid, aminoacid, polynucleotide, polyvinyl propylene, polyvinylpyrrolidone and polysiloxanes such as phenylalanine, tyrosine, isoleucine altogether.Preferred biodegradable substrate is polylactide, poly-Acetic acid, hydroxy-, bimol. cyclic ester or the polylactide substrate of one of Acetic acid, hydroxy-, bimol. cyclic ester (copolymer of lactic acid and glycolic) altogether.

The dosage of compositions as well known to those skilled in the art depends on multiple factor, for example, such as symptom to be treated, employed particular composition and other clinical factor (such as patient's body weight, size, sex and general health situation), body surface area, specific chemical compound or compositions, other medicines of granting simultaneously to be granted and grant approach.

Compositions can be united with other compositions that comprises bioactive substance or chemicals and granted, particularly be selected from least a of following chemical compound, and antibody of the present invention, and optional pharmaceutically suitable carrier and/or diluent and/or excipient: the chemical compound of anti-oxidation stress, the chemical compound of anti-apoptotic, metal-chelator, DNA repair inhibitors such as pirenzepine and metabolite, 3-amino-1-propane sulfonic acid (3APS), 1,3-third disulfonic acid (1,3PDS), the secretase activator, β-and inhibitors of gamma-secretase, tau protein, neurotransmitter, the βZhe Die disrupting agent, the antiinflammatory molecule, " atypical antipsychotic drug " (for example, such as clozapine, Ziprasidone, Risperidone, Aripiprazole or olanzapine), or cholinesterase inhibitor (ChEI) is (such as tacrine, Rivastigmine, donepezil, and/or galantamine) and other medicines and supplementary, such as vitamin B12, cysteine, the acetylcholine precursor, lecithin, choline, Semen Ginkgo, acetyl group-L-carnitine, idebenone, propentofylline or xanthine derivative; And methods for the treatment of diseases.

The protein pharmaceutically active substances can exist with the amount between every dose of 1ng to 10mg.Usually, the scheme of granting can be between the 0.1 μ g to 10mg scope according to the scope of antibody of the present invention, particularly 1.0 μ g to 1.0mg, the scope between 1.0 μ g to the 100 μ g more particularly, all single numerical value that fall into these scopes also are parts of the present invention.If grant by continuous infusion, more suitable dosage per hour can be the scope between every kg body weight 0.01 μ g to 10mg, and all single numerical value that fall into these scopes also are parts of the present invention.

Grant parenteral normally, for example grant intravenous.The goods that parenteral is granted comprise sterile aqueous or non-aqueous solution, suspension and emulsion.Non-aqueous solvent includes but not limited to propylene glycol, Polyethylene Glycol, vegetable oil (such as olive oil) and injectable organic ester (such as ethyl oleate).Aqueous solution can be selected from water, alcohol/aqueous solution, emulsion or comprise salt and the suspension of buffering medium.The carrier of parenteral comprises sodium chloride solution, woods Ge Shi dextrose, dextrose and sodium chloride, Lactated Ringer'S Solution or expressed oi.Intravenous vehicles comprises fluid and nutritional supplement, electrolyte supplements (such as based on those of woods Ge Shi dextrose) and other material.Also can there be antiseptic, for example, such as antibacterial, antioxidant, chelating agen, noble gas etc.

Pharmaceutical composition can further comprise protein carrier, for example, such as serum albumin or immunoglobulin, particularly human origin.Other bioactive substance also may reside in the pharmaceutical composition of invention, depends on its purpose purposes.

In other embodiments of the present invention, method and test kit are provided, it can be used for detecting and diagnosis amyloid related diseases or disease, diagnosis is to the quality of amyloid related diseases or disease, or small residual disease among the monitoring patient, or the prediction patient is to using according to the present invention and the reaction of antibody described above or vaccine combination treatment.These methods comprise and are generally used for detecting or quantitatively in the biological specimen or the known immunological method of the material under the original position condition.

Amyloid related diseases or disease or can finish by the immune specific bond that detects monoclonal antibody or its active fragment and sample or original position amyloid epi-position to the diagnosis of the quality of amyloid related diseases or disease, it comprises making suspects that contain antigenic sample of amyloid or given body part or body region contacts with antibody in conjunction with the amyloid epi-position, allow antibodies to amyloid antigen to form immune complex, detect the formation of immune complex, and with the existence of immune complex or do not exist with the antigenic existence of amyloid or do not exist in sample or given body part or the zone and be associated, randomly amount and the normal control value with described immune complex compares, wherein compare with the normal control value, the increase of the amount of described aggregation shows that described patient suffers from or have the risk that develops into amyloid related diseases or disease.

Can finish by the immune specific bond that detects monoclonal antibody or its active fragment and sample or original position amyloid epi-position according to using monitoring according to the patient's of antibody of the present invention or immune composition treatment small residual disease, it comprises making suspects that contain antigenic sample of amyloid or given body part or body region contacts with antibody in conjunction with the amyloid epi-position, allow antibodies to amyloid antigen to form immune complex, detect the formation of immune complex, and with the existence of immune complex or do not exist with the antigenic existence of amyloid or do not exist in sample or given body part or the zone and be associated, randomly amount and the normal control value with described immune complex compares, and the increase that the amount of wherein said aggregation is compared with the normal control value shows that described patient may still suffer from small residual disease.

The prediction patient can be by detecting in monoclonal antibody or its active fragment and the sample or the immune specific bond of original position amyloid epi-position is finished according to the reaction of the treatment of vaccine combination of the present invention to using, it comprises making suspects that contain antigenic sample of amyloid or given body part or body region contacts with antibody in conjunction with the amyloid epi-position, allow antibodies to amyloid antigen to form immune complex, detect the formation of immune complex, and with the existence of immune complex or do not exist with the antigenic existence of amyloid or do not exist in sample or given body part or the zone and be associated, randomly relatively before the treatment and the amount of described immune complex after the treatment, the minimizing of the amount of wherein said aggregation shows that described patient has treating aitiogenic high potentiality.

Can be used for diagnosing amyloid related diseases or disease, be used to diagnose to the quality of amyloid related diseases or disease or be used to monitor the small residual disease of patient or be used to predict that the patient is to using according to the present invention and the biological specimen of the reaction of antibody described above or vaccine combination treatment is a fluid for example, such as serum, blood plasma, saliva, stomachial secretion liquid, mucus, cerebrospinal fluid, lymph fluid etc.; Or the tissue or the cell sample that obtain from body, such as nerve, brain, heart or vascular tissue.For the existence that detects amyloid in sample or do not exist, can use any immunoassay known to a person of ordinary skill in the art (referring to Harlow and Lane, Antibodies:ALaboratory Manual (Cold Spring Harbor Laboratory, New York 1988555-612)), for example, utilize analysis, ELISA and immuno-precipitation and the coagulation test of the Indirect Detecting Method of having used second reagent that is used to detect.The WO96/13590 of Maertens and Stuyver for example, Zrein etc. (1998) and WO96/29605 have provided the detailed description of these methods of testing.

For in-situ diagnostics, can utilize the field known method that antibody or its any activity and funtion part are granted biology to be diagnosed, for example in the intravenous, intranasal, intraperitoneal, brain, intra-arterial injection, combine thereby between the epitope regions on antibody according to the present invention and the amyloid antigen specificity may take place.Can be by being connected to the markers tests antibody/antigen complex of antibody or its function fragment.

Can be used for diagnostic application or be applied to diagnose to the quality of amyloid related diseases or disease or be used to monitor the small residual disease of patient or be used to predict that the patient is to using according to the present invention and the immunoassay of the reaction of antibody described above or vaccine combination treatment depend on antigen, the antibody of labelling or second reagent that is used to detect usually.Albumen or reagent can comprise enzyme, radiosiotope and fluorescence, luminous and substance that show color with well known to a person skilled in the art the chemical compound labelling, include colored particle, such as gold colloidal and latex beads.In these methods, radioactive label almost can be applied to all types of tests and have maximum variations.Must avoid radioactivity or need fast as a result the time, the labelling of conjugated enzyme is particularly useful.Although fluorescent dye in use needs expensive equipment, fluorescent dye provides highstrung detection method.The antibody that is used for these detections comprises monoclonal antibody, polyclonal antibody and through the polyclonal antibody of affinity purification.

Perhaps, can be by coming traget antibody indirectly with the reaction that immunoglobulin is had a material (such as protein A or Protein G or second antibody) of affinity through labelling.Antibody can be puted together with second material, and has the 3rd material of affinity to detect with second material to being conjugated to antibody of labelling.For example, antibody can biotin-conjugated, and detects antibody-biotin conjugate with the Avidin or the streptavidin of labelling.Similarly, antibody can be puted together hapten, and detects antibody-hapten conjugate with the antihapten antibody of labelling.

As well known to those skilled in the art these and other can be used for suitable label of the present invention.The standard technique of those of ordinary skills' common general knowledge be can use and these labels and antibody or its segmental combination finished.Kennedy, J.H., etc., 1976 (Clin.Chim.Acta 70:1-31), and Schurs, A.H.W.M. etc., 1977 (Clin.Chim Acta 81:1-40) have described typical technology.The coupling technology of mentioning the latter is glutaraldehyde method, periodate method, BMI method and other method, and all these all are hereby incorporated by.

Current immunoassay utilize the double antibody method with the existing of check and analysis thing, wherein, antibody by with the second antibody reaction of detectable label labelling by indirect labelling.Second antibody preferably with the antibody of the antibodies of monoclonal antibody source animal.In other words, if monoclonal antibody is a mouse antibodies, then the second antibody of labelling is anti-mouse antibodies.For the monoclonal antibody of using in the test that describes below, this label is the pearl of antibody sandwich, particularly magnetic bead preferably.For the polyclonal antibody that will use in the immunoassay described here, preferred label is detectable molecule, such as radioactive, fluorescence or electrochemiluminescence material.

Also can use alternative double antibody system within the scope of the invention,, often be called as quick format system (fast format systems) because they are suitable for the existence of fast measuring analyte.This system needs the high-affinity between antibody and the analyte.According to one embodiment of the invention, with a pair of special to the amyloid separately antigenic existence of TPPA amyloid.One of described antigen centering is referred to herein as " detection antibody ", and another of described antigen centering is referred to herein as " trapping antibody ".Monoclonal antibody of the present invention can be used as captures or detects antibody.Monoclonal antibody of the present invention also can be used as to be captured and detects antibody one and be used from single analysis.Therefore one embodiment of the invention use double antibody sandwich method to detect amyloid antigen in biological fluid sample.In this method, analyte (amyloid antigen) is sandwiched in and detects between antibody and the trapping antibody, and trapping antibody is irreversible to be fixed on the solid support.Detect antibody and comprise detectable label, to identify sandwich the existing and therefore existing of identification of analytes of antibody-analyte.

Exemplary solid matter includes but not limited to known microtitration plate, polystyrene test tube, magnetic, plastics or glass bead and slide in radioimmunoassay and enzyme immunoassay (EIA) field.Be used for the method for antibody coupling to solid phase also be well known to a person skilled in the art.Recently, multiple porous material such as nylon, nitrocellulose, cellulose acetate, glass fibre and other porous polymer also are used as solid support.

The present invention also relates to the antigenic diagnostic kit of amyloid in the detection of biological sample, it comprises compositions defined above.In addition, the present invention relates to the diagnostic kit of back, except as compositions defined above, it also comprises detectable as defined above.Term " diagnostic kit " relates generally to the known any diagnostic kit in field.More particularly, back one term refers to as the diagnostic kit in description such as Zrein (1998).

Another object of the present invention provides new immunological probe and the test kit according to antibody of the present invention of comprising that is used to detect and diagnose amyloid related diseases and disease.For immunological probe, antibody is direct or indirect to be connected with suitable reporter molecules (for example, enzyme or radionuclide).Test kit comprises holds one or more containers according to antibody of the present invention, thereby and uses antibody to be used for making existing of immune complex with the formation that forms immune complex and detect immune complex or not existing in conjunction with amyloid antigen having or do not exist the description that is associated with amyloid antigen.

Embodiment

Be used to produce the antigen of mouse monoclonal antibody

Mice mAb	Antigen/sequence	Connector	Anchor	Adjuvant
Mice mAb	Antigen/sequence	Connector	Anchor	Adjuvant	mACI-01-Ab7	Aβ _1-16	PEG	DSPE	Lipid A
mACI-02-Ab6	Aβ _1-16(Δ14)	PEG	DSPE	Lipid A	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-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: antibody and the antigen construct that is used to produce described antibody

Embodiment 1: preparation palmitoylation A β _1-15The method of supramolecular constructs

Four (palmityl lysine)-A β 1-15 peptides are antigenic synthetic

According to the synthetic palmitoylation amyloid 1-15 peptide of reported method (Nicolau etc. 2002) before improved.This new method is included in the terminal Lys residue that on the resin Palmic acid is grafted to preformed peptide, rather than mixes the progressively solid phase synthesis of modified aminoacid 9-fluorenylmethyloxycarbonyl (Fmoc)-Lys (Pal)-OH.This new method has been improved coupling efficiency and quite more highly purified product is provided.Therefore, use [hexafluorophosphoric acid 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea cation] (HBTU) the coupling chemistry aminoacid Fmoc-Lys (the Mtt)-OH of orthogonally protect is connected to Wang resin (Wang resin).Remove the Fmoc group with 20% piperidines among the DMF, and second Fmoc-Lys of coupling (Mtt)-OH residue.Use the peptide sequence that has used Fmoc/tBu ensuing 15 aminoacid of standard automated peptide synthesis of coupling chemical and standard side chain protected group to provide then with generation SEQ IDNO:1.At last, link coupled two last aminoacid are Fmoc-Lys (Mtt)-OH.Use then that 1% trifluoroacetic acid (TFA) optionally cuts the Mtt group with the release peptide fragment in the dichloromethane, and make it be coupled to Palmic acid with HBTU.After the resin washing, remove the Fmoc group with 20% piperidines in the dimethyl formamide (DMF), and under standard conditions, carry out resin cleavage and side chain simultaneously with TFA at last and go protection.In cold diethyl ether, grind and produce white solid product.Electron spray mass spectrometry has confirmed identity (the m/z predictive value: 1097.9 ([M] 3+) of product; Experiment value: 1096.8 ([M-3H] 3+), do not detect other three-, two-or list-palmitoylation peptide.

Embodiment 2: the preparation method of supramolecular constructs

The Pegylation beta-amyloid peptide is antigenic synthetic

For the enhance immunity reaction, used another anchor/interval base to be reconstituted in the peptide in the liposome, for example Polyethylene Glycol (PEG).PEG covalently is connected to the lysine residue of peptide two ends.At another end (PEGn=70) of chain, covalently in conjunction with PHOSPHATIDYL ETHANOLAMINE with as the anchoring element in the liposome bilayer.Therefore, liposome still works as adjuvant, and can be by independent processing from bilayer peptide enough far away, and therefore compares the immunogenicity that has increased it with palmitoylation antigen.

The synthetic uniquely supermolecule construct described here of the Fmoc/tBu amino acid side chain protection of use standard.Usually, the Pegylation of peptide produces the mixture of regional isomer.At this, be used to make the C-and the special facilitated method that is connected of N-end site of PEG-lipid conjugates and A β with the indicate polypeptide of part protection.

(1-16,1-16 Δ 14 22-35), add the Lys (ivDde) of orthogonally protect to each end for those peptide sequences that include inner Lys or His residue.Adding extra Gly to the C-end synthesizes promoting.Remove the Fmoc group with 20% piperidines among the DMF, and carry out the N-acetylation with acetic anhydride.Last the selectivity cutting of finishing the ivDde group in one hour with 3% hydrazine hydrate among the DMF.2-chlorine trityl resin than the Wang resin of broader applications more preferably has more resistance because confirmed the former to hydrazinolysis.In addition, different with Wang resin, 2-chlorine trityl resin is extremely acid-sensitive sense, and therefore can separate the protection peptide.In fact, need carry out coupling reaction in mutually, because can not produce any coupled product with the coupling of the peptide of resin-bonded and pre-activated polyethylene glycol lipid reagent D SPE-PEG-SPA at solution.Therefore, (acetic acid/trifluoroethanol/dichloromethane, 1: 1: 8,1 hour, room temperature) produces inner peptide through protection from the Choice of Resin cutting under temperate condition.

In DMSO and excessive alkali, the finishing of success derived from sequence A β _1-16The peptide of (SEQ ID NO:2) and the solution coupling mutually of DSPE-PEG-SPA.Add excess ethanol amine cessation reaction by lasting 2 hours then, and lyophilizing solution.

For sequence 29-40, do not need special protection strategy.

(partly prepare anti-phase C by HPLC ₄Post) purification provides the N-and the terminal PEG-lipid conjugates of C-of 50-70% purity, and its identity confirms by MALDI (substance assistant laser desorpted ionized).Each sequence demonstrates sizable difference on the easness of coupling reaction, and corresponding regularization condition (temperature, DSPE-PEG-SPA molar equivalent number, time).In order from the product of expectation, to isolate excessive DSPE-PEG-SPA, used the HPLC purification.Can finish separating of single coupled product and double couple crosslinking product before in the end side chain goes to protect by using cation exchange chromatography.Separating of the expectation conjugate that peptide side chain subsequently goes to protect the DSPE-PEG-SPA that stops with excessive separation to cause having can accept purity.

The N-of this application protection peptide and the terminal lipid of C--antigenic synthetic method of PEG amyloid beta can be applicable to multiple peptide sequence.

The antibody that embodiment 3 draws by supramolecular constructs

3.1 anti-palmitoylation A β _1-15 The preparation of the mAbs of supramolecular constructs

With palmitoylation antigen (ACI-24, A β _1-15) be used for the immunity of C57BL/6 mice at interval with 2 time-of-weeks.With each antigen immune 10-12 animal (volume injected: 200 μ l contain the 8nmol peptide).Animal was put to death in last injection in back 4 days.After strengthening 5 times, the mice that selection has treatment titre (when 1: 5 of serum, 000 diluent was positive in ELISA) is used for merging.From through immune animal, collecting spleen cell, and produce hybridoma by spleen cell and the myeloma cell line that merges sensitization.Use well-known Kohler and Milstein (Nature 256:495-497 (1975)) and Harlow and Lane (Antibodies:A Laboratory Manual (Cold Spring HarborLaboratory, New York 1988)) method is carried out from the mice B-lymphocyte of spleen and myeloma cell line SP2-0 (ATCC, Manassas, the fusion of cell VA).

Merge by adding the Polyethylene Glycol inducing cell.The hybrid cell that obtains is cultivated 10 ± 14 days to allow clonal growth with conventional method then.Carry out initial Immune Clone Selection with limiting dilution.Select to produce the hybridoma cell clone of IgG and by ELISA test they and A β _1-42The specificity combination, and the clone who cultivates the production expectation monoclonal antibody produced.

The hybridoma that obtains like this carries out the chemistry selection by seeding cells in the selection culture medium that comprises hypoxanthine, aminopterin and thymidine (HAT).

According to the ability of the monoclonal antibody that produces anti-specific amyloid related diseases or disease hybridoma is screened subsequently.In case identified maternal clone, just with its sub-clone four times, to guarantee monoclonicity and to allow the hybrid cell stabilisation.

The clone produces the hybridoma of interested antibody, and enlarging also, freezing is used for further production.

The mouse monoclonal antibody parting kit typing of the same race antibody that can get by commerce, and be adapted to stable clone in the serum-free medium and be positioned in the bioreactor to produce antibody.

Preferred hybridoma production has IgG isotype, preferred IgG1 isotype monoclonal antibody.

3.2 anti-ethylene glycol PEG-A β _1-16 , A β _4-11 , A β _22-35 With A β _29-40 Supramolecular makes up The preparation of the mAb of body

As Nicolau etc., 2002, PNAS, 99, preparation liposome antibody as described in the 2332-37.Contain monophosphoryl lipid A (40mg/mM phospholipid) by two myristoyl phosphatidylcholines (DMPC), two myristoyl PHOSPHATIDYL ETHANOLAMINE (DMPEA), two myristoyl phosphatidyl glycerol (DMPG) and cholesterol (0.9: 0.1: 0.1: 0.7 mol ratio) reconstruction sequence PEG-A β in the construction of the liposome of making _1-16, A β _4-11, A β _22-35With A β _29-40(Fig. 1).With these antigens and Pegylation A β _1-16Interval with two weeks carries out immunity to C57BL/6.With every kind of antigen immune 10-12 animal.After strengthening 3 to 6 times, the mice of selecting to have the treatment titre (when 1: 5000 diluent of serum is positive in ELISA) is used for merging.From through immune animal, collecting spleen cell, and produce hybridoma by spleen cell and the myeloma cell line that merges sensitization.Use well-known Kohler and Milstein (Nature 256:495-497 (1975)) and Harlow and Lane (Antibodies:A Laboratory Manual (Cold Spring Harbor Laboratory, NewYork 1988)) method carry out mice B-lymphocyte and myeloma cell line SP2-0 (ATCC from spleen, Manassas, VA) fusion of cell.

Merge by adding the Polyethylene Glycol inducing cell.The hybrid cell that obtains with the conventional method clone, is for example used limiting dilution assay then.Select to produce the hybridoma cell clone of IgG and by ELISA test they and A β _1-42The specificity combination of peptide, and the clone who cultivates the production expectation monoclonal antibody that is produced.

According to the monoclonal antibody ability that produces anti-specific amyloid related diseases or disease hybridoma is screened subsequently.The clone produces the hybridoma of interested antibody, and enlarging also, freezing is used for further production.Preferred hybridoma production has the monoclonal antibody of IgG isotype, preferred IgG1 isotype.

Embodiment 4: the specific assay of antibody mACI-24-Ab4

For analyzing the specificity of antibody mACI-24-Ab4, preformed amyloid 1-42,1-40 and the 1-38 fibril of variable concentrations are put (Amersham Biosciences) on the Hybond ECL nitrocellulose filter.After with 10% milk powder and the sealing of 0.7% polysorbas20, film and 20 μ g/ml first antibodies were at room temperature hatched 2 hours.After the washing, use with the sheep anti mouse IgG antibody (Amersham Biosciences) of horseradish peroxidase and at room temperature hatched 1 hour, wash and hatch, subsequently film is exposed to the X-ray thin film with chemiluminescence solution.

For measuring combining of mAb mACI-24-Ab4 and amyloid-beta 1-42 fiber, 37 ℃ last seven days pre-A of formation β 1-42,1-40 and 1-38 fiber and with it on film.Use the antibody of 20 μ g/ml to be used to measure binding ability, and detected bonded antibody in 20 minutes by the sheep anti mouse IgG antibody exposure of horseradish peroxidase.

As proving by the Dot blot analysis, antibody mACI-24-Ab4 is attached on the different pre-formation A betas with different sensitivity.Antibody demonstrates the β with A _1-40Or A β _1-38Compare A β _1-42Fiber the highest in conjunction with sensitivity.Can detect at least 0.001 μ gA β _1-42Fiber, yet to A β _1-40Fiber, the detection of antibodies limit are at least 0.1 μ g and to A β _1-38Fiber is 1 μ g, means the type for these amyloid fibers, sensitivity be 100 times to being lower than 1000 times.These data show that antibody A CI-24-Ab4 is high at least 100 times to the sensitivity of amyloid form (1-42) (known its change by the secondary conformation becomes soluble and is the major part of amyloid speckle in AD patient's brain).

Embodiment 5: AC immunity monoclonal antibody mACI-01-Ab7 C2 is to the combination of amyloid kind in Western blotting and Dot blot

For whether the combination that detects mouse antibodies mACI-01-Ab7 C2 depends on the native conformation of A β, compared and the combining or in Dot blot, compared and combine (Fig. 2 a and 2b) with the native starch shape is proteic of linearisation amyloid by Western blotting.

Produce the amyloid monomer by dissolving A β 1-42 peptide in HFIP, and in argon evaporating solvent.Preserve exsiccant peptide thin film up to use at-80 ℃.Be the preparation monomer, the concentration of resuspended peptide thin film to 2.75 μ g/ μ l and in PBS, be diluted to 1 μ g/ μ l in DMSO.Be the preparation oligomer, the peptide thin film of resuspended drying is to 5mM in DMSO, and supersound process is also added PBS to reaching 400 μ M amyloid, adds the final concentration of SDS to 0.2% subsequently.37 ℃ hatch 6 hours after, the final concentration of thinned starch shape albumen to 100 μ M and hatched again 18 hours in water at 37 ℃.Make amyloid oligomer precipitation 1 hour at 4 ℃ with 33% ice-cold methanol, 4% acetic acid solution, 16200g rotation 10 minutes is resuspended in 5mM Na with precipitate ₂H ₂PO ₄, among the 35mM NaCl (pH7.4) to the final concentration of 1 μ g/ μ l.Be the preparation fiber, in Tris-HCl 50mM buffer, dilute the peptide thin film, and hatched 5 days at 37 ℃ to the concentration that reaches the 1mg/ml amyloid.Rotated test tube 5 minutes with 10000g, resuspended precipitate is to reaching 1 μ g/ μ l in 0.1M carbonate buffer solution (pH 9.6).

1 or 5 μ g monomers, oligomer or fiber dilution in PBS and sample-loading buffer, and are applied to 12%SDS-PAGE, gel is transferred to nitrocellulose filter.Perhaps, with 3 or 1 μ g or 100 and 10ng amyloid kind be diluted in PBS and directly putting to nitrocellulose filter, and with film at room temperature dry 1 hour.With casein solution (carrier) sealing after 30 minutes, film was used in the mACI-01-Ab7 C2 that is diluted to 1 μ g/ml in the casein solution or 6E10 (Chemicon) antibody incubation 30 minutes.After casein solution washing 3 times, film is used in the anti-mice IgG of the HRP labelled goat of diluting in the casein solution (Dako Cytomation) at room temperature hatched 30 minutes, wash 3 times and use DAB substrate (Dako Cytomation) development.

In Dot blot, as positive control antibody 6E10, monoclonal mouse antibody mACI-01-Ab7 C2 is attached to monomer, oligomer and fiber specifically.On the contrary, the linearizing amyloid kind of mACI-01-Ab7 C2 antibody nonrecognition in Western blotting, and 6E10 antibody is clearly discerned all linearisation peptides.This result confirms that mACI-01-Ab7 C2 and combining of amyloid depend on the native conformation of amyloid.

Embodiment 6:mACI-01Ab7 C2-A β _1-42Interact

The main antibody mACI-01-Ab7 C2 (mC2) and amyloid peptide A β of application surface plasma resonance research AC immunity _1-42Between interaction.Mouse antibodies mACI-01-Ab7 C2 and A β have been measured _1-42The combination of monomer or fiber.

All SPR experiments are all carried out on Biacore X instrument (Biacore AB).The reagent that is used for fixing (EDC, NHS and ethanolamine), induction chip CM5 and SA and running buffer and sample buffer HBS-EP are all available from Biacore AB.Use sodium acetate (10mM, pH 5.0) as coupling buffer to increase the coupling productive rate.By adding the PBS buffer to A β _1-42Reach the final concentration of 3mg/ml and place 37 ℃ to last and prepare fibril A β over 7 days bottle _1-42(BAchem).With fibril A β _1-42Be coupled on the CM5 induction chip that comprises surface combination Sensor Chip CM 5 substrate.With biotinylated monomer A β _1-42(Bachem) be coupled to the induction chip SA that comprises with the covalently bound Sensor Chip CM 5 substrate of streptavidin.Usually by analyze the mAb of four to five concentration with the running buffer serial dilution.Begin to inject from minimum concentration, and last 3 minutes through fc 1 and fc 2 with 30 μ L/ minutes speed.Flow cell 2 is by derivatization, and deducts reaction with noise and the overall refraction index changing of rectifying an instrument from fc 1.After finishing injection, used the running buffer washing surface immediately 5 minutes.For from A β _1-42Remove remaining binding antibody in the fibril, carry out surface regeneration by injection 10mM NaOH pulse.Use BIAevaluation 3.0 to carry out dynamic analysis with numerical integration and unitary analysis algorithm.Covering is at the resulting curve of the injection of variable concentrations analyte and baseline adjustment is extremely zero.For curve fitting, simultaneously all data fittings are become 1: 1 homojunction combined.

Mice mACI-01-Ab7 C2 antibody is measured as relative stronger with the combination of amyloid.As indicated at table 2, mouse antibodies mACI-01-Ab7 C2 is with 3.8 * 10 ^-4The average association constant of M/s (ka), 1.1 * 10 ^-3s ^-1Dissociation constant (kd) and therefore obtain 3.5 * 10 ^-8The average KD of M and specificity is attached to fixed A β _1-42Fiber.The association class of mACI-01-Ab7 C2 and A beta monomers like or slightly fast, have 1.6 * 10 ^-4The average ka of M/s, but it is faster to dissociate, and obtains 2.5 * 10 ^-7The KD of M.

Table 2:

Embodiment 7:mACI-01-Ab7 C2 monoclonal antibody combines with the amyloid fiber

For analyzing the molecule binding site that forms antibody on the fiber pre-, carried out negative contrast transmission electron microscopy (TEM) (Fig. 3 a and 3b).

According to ⁴, ⁵, with antibody mACI-01-Ab7 C2 and the coupling of 8nm gold colloidal.For hatching altogether of amyloid 1-42 (A β 1-42) fiber, the antibody of 6.65 μ M fibers and golden labelling was at room temperature hatched 24 hours with 1: 100 mol ratio.Subsequently 5 μ l samples were hatched 45 seconds in the fresh glow discharge Cu grid (200 order) of sending sieve pyridine (parlodium)/C thin film to cover, wash with water 3 times and freshly wash 1 time through dilution and filtering uranyl acetate with 2%.Sample is dyeed in 2% uranyl acetate 15-20 second.Stain excessive in the grid and so air drying are gone in suction.3 grids that prepared each sample.In transmission electron microscope Hitachi 7000, analyze grid.

Monoclonal antibody mACI-01-Ab7 C2 directly is attached to A β _1-42Fiber.What is interesting is that antibody does not show the symmetry combination with respect to single fiber axis, but be attached to the specific rather than All Ranges of fleece side shoot.As if the specific region in the antibody target side shoot.Possible explanation is the specific secondary structure that only exists in this specific side shoot.This hypothesis has obtained the support of NMR data, and the NMR data show that the antibody induction conformation transforms also the conformation that therefore its combination may depend on the amyloid fiber that comprises the beta sheet structure.

Embodiment 8: by the fractionated of density gradient ultracentrifugation

By based on the density gradient ultracentrifugation (Rzepecki etc., 2004) of the principle of the peptide fiber that obtains after the hatching between the different sizes that distribute, pass through at prefabricated gradient (OptiPrep subsequently with antibody and different antibodies ^TM) on SDS-PAGE precipitation research monoclonal antibody suppress A β _1-42Fiber polymerization and depolymerization A β _1-42The characteristic of fiber.The depolymerization of the total amount of the A of the pre-formation of analysis simultaneously beta, the antibody of hatching altogether and gathering inhibition activity and antibody are the tangible advantages of this method with combining of fiber.

All anti-A β in the depolymerization algoscopy, have been analyzed _1-16(mACI-01-Ab7 C2), A β _{1-16 (Δ 14)}(mACI-02-Ab6), A β _1-15(mACI-24-Ab4), A β _22-35(mACI-11-Ab9) and A β _29-40(mACI-12-Ab11) monoclonal antibody of Chan Shenging has wherein only been studied the gathering inhibition activity to monoclonal antibody mACI-02-Ab6, mACI-24-Ab4 and mACI-01-Ab7 C2.

For A β _1-42Accumulative inhibition makes A β _1-42Monomer and mAb are with two different mol ratio (monomer A β _1-42Mol ratio than mAb Senior Three ten or 100 times) under the A β of 50 μ M final concentration, hatches.37 ℃ hatch 24 hours after, at Optiprep ^TMDiscontinuous gradient on cover sample, and 4 ℃ with 259000g will manage the rotation 3 hours.Collected 15 fraction, fraction 1 is the minimum fraction from the vertical density of gradient, and fraction 15 is the fraction from the density maximum of gradient bottom.Also obtained precipitate.Dye the fraction of having analyzed collection with silver by SDS-PAGE.The A β that is used for inhibition analysis _1-42Concentration ratio be used for the A β that depolymerization is analyzed _1-42Concentration low five times, this has reduced amyloid aggregation kinetics and has guaranteed measures in linear phase scope.

For by hatch (mAb+ monomer A β altogether with mAb _1-421: 30 with two of 1: 100 different mol ratios, the final concentration of A β is 246 μ M) and depolymerization forms A β in advance _1-42Fibril is hatched sample 24 hours at 37 ℃.After 24 hours, by the ultracentrifugation fractionated and by above and before (Rzepecki etc., 2004) description the SDS-PAGE separating sample.

8.1 suppress A β _1-42Accumulative method of testing

Can prove that when not adding mAb, A β peptide is assembled behind 24 hours incubation time, and most of albumen appears at fraction 13-15, the complete polymerization of this proof A β peptide monomer.Success and significant the gathering suppresses cause littler fiber or polymeric soluble amyloid-beta (A β) albumen, and it should appear at the fraction (10-13) with less dense.The transfer of this band can obtain definite confirmation in comprising the gathering method of testing of mACI-01-Ab7 C2, and it demonstrates A β peptide and is distributed in fraction 11,12 and 13.

This is obtaining confirmation in experiment for the second time, and wherein mACI-01-Ab7 C2 causes the transfer of most bands (the strongest band) from 14 to 13 once more, and to entering the remarkable dissolving of fraction 14 to sedimentary band.This means that mACI-01-Ab7 C2 shows strong inhibition A β peptide monomer and aggregates into the ability of fiber and demonstrate specific bond (at fraction 13) to the A beta.

When using antibody mACI-24-Ab4 and mACI-02-Ab6, also carried out same observation.When not adding mAb, A β peptide is assembled behind 24 hours incubation times and most of albumen appears at fraction 13 (it is few being deposited in 12) to the precipitation, shows the complete polymerization of A β peptide monomer.Success and significant the gathering suppress cause littler fiber or polymeric soluble amyloid-beta (A β) albumen, and they should appear in the fraction with less dense.In assembling the inhibition method of testing, mACI-24-Ab4 causes the transfer of most bands (the strongest band) from 13 to 11 and 12 and enters the remarkable dissolving of fraction 13 to sedimentary band, and mACI-02-Ab6 causes the transfer of band from 13 to 10 but the extra inhibition fully that big fiber is formed (classification at fraction 13 to precipitation).These data show that mACI-24-Ab4 and mACI-02-Ab6 show stronger inhibition A β peptide monomer and aggregate into the ability of fiber and demonstrate specific bond (in fraction 11 and 12) to the A beta.

On the contrary, 1: 30 the gathering method of testing that comprises mACI-11-Ab9 of mol ratio shows the big aggregation that is distributed between the fraction 12-15 and in the precipitation.When mACI-12-Ab11 existed with mol ratio at 1: 30, aggregation appeared in fraction 11-15 and the precipitation, but has the strongest signal in fraction 11 and 12.This means that mACI-01-Ab7 C2 and mACI-24-Ab4 show the ability that the strongest inhibition A β peptide monomer aggregates into fiber.MACI-12-Ab11 has the remarkable lower rejection characteristic than mACI-01-Ab7 C2, for obtaining the inhibition activity a little less than this, need be higher than 3 times mol ratio.When comparing, still can observe slight inhibition with not suppressing the accumulative mACI-11-Ab9 of A β peptide fiber.All mAb demonstrate the specificity of A beta in conjunction with (for mACI-01-Ab7 C2, in fraction 11+12; For mACI-11-Ab9, in fraction 12 and a little less than in fraction 13; For mACI-12-Ab11, in fraction 11 and 12).

In all inhibition methods of testing, all detect peptide at the bottom fraction.Unconjugated mAb (37kDa, 95kDa and greater than 120kDa) appears at the first half (being respectively fraction 3-9 and 4-8) of gradient.

8.2A β _1-42 The depolymerization method of testing of fiber

Because incomplete fiber polymerization, A β separately _1-42Fibriilar distribution is shown in the fraction (11-15) of wider scope.The proof of the success of antibody and significant depolymerization characteristic is than more difficult in the analysis of agglomeration when therefore, hatching altogether with pre-formation fiber.Have only most fibers to show aggregation activity to less dense but still the migration energy in independent amyloid fraction scope; For independent amyloid, main band is a fraction 12.When comparing with independent amyloid, add mACI-01-Ab7 C2 at 1: 100 with mol ratio and do not demonstrate of the migration (still among fraction 11-15s) of amyloid fiber, but peak signal is transferred to 11 from 12 in the fraction scope to the less dense fraction.Although be not the suitable environment that fiber not exclusively forms, but mACI-01-Ab7 C2 demonstrates low detectable depolymerization activity.

On the contrary, when being similar to mACI-01-Ab7 C2 with identical amyloid peptide: when the antibody mol ratio is hatched, form A β in advance _1-42Hatching altogether of fibril and mACI-02-Ab6 do not demonstrate the migration of band to the less dense fraction.Have only when high 3 times 1: 30 mol ratio of use, the amyloid fiber migrates to fraction 11-15 from 12-15 (the independent amyloid that does not have antibody to hatch altogether).Therefore, as if mACI-01-Ab7 C2 has the depolymerization character slightly higher than mACI-02-Ab6.

The latter half in gradient detects the corresponding band proof mACI-02-Ab6 of mAb and mACI-01-Ab7 C2 and A β _1-42Fibriilar combination (for two mAb, all being fraction 11 to 15).

The depolymerization characteristic of mACI-01-Ab7 C2 can confirm in other experiment, wherein can pass through A β when lacking antibody _1-42Fibril proves fiber polymerization completely in the distribution of fraction 13 to P (precipitations).Here, fiber shows the depolymerization activity of antibody when hatching altogether with pre-formation fiber to the migration than the low-density fraction.Add mACI-01-Ab7 C2 with mol ratio 1: 100 and show that most of amyloid fiber migrates to 12 from 13, and in addition the band of least density from 13 to 11 migrations.Therefore, mACI-01-Ab7 C2 also demonstrates strong depolymerization activity.

The latter half in gradient detects corresponding band proof mACI-01-Ab7 C2 of mAb and A β _1-42Fibriilar combination (fraction 11 is to P), and show not combination of antibody in the respective strap of the antibody of fraction 4 to 7.

Sum up these results, the monoclonal antibody mACI-01-Ab7 C2 of proof targeting amyloid A β peptide that can be successful is attached to also can be at gathering and the depolymerization preformed fiber of vitro inhibition from monomer A β peptide to fiber on the pre-formation fiber.

When using mACI-24-Ab4, carried out similar observation.Similar with analysis of agglomeration, can pass through independent A β _1-42Fibril confirms complete fiber polymerization in the distribution of fraction 12 to P (precipitations).Here, fiber shows the depolymerization activity of antibody when hatching altogether with pre-formation fiber to the migration than the low-density fraction.Add mACI-24-Ab4 at 1: 100 with mol ratio and show that most of amyloid fiber migrates to 11 from 12.Therefore, mACI-24-Ab4 also demonstrates the poly-activity of strong solution.

Embodiment 9: suppress A β by hatching assessment altogether with mAb _1-42Fibril is assembled and depolymerization forms A β in advance _1-42The fluorometric investigation method of fibril

BIS-ANS fluorometric investigation method

For the rejection characteristic of assessment mAb, detection monomer or non-fibrous A β have been used _1-42The BIS-ANS of fibril colony (LeVine, 2002) fluorometric investigation method.Before fluorescence measurement, with A β _1-42Monomer and buffer (in contrast) or mAb (mAb and A β _1-42The mol ratio of peptide 1: 100) 37 ℃ of preincubate 14 hours.Automatically record relative fluorescence unit and with respect to the variation percentage of contrast ecbatic recently.

Compared with the control, mACI-02-Ab6 demonstrates slight inhibition ability (125.8 ± 28.5% pairs 100 ± 29.5%).As if mACI-01-Ab7 C2 has more weak activity (108.0 ± 30.0%), and compared with the control, not observing mAb mACI-11-Ab9 and mACI-12-Ab11 has raising (93.5 ± 21.9% and 73.2 ± 47.7%).This result has confirmed the ultracentrifugation data, and wherein mACI-01-Ab7 C2 demonstrates than mACI-11-Ab9 and the bigger inhibition ability of mACI-12-Ab11.

Embodiment 10: thio-flavin T (Th-T) fluorometric investigation method

For measuring gathering inhibition and the depolymerization characteristic of mAb, used thio-flavin T (Th-T) fluorometric investigation method, its specific bond is to fibril A β _1-42The A β that exists in molecule and fluorescent emission intensity subsequently and the solution _1-42The fibril amount is associated.

Before fluorescence measurement, with A β _1-42Monomer and buffer (in contrast) or mAb (mAb and A β _1-42The mol ratio of peptide 1: 100) 37 ℃ of preincubate 48 hours.Automatically record relative fluorescence unit and with respect to the variation percentage of contrast ecbatic recently.

Compared with the control, mACI-01-Ab7 C2 demonstrates significant inhibition ability (11.03 ± 20.7% pairs 100 ± 40.5%).This result has confirmed the ultracentrifugation data, and wherein mACI-01-Ab7 C2 demonstrates the inhibition ability.

For measuring the depolymerization characteristic of mAb, used thio-flavin T (Th-T) fluorometric investigation method, its specific bond is to fibril A β _1-42The A β that exists in molecule and fluorescent emission intensity subsequently and the solution _1-42The fibril amount is associated.Before measuring, form the A beta in advance and (mAb was than A β with 1: 100 mol ratio with mAb or buffer (negative control) subsequently through 7 days (37 ℃, in PBS, pH 7.1) _1-42) hatched altogether 24 hours at 37 ℃.Automatically write down relative fluorescence unit and with variation percentage ecbatic recently by ELISA microtitration plate reader with respect to contrast.

With the ultracentrifugation data consistent, mACI-01-Ab7 C2 also demonstrates optkmal characteristics in two independent experiments in the Th-T depolymerization is analyzed, and depolymerization ability compared with the control is respectively 35 ± 11% and 64.57 ± 13.58% (comparing with 100.0 ± 15.37%).

In the Th-T method of testing, mACI-24-Ab4 also demonstrates (62.99 ± 10.34% pairs 100.0 ± 10.03% of significant depolymerization characteristics; P＜0.0001).

MACI-11-Ab9 activity low slightly (28 ± 14%), and ACI-02-Ab6 and ACI-12-Ab11 do not demonstrate significant depolymerization characteristic (being respectively 17 ± 12% and 13 ± 11%).

But when summing up ultracentrifugation and fluorometric investigation method, mACI-01-Ab7 C2, mACI-01-Ab6 and mACI-24-Ab4 demonstrate in centrefuge experiment and suppress fibril aggregation and shorten pre-formation A β _1-42The difunctional ability of fibril, this can be confirmed in the fluorometric investigation method.In addition, centrefuge experiment has confirmed that mAb combines with the specificity of amyloid.

When comparing with mACI-01-Ab7 C2, mACI-11-Ab9 even when 3 times of higher concentrations, mACI-11-Ab9 demonstrates significantly lower inhibition ability in ultracentrifugation experiments, and this can be confirmed in BIS-ANS.For the depolymerization analysis, mACI-01-Ab7 C2 demonstrates and shortens pre-formation A β in centrifugal analysis and ThT method of testing _1-42The characteristic of fibril.The mACI-02-Ab6 of three times of higher concentrations is positive in two methods of testing in centrefuge experiment, but stronger in centrefuge experiment.

From above result, obviously mACI-01-Ab7 C2 and mACI-02-Ab6 demonstrate the β with A _1-42Fibril interaction, inhibition gathering and depolymerization form difunctional active only antibody of fiber in advance.

Embodiment 11:mACI-01-Ab7 C2 monoclonal antibody with ¹³The NMR and the fluorescent characteristic of the amyloid beta 1-42 peptide interaction of C labelling

Form fiber or suppress fibroplastic possibility mechanism for assessment mAb dissolving is pre-, carried out U- ¹³The Th-T fluorometric investigation method of the beta amyloid albumen 1-42 peptide of C Tyr10 and Val12 labelling and the parallel experiment (Fig. 4) between the solid state NMR.Therefore, the purpose of this research is to follow the tracks of in beta amyloid albumen and the transformation of beta sheet under the situation that monoclonal antibody exists is being arranged by the solid state NMR spectrographic method, and directly with this with the depolymerization ability that records by Th-T fluorometric investigation method relatively.

The solid state NMR spectrographic method not only detects the transformation of secondary structure, and can locate the A β that predominate structure changes _1-42Peptide domain.Solid state NMR has proved its suitability for this problem, because it is to A β _1-42The structure determination of fiber contribute (Petkova etc., 2004, Petkova etc., 2002).Particularly, ¹³C _αWith ¹³C _βChemical shift and the dependency between the secondary structure (Cornilescu etc., 1999, Luca etc., 2001, Iwadate etc., 1999) be the valuable instrument of secondary structural change in the test peptides.

Be included in 12 by the Fmoc synthetic schemes ¹³The valine of C preliminary making ( ¹²Val) with at 10 ¹³The tyrosine of C preliminary making ( ¹⁰Synthesizing of the peptide of labelling Tyr).Confirm the identity and the purity of peptide by the MALDI mass spectral analysis.The beta amyloid protein peptide (1-42) of usage flag is by hatching peptide solution 1 week generation fiber at 37 ℃ in the PBS buffer.Main problem, promptly the relatively poor dissolubility of amyloid-beta peptide in the PBS buffer can solve by following mode: the temporary transient rising with the dissolving amyloid-beta peptide of pH value that makes the PBS buffer by micro-ammonia.Utilize the character of ammonia volatilization, by under the situation that has bigger PBS buffering bath of liquid to exist, hatching sample to regain the original ph of PBS buffer.

Be to measure βZhe Die and destroy the effect of antibody, fiber solution and antibody are hatched at 37 ℃ be used for NMR and Th-T method of testing in 24 hours.Be real-time comparison, the aliquot of same solution is used for Th-T fluorometric investigation method and the lyophilizing surplus solution is used for the NMR measurement.

At first by with pre-formation ¹³After the amyloid beta of C labelling is hatched altogether and measured the depolymerization ability of mACI-01-Ab7 C2 with Th-T fluorometric investigation method, can demonstrate mAb and make fiber depolymerization 38%.Carried out spectrum analysis then.

Be research PBS (contrast) and different between mAb is hatched, usefulness PeakFit each spectrum of deconvolution (http://www.systat.com/products/PeakFit).Make the line matched well by application mix Lorentzian/Gaussian fit procedure, it the results are shown in Fig. 4.The result is summarised in the table 3, and still evident difference is the integrated intensity of the two bimodal required colonies around the match 30-33ppm.The peak at c33ppm place is corresponding to the βZhe Die of fiber, and the peak at 30ppm place is the result of random-coil conformation.The sample of hatching in PBS demonstrates most of labelling (81.7%) (Fig. 2 top) in the βZhe Die conformation, and when sample was hatched with mACI-01-Ab7 C2, the labelling in the βZhe Die conformation had reduced (53.5%) (bottom Fig. 4).As measuring by Val 12 C β research like that, the minimizing of the βZhe Die conformation total amount with regard to random-coil conformation is about 35%, and therefore approaching consistent with the minimizing total amount of using fluorescence measurement.

The comparison of the fitting parameter of two kinds of conformations of table 3:Val12 C β.The match chemical shift of two kinds of conformations is very similar but integrated intensity is very different, and this reflects that original βZhe Die conformation has reduced about 35% (1-(53.5/81.7)).This is quite consistent with the numerical value that derives from fluorescence measurement.

As confirming by the density gradient ultracentrifugation experiment with by Th-T fluorometric investigation method, sum up these results, the monoclonal antibody mACI-01-Ab7 C2 in the N-terminal 1-16 zone of confirmation targeting amyloid A β peptide that can be successful forms fiber and can become fiber and depolymerization to form fiber in advance at vitro inhibition monomer A β peptide aggregation in conjunction with pre-.This antibody can also be induced the transformation of the most conformations of βZhe Die of Val12 to random coil secondary conformation environment except forming the fiber in conjunction with pre-.This may be machine-processed by the possibility that can dissolve fiber in conjunction with monoclonal mACI-01-Ab7 C2 antibody, has also reduced 35% because the labor at Val 12 C β peaks shows the βZhe Die component, and this is approaching consistent with fluorescence data (38%).

Embodiment 12:mACI-01-Ab7 C2 is functional to the amyloid fiber

12.1 after in conjunction with mACI-01-Ab7 C2 antibody, the conformational change of A β 1-42 fiber and the initiation of depolymerization

For assessment antibody can depolymerization form beta amyloid albumen (A β in advance _1-42) mechanism of fiber, the U-that has carried out measuring thio-flavin T (Th-T) the fluorometric investigation method of depolymerization and analyzed secondary structure ¹³The parallel comparison of the solid state nmr (NMR) of the A β 1-42 peptide of C tyrosine 10 and valine 12 labellings.Antibody has dissolved 35.4% pre-formation A β 1-42 fiber and has induced the secondary conformation from the transformation of βZhe Die to random coil simultaneously.With regard to random coil, the minimizing of βZhe Die conformation total amount is about 35%, and therefore approaching consistent with the minimizing total amount of measuring with fluorescence Th-T method of testing.These data show the transformation in conjunction with the initiation secondary structure of antibody, the instability that it may cause the parallel intermolecular arrangement of βZhe Die, and the fibrous fracture that causes prolonging becomes less fragment.

12.2 the conformation dependent binding affinity of mACI-01-Ab7 C2 antibody

Because known partial antibody in the scientific literature-antigenic energy dependence change that can be used for the antigen conformation in conjunction with energy ⁶, therefore carry out mACI-01-Ab7 C2 antibodies to whole A β _1-42The comparative experiments of binding affinity of albumen and the less peptide of the epi-position that comprises antibody that is attached to 9 amino acid longs.For carrying out this relatively, use peptide (the A β of the whole aminoacid sequences of epi-position of biotinylated covering mACI-01-Ab7 C2 by ELISA _1-42The amino acid/11 3-21 of sequence, Mimotopes produce, available from ANAWA Trading SA) and biotinylated complete A β 1-42 peptide (Bachem) analyzed the affinity of antibody mACI-01-Ab7 C2.Explanation according to manufacturer (Mimotopes) is analyzed.Antibody with comprise its specific epitopes (A β _1-42The binding affinity ratio and the whole A β of the peptide amino acid/11 3-21 of sequence) _1-42Proteic binding affinity is high by 38.40%.The energy expenditure sex reversal of therefore pointing out the difference of binding affinity ability to be used to amyloid secondary conformation is to present antigen on the more acceptable position for antibody interacts.It is low that this can be interpreted as the affinity what antibody compares isolating subunit to the affinity of native antigen (whole amyloid peptide).

Embodiment 13:mACI-01-Ab7 C2 combines with the conformation specific of inhomogeneous amyloid

In order to assess mACI-01-Ab7 C2 to the polymeric amyloid of different phase, promptly, carried out being coated with the proteic ELISA of the polymeric beta amyloid of these different phases to the specificity of monomer and polymeric soluble starch shape albumen, particularly amyloid-beta (A β) albumen and fibril amyloid.According to what revise ⁷Disclosed method prepares monomer, according to ⁸Prepare polymeric soluble starch shape albumen, particularly amyloid-beta (A β), and pass through amyloid (Bachem, Switzerland) hatched 5 days at 37 ℃ with the final concentration of 1 μ g/ μ l in Tris/HCl (pH 7.4), centrifugal subsequently (10,000rpm, 5 minutes) prepare fiber.Then the final concentration of amyloid polymer with 55 μ g/ml is coated on the elisa plate, and the anti-mice IgG monoclonal antibody by using the alkaline phosphate labelling (Jackson ImmunoResearch Laboratories Inc.) carries out binding affinity ELISA.The soluble amyloid-beta of antibody and polymerization (A β) proteic binding affinity (IC50=2.53nM) is than higher with the binding affinity (IC50=5.27nM) of fiber, and with monomeric binding affinity minimum (IC50=8.3nM).These data show that the combination of antibody is except that being subjected to its epi-position influence, and the combination of antibody also is subjected to the influence of the conformation of different amyloid aggregation things.

Embodiment 14: the epitope mapping of monoclonal antibody mACI-01-Ab7 C2

Use three kinds of different peptide libraries to carry out the epitope mapping of monoclonal antibody mACI-01-Ab7 C2 by ELISA.Library comprises altogether complete aminoacid (aa) sequence of 33 biotinylated covering A β 1-42 and (is made by Mimotopes, available from ANAWA Trading SA) peptide, second library comprises biotinylated use from the peptide 12 (aa12-20 of A β) of first peptide library and with each amino acid whose peptide (seeing table 4) in the alanine alternative sequence, and the 3rd library comprises biotinylated peptide 13,14 or 15 (the aa 13-21 of A β, 14-22 or 15-23) and be alanine in each case with last amino acid replacement or will be that the aa 21 of alanine is replaced by glycine (seeing table 5).The complete A β 1-42 peptide of applicating biotinization is as positive control (Bachem).Epitope mapping is carried out in explanation according to manufacturer (Mimotopes).Briefly, will spend the night with the sealing of the 0.1%BSA among the PBS at 4 ℃ through the plate (NUNC) of streptavidin bag quilt.With after the PBS-0.05% polysorbas20 washing, use different peptides from the library at room temperature to wrap by plate 1 hour, peptide is diluted to 10 μ M final concentrations in 0.1%BSA, 0.1% Sodium Azide in PBS.After the washing, culture plate and mACI-01-Ab7C2 antibody or isotype control mice IgG2b antibody were at room temperature hatched 1 hour, antibody is diluted to 10 μ g/ml in 2%BSA, 0.1% Sodium Azide in PBS.Wash plate once more, and at room temperature hatched 1 hour with the link coupled goat anti-mouse IgG of alkali phosphatase.After final washing, with plate and phosphatase substrate (pNPP) is hatched and with the elisa plate reader at the 405nm reading.

The peptide 12,13,14 and 15 that shows first peptide library of monoclonal antibody mACI-01-Ab7 C2 specific bond.These 4 peptides comprise aa 12-20 (VHHQKLVFF), 13-21 (HHQKLVFFA), 14-22 (HQKLVFFAE) and the 15-23 (QKLVFFAED) of A β 1-42, and this prompting epi-position is positioned at the regional 12-23 of A β.The crucial aa that is used to measure WHWTNWGKTSPA 2-20 (VHHQKLVFF) with alternate second library of alanine.After aa16,17,19 or 20 was substituted by alanine, the combination of mACI-01-Ab7 C2 antibody completely lost, and showed that these aa are absolute crucial for antibody and combining of A β.As aa15 and 18 when replaced, the forfeiture of the bound fraction of mACI-01-Ab7 C2 antibody.

When aa14 is replaced with alanine,, show that aa14 is very important also to combination in conjunction with also almost completely forfeiture.

Whether at last, use the 3rd library is crucial with the combination of measuring 21,21 or 23 pairs of epi-positions of aminoacid.When aa 23 was substituted by alanine, antibody had reduced with combining of aa 15-23, shows that 23 pairs of combinations of aa also are important.When aa 21 is substituted by glycine, the bound fraction forfeiture, and when aa 22 is substituted by alanine, in conjunction with slightly forfeiture.

Table 4: the general introduction of second used peptide in library

With italic and underscore labelling in conjunction with important aa, and with italic and runic and underscore labelling in conjunction with absolute crucial aa.

p12-20 V H H Q K L V F F

A12 A H H Q K L V F F

A13 V A H Q K L V F F

A14 V H A Q K L V F F

A15 V H H A K L V F F

A16 V H H Q A L V F F

A17 V H H Q K A V F F

A18 V H H Q K L A F F

A19 V H H Q K L V A F

A20 V H H Q K L V F A

Aa numbers 12 13 14 1516 17 1819 20

Table 5: the general introduction of the 3rd the used peptide in library

p13-21 H H Q K L V F F A

p13-21 G21 H H Q K L V F F G

p14-22 H Q K L V F F A E

p14-22 A22 H Q K L V F F A A

p15-23 Q K L V F F A E D

p15-23 A23 Q K L V F F A E A

Aa numbers 13 14 1516 17 1819 20 21 22 23

Embodiment 15: use the passive inoculation of mACI-01-Ab7 C2 to amyloid effects of load in the single transgene hAPP mouse brain

Clear out of the ability of brain for assessing in vivo mACI-01-Ab7 C2 monoclonal antibody in conjunction with soluble starch shape albumen and with it, use single hAPP mice at 6 monthly ages of sex and age-matched ⁹Be used for the passive immunity research of various dose.When research finishes, the brain by collecting animal and carry out A β 1-40 and A β 1-42 specific ELISA (TGC, Germany) is analyzed soluble starch shape protein-bearing.

Every group of 8-13 animal accepted the injection of twice 100,300 and 1000 μ g monoclonal antibody in 200 μ l PBS with the interval in a week, and only uses the PBS injection in contrast.After injecting one day for the second time, put to death the biochemical analysis that animal is used for soluble starch shape protein part.For people A β 1-40 in quantitative brain homogenate soluble fraction and/or the cerebrospinal fluid (CSF) and the amount of people A β 1-42, used Enzyme Linked Immunoadsorbent Assay (ELISA) test kit (people's amyloid-beta 40 or β 42ELISA, hypersensitivity, TGC, Switzerland).Scheme according to manufacturer is carried out ELISA.Briefly, preparation standard product (diluent of synthetic A β 1-40 or A β 1-42) and sample in the 96 hole polypropylene boards (Greiner, Germany) that do not have the protein binding ability.The preparation final concentration is 1000,500,250,125,62.5,31.3 and standard substance diluent and the sample of 15.6pg/ml in sample diluent, and assembling ELISA test kit reaches the final volume of 60 μ l.Because the level of amyloid increases with the increase at mice age, and because actual assessment requires the reading of sample in the linear segment of standard curve, the sample that therefore is used for A β 40 analyses is used for the not dilution of sample that A β 42 analyzes with dilution in 2: 3.

Sample, standard substance and blank (50 μ l) are added to the polystyrene board (the trapping antibody selectivity is discerned antigenic C-terminal) of wrapping quilt through anti-A β, add selectivity anti-amyloid beta antibodies conjugate (biotinylated detection antibody) in addition, and form antibody-amyloid-antibody-complex with permission 4 ℃ of overnight incubation.Second day, add streptavidin-peroxidase-conjugate, add the TMB/ peroxide mixture after 30 minutes, cause substrate to change coloured product into, and measure color intensity with ELISA reader with 450nm optical filter by photometry.By with the standard curve comparison absorbance of making of synthetic A β 1-40 or A β 1-42 obtain A β content in the sample quantitatively.The ecbatic of change respectively (with the percentage ratio of relative comparison) with relative average control value.

When with twice peritoneal injection monoclonal antibody of dosage ACI-01-Ab7 C2 of 300 μ g to single hAPP mice passive immunity after, the minimizing of remarkable minimizing of the total amount of A β 40 and A β 42 total amounts remarkable substantially (A β 40:-27.3 ± 13.9%, p＜0.05 in its brain homogenate; A β 42:-8.6 ± 22.4, p=0.56; Non-matching Student T checks), and 100 and 1000 μ g do not reach significance.Increase (A β 40:32.3 ± 36.8% that causes A β 40 and A β 42 in the brain homogenate with 100 μ g immunity; A β 42:38.3 ± 51.4%), and handle to cause the correct trend that the amyloid load reduces with 1000 μ g, and when every treated animal number increases potential effectively (A β 40:-2.2 ± 26.0%; A β 42:-9.3 ± 15.9%).These data acknowledgements are in acute immunization protocol, and antibody mACI-01-Ab7 C2 can reduce the total amount of solvable A β in the brain of these Muridaes AD model.As if what is interesting is that dose relationship is moment, the research that still need carry out more bigger group is to obtain significant data.

Embodiment 16: the chronic passive mACI-01-Ab7 C2 that grants is to double transgenic hAPPxPS1 mice speckle effects of load

For mACI-01-Ab7 C2 monoclonal antibody in the assessment body in conjunction with and reduce the ability of amyloid speckle in the brain, the double transgenic hAPPxPS1 mice 10 of using 3.5 monthly ages of sex and age-matched is used for long chronic passive immunity research in 4 months.When research finishes, analyze the amyloid speckle by the histochemistry of animal brain by the combination of thio-flavin S.

15 transgenic animal are accepted the injection weekly of 16 times 500 μ g monoclonal antibodies.Only in contrast with 15 animals of PBS injection.All injections are intraperitoneal and give.During execution, wash from cerebrovascular, to remove blood through heart at 4 ℃ with mouse anesthesia and with physiological serum.Subsequently, from cranium, remove brain, and separate forebrain and hindbrain in hat/volume plane cutting.By using the cutting of center line sagittal that forebrain evenly is divided into a left side and right hemisphere.To be fixed in behind the hemisphere in 4% paraformaldehyde of histology's use.Cutting sagittal vibratome section (40 μ m) is used for free-floating and hatches and be stored in 4 ℃ up to dyeing with 1% Hydrazoic acid,sodium salt at PBS.For intensive speckle, with the section of five varying levels of thio-flavin S dyeing.The section of all animals is dyeed at random and is blind quantitative.Obtain image with the Leica DMR microscope that is equipped with the SonyDXC-9100P camera, and use the computer analysis of Leica Q-Win software.In image acquisition procedures, microscopical light intensity and condenser setting remain constant.The image of all acquisitions is all handled through the same computer subprogram, to minimize the deviation of researcher.The application density segmentation threshold of unanimity during whole analysis.Select the subiculum zone to be used for the automatic ration of loading thio-flavin S dyeing amyloid.

To two hAPP/PS1 mice passive immunitys in the time of 4 months, the total speckle load and the number of speckle can reduce significantly in the subiculum zone as described above.In the speckle load, can reach 31% remarkable reduction (mACI-01-Ab7 C2:1.11 ± 0.21%, and contrast: 1.61 ± 0.35%; P=0.003, Mann-Whitney U-check), and chronic passive immunity makes plaque volumes significantly reduce by 19% (mACI-01-Ab7 C2:8.73 ± 1.36 and contrast: 10.78 ± 1.36; P=0.006, Mann-Whitney U-Test), show that the dissolved occurrence degree of speckle is lower than the speckle cracking slightly.

Embodiment 17: with the influence of the passive inoculation of mACI-01-Ab7 C2 to single transgene hAPP mouse memory ability

In order to analyze the ability that mACI-01-Ab7 C2 antibody in the body changes or increase cognitive function, single hAPP mice of using 9 monthly ages of sex and age-matched carries out passive immunity research.When duration of immunity finishes, measure the non-space cognition by new object identification task (ORT).

The peritoneal injection of 400 μ g monoclonal antibodies among twice 200 μ l of every group 12 the animals received PBS, and only to inject PBS in contrast.Inject one day after for the second time, in new object identification task (ORT) ^12,13Middle research cognitive competence.For the ORT registration, mice was placed the behavior place 10 minutes and faced new unknown object.The record exploration time.After three hours, identical mice is placed identical second period of place experience again, but in the face of previous object of having explored and extra new object.The time of two objects explored in record once more, and with time of exploring new object ratio calculation with respect to total exploration time, and change with the ratio of relative comparison and to represent cognitive coefficient.

With the passive inoculation of mACI-01-Ab7 C2 cause the cognitive memory ability of single transgene AD mice remarkable increase (mACI-01-Ab7 C2:131.6 ± 9.1%, and contrast: 100.0 ± 9.2%, p＜0.05; Non-matching Student T check, and respectively organize n=12).

Preservation:

Following hybridoma cell line is deposited in according to budapest treaty and is positioned at German Braun this cuts lattice Wei, this cuts lattice Wei 38124 Brauns, Maas Qie Er Mark Odell Wei lattice 1B " Germany microbial preservation center (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH; DSMZ) ".

The hybridoma cell strains title	The antibody title	Preservation day	Preserving number
The hybridoma cell strains title	The antibody title	Preservation day	Preserving number	FP 12H3	mACI-01-Ab7	On December 1st, 2005	DSM ACC2752
FP 12H3-C2	mACI-01-Ab7C2	On December 1st, 2005	DSM ACC2750	FP 12H3	mACI-01-Ab7	On December 1st, 2005	DSM ACC2752
FP 12H3-C2	mACI-01-Ab7C2	On December 1st, 2005	DSM ACC2750	FP 12H3-G2	mACI-01-Ab7G2	On December 1st, 2005	DSM ACC2751
ET 7E3	mACI-02-Ab6	On December 8th, 2005	DSM ACC2755	FP 12H3-G2	mACI-01-Ab7G2	On December 1st, 2005	DSM ACC2751
ET 7E3	mACI-02-Ab6	On December 8th, 2005	DSM ACC2755	EJ 7H3	mACI-24-Ab4	On December 8th, 2005	DSM ACC2756

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WO 2004/058258

WO96/1359

WO96/29605

Microorganism or the relevant statement of other biomaterial with preservation

(PCT detailed rules and regulations the 13rd 2)

A. following indication relates to microorganism or other biomaterial of the preservation of the 39th page of the 7th capable indication of description
	B. preservation identifies that other preservation thing is at another page evaluation
Preservation organization names DSMZ-Germany microbial preservation center
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Preservation day preserving number December in 2005 DSM ACC2750 on the 1st
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The applicant has utilized detailed rules and regulations 28 (4) EPC
	D. state the designated state (can not vacate) that is suitable for if having
EP
EP	E. statement provides (can not vacate if having) separately
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	Only being used to accept mechanism only is used for this page or leaf of international office and receives that together with international application the international office received this page or leaf on the 23rd at December in 2006
Authorize official to authorize official Wei Muerpeite

PCT/RO/134 shows (in July, 1998; Reprint in January, 2004)

Microorganism or the relevant statement of other biomaterial with preservation

(PCT detailed rules and regulations the 13rd 2)

A. following indication relates to microorganism or other biomaterial of the preservation of the 39th page of the 7th capable indication of description
	B. preservation identifies that other preservation thing is at another page evaluation
Preservation organization names DSMZ-Germany microbial preservation center
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Preservation day preserving number December in 2005 DSM ACC2751 on the 1st
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	Only being used to accept mechanism only is used for this page or leaf of international office and receives that together with international application the international office received this page or leaf on the 23rd at December in 2006
Authorize official to authorize official Wei Muerpeite

PCT/RO/134 shows (in July, 1998; Reprint in January, 2004)

Microorganism or the relevant statement of other biomaterial with preservation

(PCT detailed rules and regulations the 13rd 2)

A. following indication relates to microorganism or other biomaterial of the preservation of the 39th page of the 7th capable indication of description
	B. preservation identifies that other preservation thing is at another page evaluation
Preservation organization names DSMZ-Germany microbial preservation center
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Preservation day preserving number December in 2005 DSM ACC2752 on the 1st
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The applicant has utilized detailed rules and regulations 28 (4) EPC
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Authorize official to authorize official Wei Muerpeite

PCT/RO/134 shows (in July, 1998; Reprint in January, 2004)

Microorganism or the relevant statement of other biomaterial with preservation

(PCT detailed rules and regulations the 13rd 2)

A. following indication relates to microorganism or other biomaterial of the preservation of the 39th page of the 23rd capable indication of description
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Preservation organization names DSMZ-Germany microbial preservation center
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Authorize official to authorize official Wei Muerpeite

PCT/RO/134 shows (in July, 1998; Reprint in January, 2004)

Microorganism or the relevant statement of other biomaterial with preservation

(PCT detailed rules and regulations the 13rd 2)

A. following indication relates to microorganism or other biomaterial of the preservation of the 40th page of the 3rd capable indication of description
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Preservation organization names DSMZ-Germany microbial preservation center
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Preservation day preserving number December in 2005 DSM ACC2756 on the 8th
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Authorize official to authorize official Wei Muerpeite

PCT/RO/134 shows (in July, 1998; Reprint in January, 2004)

Sequence table

＜110〉Ac Immune S. A.

＜120〉has the A β 1-42 monoclonal antibody specific of therapeutic properties

<130>L3017 PCT

<150>EP 05027092.5

<151>2005-12-12

<150>EP 06014729.5

<151>2006-07-14

<150>EP 06020766.9

<151>2006-10-02

<160>22

＜170〉PatentIn version 3 .3

<210>1

<211>15

<212>PRT

＜213〉people (Homo sapiens)

<220>

＜223〉antigenic peptides A β _1-15

<400>1

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

1 5 10 15

<210>2

<211>16

<212>PRT

＜213〉people

<220>

＜223〉antigenic peptides A β _1-16

<400>2

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

1 5 10 15

<210>3

<211>15

<212>PRT

＜213〉people

<220>

＜223〉antigenic peptides A β _{1-16 (Δ 14)}

<400>3

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

1 5 10 15

<210>4

<211>14

<212>PRT

＜213〉people

<220>

＜223〉antigenic peptides A β _22-35

<400>4

Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met

1 5 10

<210>5

<211>12

<212>PRT

＜213〉people

<220>

＜223〉antigenic peptides A β _29-40

<400>5

Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val

1 5 10

<210>6

<211>17

<212>PRT

＜213〉people

<220>

＜223〉antigenic peptides A β _1-17

<400>6

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

1 5 10 15

<210>7

<211>112

<212>PRT

＜213〉house mouse (Mus musculus)

<400>7

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser

20 25 30

Asn Gly Asp Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>8

<211>112

<212>PRT

＜213〉house mouse

<400>8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Leu Val

35 40 45

Ala Ser Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ser Gly Asp Tyr Trp Gly Gln Gly Ser Thr Leu Thr Val Ser Ser

100 105 110

<210>9

<211>336

<212>DNA

＜213〉house mouse

<400>9

gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagccttgta tatagtaatg gagacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttcct 300

tggacgttcg gtggaggcac caagctagaa atcaaa 336

<210>10

<211>417

<212>DNA

＜213〉house mouse

<400>10

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60

gttgtgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcagat ctagtcagag ccttgtatat agtaatggag acacctattt acattggtac 180

ctgcagaagc caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 240

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300

agagtggagg ctgaggatct gggagtttat ttctgctctc aaagtacaca tgttccttgg 360

acgttcggtg gaggcaccaa gctagaaatc aaacgggctg atgctgcacc aactgta 417

<210>11

<211>336

<212>DNA

＜213〉house mouse

<400>11

gaggtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt agctatggca tgtcttgggt tcgccagact 120

ccagacaaga ggctggaatt ggtcgcaagc atcaatagta atggtggtag cacctattat 180

ccagacagtg tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240

ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagtggtgac 300

tactggggcc aaggctccac tctcacagtc tcctca 336

<210>12

<211>408

<212>DNA

＜213〉house mouse

<400>12

atgrasttsg ggytcagmtt grttttcctt gcccttattt taaaaggtgt ccaatgtgag 60

gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 120

tgtgcagcct ctggattcac tttcagtagc tatggcatgt cttgggttcg ccagactcca 180

gacaagaggc tggaattggt cgcaagcatc aatagtaatg gtggtagcac ctattatcca 240

gacagtgtga agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300

caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgcaag tggtgactac 360

tggggccaag gctccactct cacagtctcc tcagccaaaa caacaccc 408

<210>13

<211>10

<212>PRT

＜213〉people

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa can be Ash or Gln

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa can be Ala, Val, Leu, nor-leucine, Met, Phe, or Ile

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa can be Ala, Val, Leu, nor-leucine, Met, Phe, or Ile

<220>

<221>misc_feature

<222>(7)..(7)

＜223〉Xaa can be Ala, Val, Leu, nor-leucine, Met, Phe, or Ile

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa can be Ala, Val, Leu, Ser or Ile

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa can be Glu or Asp

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa can be Glu or Asp

<400>13

His Xaa Lys Leu Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10

<210>14

<211>11

<212>PRT

＜213〉people

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa can be His, Asn, Gln, Lys or Arg

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa can be Asn or Gln

<220>

<221>misc_feature

<222>(4)..(4)

＜223〉Xaa can be His, Asn, Gln, Lys or Arg

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa can be Ala, Val, Leu, Ser or Ile

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa can be Ala, Val, Leu, Ser or Ile

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa can be Ala, Val, Leu, Ser or Ile

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa can be Glu or Asp

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉Xaa can be Glu or Asp

<400>14

Xaa His Xaa Xaa Xaa Xaa Phe Phe Xaa Xaa Xaa

1 5 10

<210>15

<211>10

<212>PRT

＜213〉people

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa can be His, Asn, Gln, Lys or Arg

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa can be Asn or Gln

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa can be Ala, Val, Leu, nor-leucine, Met, Phe, or Ile

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa can be Ala, Val, Leu, Ser or Ile

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa can be Glu or Asp

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa can be Glu or Asp

<400>15

Xaa Xaa Lys Leu Xaa Phe Phe Xaa Xaa Xaa

1 5 10

<210>16

<211>10

<212>PRT

＜213〉people

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa can be Asn or Gln

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa can be Ala, Val, Leu, nor-leucine, Met, Phe, or Ile

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa can be Ala, Val, Leu, Ser or Ile

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa can be Glu or Asp

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa can be Glu or Asp

<400>16

His Xaa Lys Leu Xaa Phe Phe Xaa Xaa Xaa

1 5 10

<210>17

<211>10

<212>PRT

＜213〉people

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa can be His, Asn, Gln, Lys or Arg

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa can be Asn or Gln

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa can be Ala, Val, Leu, nor-leucine, Met, Phe, or Ile

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa can be Ala, Val, Leu, Ser or Ile

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa can be Glu or Asp

<400>17

Xaa Xaa Lys Leu Xaa Phe Phe Xaa Xaa Asp

1 5 10

<210>18

<211>10

<212>PRT

＜213〉people

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa can be Asn or Gln

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa can be Ala, Val, Leu, nor-leucine, Met, Phe, or Ile

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa can be Ala, Val, Leu, Ser or Ile

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa can be Glu or Asp

<400>18

His Xaa Lys Leu Xaa Phe Phe Xaa Xaa Asp

1 5 10

<210>19

<211>10

<212>PRT

＜213〉people

<220>

<221>misc_feature

<222>(1)..(1)

＜223〉Xaa can be His, Asn, Gln, Lys or Arg

<220>

<221>misc_feature

<222>(2)..(2)

＜223〉Xaa can be Asn or Gln

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa can be Lys, His, Asn, Gln or Arg

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉Xaa can be Ala, Val, Leu, nor-leucine, Met, Phe, or Ile

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉Xaa can be Ala, Val, Leu, Ser or Ile

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉Xaa can be Glu or Asp

<220>

<221>misc_feature

<222>(10)..(10)

＜223〉Xaa can be Glu or Asp

<400>19

Xaa Xaa Xaa Leu Xaa Phe Phe Xaa Xaa Xaa

1 5 10

<210>20

<211>11

<212>PRT

＜213〉people

<400>20

Val His His Lys Leu Val Phe Phe Ala Glu Asp

1 5 10

<210>21

<211>219

<212>PRT

＜213〉house mouse

<400>21

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser

20 25 30

Asn Gly Asp Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

130 135 140

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

145 150 155 160

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

180 185 190

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200 205

Pro Ile Val Lys Ser Phe Ash Arg Asn Glu Cys

210 215

<210>22

<211>448

<212>PRT

＜213〉house mouse

<400>22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Leu Val

35 40 45

Ala Ser Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ser Gly Asp Tyr Trp Gly Gln Gly Ser Thr Leu Thr Val Ser Ser

100 105 110

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly

115 120 125

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

130 135 140

Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

145 150 155 160

Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met

165 170 175

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val

180 185 190

Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys

195 200 205

Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys

210 215 220

Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu

245 250 255

Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

260 265 270

Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val

290 295 300

Ser Thr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

305 310 315 320

Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr

325 330 335

Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu

340 345 350

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

355 360 365

Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser

370 375 380

Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser

405 410 415

Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly

420 425 430

Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys

435 440 445

Claims

1. the monoclonal antibody that comprises any function equivalent antibody or its funtion part, described antibody with amyloid monomeric peptide, particularly amyloid beta monomeric peptide, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After monomeric peptide is hatched altogether, suppress the A beta monomers and be gathered into high molecular polymerization fibril.

2. according to the monoclonal antibody of comprising of claim 1 any function equivalent antibody or its funtion part, compare with other amyloid peptide monomer (contrast) of the branch of in buffer, hatching, described antibody is gathered into high molecular polymerization fibril to the A beta monomers and has suppressed at least 50%, especially at least 60%, especially at least 65%, more particularly at least 75%, even more particularly at least 80%, but 85%-90% is perhaps more especially at least.

3. according to the monoclonal antibody of comprising of claim 1 or 2 any function equivalent antibody or its funtion part, described antibody is hatched altogether with molar concentration rate up to 1: 100 and amyloid monomeric peptide.

4. according to the monoclonal antibody of comprising of claim 3 any function equivalent antibody or its funtion part, described antibody between with 1: 30 to 1: 100 molar concentration rate and after the amyloid monomeric peptide hatches altogether, show its gathering rejection characteristic.

5. according to the monoclonal antibody of comprising of claim 3 or 4 any function equivalent antibody or its funtion part, described antibody shows its gathering rejection characteristic after 37 ℃ temperature and amyloid monomeric peptide are hatched 48 hours altogether.

6. according to the monoclonal antibody of comprising of claim 5 any function equivalent antibody or its funtion part, the molar concentration rate of described antibody between with 1: 30 to 1: 100, particularly with 1: 100 molar concentration rate, with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched altogether, can make preformed polymerization fibril or fibril depolymerization at least 35%, especially at least 40%, more particularly at least 50%, even especially at least 60%, but especially at least 70% or higher.

7. according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 1-6, the gathering inhibition of described antibody and depolymerization potentiality are then determined by the SDS-PAGE sedimentation analysis on preformed gradient respectively by the density gradient ultracentrifugation method.

8. according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 1-6, the gathering inhibition of described antibody and depolymerization potentiality are determined by thio-flavin T (Th-T) fluorimetry respectively.

9. the monoclonal antibody that comprises any function equivalent antibody or its funtion part, described antibody with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched altogether, can induce the βZhe Die conformation to change to the random-coil conformation in the intramolecularly given area, this has caused with the βZhe Die conformation is the increase of random-coil conformation of cost and the improved dissolution that forms high molecular polymerization amyloid fibril or fibril in advance.

10. according to the monoclonal antibody of comprising of claim 9 any function equivalent antibody or its funtion part, it is the increase of the random-coil conformation of cost that described antibody can be induced with the βZhe Die conformation, when other forms amyloid polymerization fibril or fibril (contrast) when comparing in advance with the branch of hatching in buffer, the βZhe Die conformation has reduced at least 30%, especially at least 35%, and more particularly at least 40% and more.

11. according to claim 9 or the 10 described monoclonal antibodies that comprise any function equivalent antibody or its funtion part, wherein the transformation of secondary conformation occurs in Val 12 environment of a.

12., be to carry out 24 hours wherein 37 ℃ temperature with hatching altogether of pre-formation high molecular polymerization amyloid fibril or fibril according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 6-11.

13. according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 9-12, wherein the potentiality of antibody induction secondary structure transformation are determined by solid state NMR spectroscopy.

14. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody and amyloid monomeric peptide, particularly amyloid beta monomeric peptide, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After monomeric peptide is hatched altogether, suppressed the A beta monomers and be gathered into high molecular polymerization fibril or fibril, and in addition, with by the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42After pre-formation high molecular polymerization amyloid fibril that the gathering of monomeric peptide forms or fibril are hatched altogether, can preformed polymerization fibril of depolymerization or fibril.

15., wherein carry out with molar concentration rate up to 1: 100 with amyloid monomeric peptide and pre-formation high molecular polymerization amyloid fibril or hatching altogether respectively of fibril according to the monoclonal antibody of comprising of claim 14 any function equivalent antibody or its funtion part.

16. monoclonal antibody according to comprising of claim 15 any function equivalent antibody or its funtion part, wherein and amyloid monomeric peptide and pre-form the hatching altogether respectively of high molecular polymerization amyloid fibril or fibril, carry out with 1: 100 molar concentration rate especially with the molar concentration rate between 1: 30 to 1: 100.

17., wherein carried out respectively 48 hours and 24 hours with amyloid monomeric peptide and pre-37 ℃ the temperature of being incubated in altogether that forms high molecular polymerization amyloid fibril or fibril according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 14-16.

18. according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 14-17, described antibody can make preformed polymerization fibril or fibril depolymerization at least 10%, especially at least 25%, more particularly at least 35%, even more particularly at least 50%, but 60-70% or more especially at least.

19. according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 14-17, compare with other amyloid peptide monomer (contrast) of the branch of in buffer, hatching, described antibody is to the amyloid monomeric peptide, amyloid beta monomeric peptide particularly, for example, such as A beta monomers peptide 1-39,1-40,1-41,1-42 or 1-43, but A β especially _1-42The gathering of monomeric peptide has suppressed at least 50%, and especially at least 65%, more particularly at least 75%, even more particularly at least 80%, but 85%-90% especially at least is perhaps more.

20. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has been incorporated into according to claim 1-5 and each described gathering rejection characteristic of 14-19 with according to claim 6-8,12 and each described depolymerization characteristic of 14-19, and according to the characteristic of each described destruction βZhe Die of claim 9-13.

21. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody by targeting by amino acid residue aa _n-aa _mThe epi-position district of the A beta polypeptides of restriction directly and specifically is bonded to the A beta; Wherein said aa _n-aa _mIn n be integer between 13 to 15, but especially 14; And m is the integer between 22 to 24, but especially 23; Wherein n and m can not be that identical numeral and n must always little than m numerals, poor 〉=2 between n and the m.

22. monoclonal antibody according to comprising of aforementioned arbitrary claim any function equivalent antibody or its funtion part, the native conformation of its identification amyloid, because it is bonded to amyloid oligomer and fiber specifically, rather than be bonded to nonlinearized amyloid kind.

23. according to the monoclonal antibody of comprising of aforementioned arbitrary claim any function equivalent antibody or its funtion part, described antibody or fragment are with at least about 1 * 10 ^-6Extremely at least about 1 * 10 ^-8, especially at least about 1 * 10 ^-6Extremely at least about 1 * 10 ^-7, more particularly at least about 1 * 10 ^-7Extremely at least about 1 * 10 ^-8, even more particularly at least about 1 * 10 ^-7Extremely at least about 4 * 10 ^-7Binding affinity be attached to the A beta monomers, but preferably do not demonstrate the cross reactivity of any significant and amyloid precursor protein (APP).

24. according to the monoclonal antibody of comprising of aforementioned each claim any function equivalent antibody or its funtion part, wherein antibody or fragment are with at least about 1 * 10 ^-7Extremely at least about 1 * 10 ^-9, especially at least about 1 * 10 ^-7Extremely at least about 1 * 10 ^-8, more particularly at least about 1 * 10 ^-8Extremely at least about 1 * 10 ^-9, even more particularly at least about 1 * 10 ^-8Extremely at least about 5 * 10 ^-8Binding affinity be attached to A beta, fibril or fibril, but preferably do not demonstrate the cross reactivity of any significant and amyloid precursor protein (APP).

25. monoclonal antibody according to comprising of aforementioned each claim any function equivalent antibody or its funtion part, described antibody or fragment show than with at least 5 times of the binding affinity height of A beta monomers, high especially at least 10 times, more particularly high at least 15 times and binding affinity A beta, fibril or fibril.

26. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has been incorporated at least a according to each described characteristic of claim 1 to 25, the epi-position district that promptly assemble inhibition, depolymerization, induce conformation transition, identification also directly is bonded to the A beta polypeptides, but two kinds or above combination of especially described characteristic.

27. according to the monoclonal antibody of comprising of aforementioned each claim any function equivalent antibody or its funtion part, described antibody is to A β _1-42Monomeric peptide shows high specific, but to A β _1-38, A β _1-39, A β _1-40And/or A β _1-41Monomeric peptide demonstrates does not have cross reactivity in fact.

28. according to the monoclonal antibody of comprising of claim 27 any function equivalent antibody or its funtion part, described antibody is to amyloid peptide A β _1-42Comparison A β _1-38, A β _1-39, A β _1-40, A β _1-41More responsive to 100 times, 50 to 100 times especially, more particularly 80 to 100 times, but especially 100 times, and can suppress to form the gathering of the monomeric peptide of amyloid in vitro and in vivo.

29. according to the monoclonal antibody of comprising of claim 27 or 28 any function equivalent antibody or its funtion part, described antibody has the β to amyloid peptide A _1-42Height in conjunction with sensitivity, and can detect concentration, but especially between 0.5 μ g to 0.01 μ g, the concentration range between 0.1 μ g to 0.01 μ g more particularly, but the A β of 0.01 μ g concentration especially up to 0.01 μ g _1-42Fiber.

30. according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of aforementioned claim, described antibody can reduce to suffer from and causes solvable A β concentration increases in the brain the disease or the animal of disease, mammal particularly, but the total amount of solvable A β in the especially human brain.

31. according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of aforementioned claim, described antibody can destroy speckle, therefore reduce to suffer from and cause the speckle load increases in the brain the disease or the animal of disease, mammal particularly, but the speckle load in the especially human brain.

32. according to the antibody of comprising of claim 31 any function equivalent antibody or its funtion part, described antibody makes in the brain speckle load reduce at least 20%, and especially at least 25%, more particularly at least 30%, even more particularly more than 30%.

33. monoclonal according to comprising of claim 31 any function equivalent antibody or its funtion part, described antibody can dissolve speckle, cause suffering from the disease or the animal of disease, particularly mammal that cause speckle load increase in the brain, but the minimizing of plaque volumes in the especially human brain.

34. according to the antibody of comprising of claim 31 any function equivalent antibody or its funtion part, described antibody makes that plaque volumes has reduced at least 10% in the brain, and especially at least 15%, more particularly at least 20%.

35. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has been incorporated at least a characteristic that is selected from the following properties into: assemble inhibition, depolymerization, induce conformation transition, discern and directly be attached to epi-position, particularly at 14-23, particularly the discontinuous epi-position of the conformation in 14-20 zone, prevent or slow down the amyloid speckle formation, reduce soluble A β total amount in the brain, reduce speckle load in the brain, reduce in the brain plaque volumes, maintenance or improve cognitive memory ability, but two kinds or above combination of especially described characteristic.

36. monoclonal antibody according to comprising of claim 35 any function equivalent antibody or its funtion part, described antibody has been incorporated at least 2 kinds, at least 3 kinds especially, more particularly at least 4 kinds even more particularly at least 5,6,7 or 8 kind into, but all characteristics above-mentioned especially.

37. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody recognition also is bonded at least one unique binding site on the amyloid beta, the binding site of at least two uniquenesses particularly, the binding site of wherein said at least one or described at least two uniquenesses comprises at least one amino acid residue and at least two successive amino acid residues of main participation antibodies respectively, wherein in the specific embodiment of the present invention, at least one residue that constitutes first unique combination position is the Leu that is inserted in the following core sequence, and at least two successive amino acid residues that constitute second unique combination position are inserted in the following core sequence-Phe-Phe-:

Xaa ₁-Xaa ₂-Xaa ₃-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Xaa ₇-

Wherein

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

Xaa ₇It is the amino acid residue that is selected from Glu and Asp.

38. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody recognition also is bonded at least one unique binding site on the amyloid beta, the binding site of at least two uniquenesses especially, the binding site of at least three uniquenesses more particularly, the binding site of wherein said uniqueness comprises the amino acid residue of at least one and at least two successive main participation antibodies respectively, wherein do not participated in antibodies by at least one or compare with the amino acid residue of main participation antibodies participate in the antibodies degree significantly at least one and at least two the successive aminoacid that separate of less amino acid residue be inserted in respectively in the following core sequence-His-and-Lys-Leu-:

-His-Xaa ₂-Lys-Leu-Xaa ₃-Xaa ₄-Xaa ₅-Xaa ₆-Xaa ₇-Xaa ₈-

Wherein

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₇It is the amino acid residue that is selected from Glu and Asp;

Xaa ₈It is the amino acid residue that is selected from Glu and Asp;

39. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody recognition also is bonded at least one unique binding site on the amyloid beta, the binding site of at least two uniquenesses especially, the binding site of at least three uniquenesses more particularly, the binding site of wherein said uniqueness comprises the amino acid residue of at least one and at least two successive main participation antibodies respectively, wherein represent at least two successive amino acid residues of first binding site be inserted in the following core sequence-Phe-Phe-and at least one amino acid residue be inserted in the following core sequence-His-:

-Xaa ₁-His-Xaa ₃-Xaa ₄-Xaa ₅-Xaa ₆-Phe-Phe-Xaa ₇-Xaa ₈-Xaa ₉-

Wherein,

Xaa ₃It is the amino acid residue that is selected from Asn and Gln;

Xaa ₈It is the amino acid residue that is selected from Glu and Asp;

Xaa ₉It is the amino acid residue that is selected from Glu and Asp;

And wherein said amino acid residue Xaa ₁, Xaa ₃, Xaa ₆, Xaa ₇, Xaa ₈And Xaa ₉Do not participate in antibodies or compare with-Phe-Phe-binding site with His that to participate in the antibodies degree significantly less.

40. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody recognition also is bonded at least one unique binding site on the amyloid beta, the binding site of at least two uniquenesses especially, the binding site of at least three uniquenesses more particularly, the binding site of wherein said uniqueness comprises the amino acid residue of at least one and at least two successive main participation antibodies respectively, wherein the amino acid residue of first kind at least two successive main participation antibodies be inserted in the following core sequence-Lys-and-Leu-, and second kind at least two successive amino acid residues are inserted in the following core sequence-Phe-Phe-:

Xaa ₁-Xaa ₂-Lys-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Xaa ₇-

Wherein

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

Xaa ₇It is the amino acid residue that is selected from Glu and Asp;

And wherein said amino acid residue Xaa ₂, Xaa ₃, Xaa ₄, Xaa ₅, Xaa ₆And Xaa ₇Do not participate in antibodies or compare with-Phe-Phe-binding site with Lys-Leu that to participate in the antibodies degree significantly less.

41. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody recognition also is bonded at least one unique binding site on the amyloid beta, the binding site of at least two uniquenesses especially, the binding site of at least three uniquenesses more particularly, the binding site of wherein said uniqueness comprises the amino acid residue of at least one and at least two successive main participation antibodies respectively, wherein the amino acid residue of first kind at least two successive main participation antibodies is inserted in the following core sequence-Lys-Leu-, and second kind at least two successive amino acid residues are inserted in the following core sequence-Phe-Phe-; And the third at least one amino acid residue is inserted in the following core sequence-His-:

-His-Xaa ₂-Lys-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Xaa ₇

Wherein,

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

Xaa ₇It is the amino acid residue that is selected from Glu and Asp;

42. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody recognition also is bonded at least one unique binding site on the amyloid beta, the binding site of at least two uniquenesses especially, the binding site of at least three uniquenesses more particularly, the binding site of wherein said uniqueness comprises the amino acid residue of at least one and at least two successive main participation antibodies respectively, wherein the amino acid residue of first kind at least two successive main participation antibodies is inserted in the following core sequence-Lys-Leu-, and second kind at least two successive amino acid residues are inserted in the following core sequence-Phe-Phe-; And the third at least one amino acid residue is inserted in the following core sequence-Asp-:

-Xaa ₁-Xaa ₂-Lys-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Asp.-

Wherein,

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

43. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, the binding site of 4 uniquenesses of described antibodies to the amyloid beta, the binding site of wherein said 4 uniquenesses comprises an amino acid residue and two successive amino acid residues respectively, described residue mainly participates in antibodies, the binding site of wherein said 4 uniquenesses is closely closely located on antigen each other, do not participated in antibodies by at least one respectively or compare participating in the significantly less amino acid residue of antibodies degree and separating, thereby form the discontinuous epi-position of conformation with the described amino acid residue of the binding site of 4 uniquenesses and described two successive amino acid residues.

44. monoclonal antibody according to comprising of claim 43 any function equivalent antibody or its funtion part, wherein the amino acid residue of first kind of two successive main participation antibodies is inserted in the following core sequence-Lys-Leu-, and second kind at least two successive amino acid residues are inserted in the following core sequence-Phe-Phe-; First kind of monamino acid residue be inserted in the following core sequence-and His-and second kind of monamino acid residue be inserted in the following core sequence-Asp-:

-His-Xaa ₂-Lys-Leu-Xaa ₄-Phe-Phe-Xaa ₅-Xaa ₆-Asp-

Wherein,

Xaa ₂It is the amino acid residue that is selected from Asn and Gln;

Xaa ₆It is the amino acid residue that is selected from Glu and Asp;

45. according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 37-44, the bonded continuous amino acid residue of wherein said main participation amyloid beta, particularly in the position 16 and 17-Lys-Leu-and in the position 19 and 20-Phe-Phe-, be embedded in 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。

46. according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 1-36, described antibody recognition also is bonded to the discontinuous epi-position of conformation of each definition of claim 37 to 45.

47. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, it comprises the variable region of light chain (LCVR) of SEQID NO:7.

48. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, it comprises the variable region of heavy chain (HCVR) of SEQID NO:8.

49. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, its comprise the variable region of heavy chain (HCVR) of SEQID NO:8 and SEQ ID NO:7 variable region of light chain (LCVR) the two.

50. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, its comprise variable region of light chain (LCVR) or variable region of heavy chain (HCVR) or variable region of light chain (LCVR) and variable region of heavy chain (HCVR) the two, arbitrary peptide homology that variable region of light chain (LCVR) and variable region of heavy chain (HCVR) provide with SEQ IDNO:7 and 8 respectively.

51. according to the monoclonal antibody of comprising of claim 50 any function equivalent antibody or its funtion part, wherein variable region of light chain (LCVR) has the same aminoacid sequence of the sequence that provides with SEQ ID NO:7 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

52. according to the monoclonal antibody of comprising of claim 50 any function equivalent antibody or its funtion part, wherein variable region of heavy chain (HCVR) has the same aminoacid sequence of the sequence that provides with SEQ ID NO:8 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

53. according to the monoclonal antibody of comprising of claim 50 any function equivalent antibody or its funtion part, wherein variable region of light chain (LCVR) and variable region of heavy chain (HCVR) have the same aminoacid sequence of the sequence that provides with SEQID NO:7 and 8 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% together.

54. according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 47-53, described antibody recognition also is bonded to the discontinuous epi-position of conformation of each definition of claim 37 to 45, and shows the special characteristic according to each described antibody of claim 1-36.

55. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has the features characteristic by the antibody that produces with the hybridoma cell line FP 12H3 of DSM ACC2752 preservation respectively at December in 2005 1 day and December in 2005 9 days.

56. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has the features characteristic by the antibody that produces with the hybridoma cell line FP 12H3-C2 of DSM ACC 2750 preservations respectively at December in 2005 1 day and December in 2005 9 days.

57. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has the features characteristic by the antibody that produces with the hybridoma cell line FP 12H3-G2 of DSM ACC2751 preservation respectively at December in 2005 1 day and December in 2005 9 days.

58. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has by December in 2005 features characteristic with the antibody of the hybridoma cell line ET 7E3 generation of DSM ACC2755 preservation on the 8th.

59. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has by December in 2005 features characteristic with the antibody of the hybridoma cell line EJ 7H3 generation of DSM ACC2756 preservation on the 8th.

60. by the monoclonal antibody that produced with the hybridoma cell line FP 12H3 of DSM ACC2752 preservation in 9th respectively at December in 2005 1 day and December in 2005, described monoclonal antibody comprises any function equivalent antibody or its funtion part.

61. by the monoclonal antibody that produced with the hybridoma cell line FP 12H3-C2 of DSM ACC2750 preservation in 9th respectively at December in 2005 1 day and December in 2005, described monoclonal antibody comprises any function equivalent antibody or its funtion part.

62. by the monoclonal antibody that produced with the hybridoma cell line FP 12H3-G2 of DSM ACC2751 preservation in 9th respectively at December in 2005 1 day and December in 2005, described monoclonal antibody comprises any function equivalent antibody or its funtion part.

63. by December in 2005 monoclonal antibody with the hybridoma cell line ET7E3 generation of DSM ACC2755 preservation on the 8th, described monoclonal antibody comprises any function equivalent antibody or its funtion part.

64. by December in 2005 monoclonal antibody with the hybridoma cell line EJ7H3 generation of DSM ACC2756 preservation on the 8th, described monoclonal antibody comprises any function equivalent antibody or its funtion part.

65. comprise the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody resists supramolecular constructs and produces, described supramolecular constructs comprises and beta-amyloid peptide, particularly beta-amyloid peptide A β _1-15, A β _1-16With A β _{1-16 (Δ 14)}The corresponding antigenic peptides of aminoacid sequence, described antigenic peptides hydrophobic part, for example, such as Palmic acid, or hydrophilic segment, for example, such as Polyethylene Glycol (PEG), or both combinations modify, and wherein said hydrophobic and hydrophilic segment passes through aminoacid respectively, for example, such as lysine or any other can covalently be connected to each end as the suitable aminoacid of link molecule or amino acid analogue.

The polynucleotide of the nucleotide sequence of the monoclonal antibody that 66. comprising encodes comprises any function equivalent antibody or its funtion part, it comprises:

C) nucleotide sequence that has (a) and complementary series (b); Or

D) fragment of (a) and (b) or nucleotide sequence (c), it comprises and is selected from following continuous nucleotide section: at least 20 successive nucleotide, at least 25 successive nucleotide, at least 30 successive nucleotide, at least 35 successive nucleotide, at least 40 successive nucleotide, at least 45 successive nucleotide and at least 50 successive nucleotide.

The polynucleotide of the nucleotide sequence of the monoclonal antibody that 67. comprising encodes comprises any function equivalent antibody or its funtion part, it comprises:

A) nucleotide sequence of the light chain of SEQ ID NO:10 at least;

C) nucleotide sequence that has (a) and complementary series (b); Or

The polynucleotide of the nucleotide sequence of the monoclonal antibody that 68. comprising encodes comprises any function equivalent antibody or its funtion part, it comprises:

C) nucleotide sequence that has (a) and complementary series (b); Or

The polynucleotide of the nucleotide sequence of the monoclonal antibody that 69. comprising encodes comprises any function equivalent antibody or its funtion part, it comprises:

A) nucleotide sequence of the heavy chain of SEQ ID NO:12 at least;

C) nucleotide sequence that has (a) and complementary series (b); Or

70. comprise polynucleotide with the nucleotide sequence of each described nucleotide sequence hybridization of claim 66-69.

71. according to the polynucleotide of claim 70, wherein said nucleotides sequence is listed under the conventional hybridization condition, under stringent hybridization condition, hybridization is extremely according to each described nucleotide sequence of claim 66-69 especially.

72.SEQ the variable region of light chain of ID NO:7 (LCVR).

73.SEQ the variable region of heavy chain of ID NO:8 (HCVR).

74. the polynucleotide of the variable region of light chain (LCVR) of coding SEQ ID NO:7.

75. the polynucleotide of the variable region of heavy chain (HCVR) of coding SEQ ID NO:8.

76. compositions, its comprise the treatment effective dose according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 1 to 65.

77. according to the compositions of claim 76, it is the pharmaceutical composition that randomly also comprises pharmaceutically suitable carrier.

78. according to the compositions of claim 77, it comprises the monoclonal antibody for the treatment of effective dose.

79. according to each described compositions of claim 76 to 78, be used for the treatment of cause by amyloid or amyloid sample albumen or relative disease and disease, described disease and disease comprise amyloidosis, and this is one group of disease and disease of being correlated with amyloid or amyloid sample albumen such as a relevant with Alzheimer.

80. mixture, its comprise the treatment effective dose according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 1 to 65, and the bioactive substance and/or pharmaceutically suitable carrier and/or diluent and/or the excipient that randomly also comprise other.

81. mixture according to claim 79, wherein other bioactive substance is to be used for the treatment of by amyloid or amyloid sample albumen chemical compound that cause or relative disease and disease, described disease and disease comprise amyloidosis, and this is one group of disease and disease of being correlated with amyloid or amyloid sample albumen such as a relevant with Alzheimer.

82. 0 or 81 mixture according to Claim 8, except according to each described monoclonal antibody that comprises any function equivalent antibody or its funtion part of claim 1 to 65, also comprise and be selected from least a of following chemical compound: the chemical compound of anti-oxidation stress, the chemical compound of anti-apoptotic, metal-chelator, DNA repair inhibitors such as pirenzepine and metabolite, 3-amino-1-propane sulfonic acid (3APS), 1,3-third disulfonic acid (1,3PDS), the secretase activator, β-and inhibitors of gamma-secretase, tau protein, neurotransmitter, the βZhe Die disrupting agent, the antiinflammatory molecule, or cholinesterase inhibitor (ChEI), such as tacrine, Rivastigmine, donepezil, and/or galantamine and other medicines and supplementary, with antibody according to the present invention, and optional pharmaceutically suitable carrier and/or diluent and/or excipient.

83. 2 mixture according to Claim 8, wherein chemical compound is cholinesterase inhibitor (ChEI).

84. 2 mixture according to Claim 8, wherein chemical compound is selected from tacrine, Rivastigmine, donepezil, galantamine, nicotinic acid, Memantine hydrochloride.

85. 0 to 84 each described mixture according to Claim 8, it comprises the monoclonal antibody and/or the bioactive substance for the treatment of effective dose.

86. produce according to each described monoclonal antibody method that comprises any function equivalent antibody or its funtion part of claim 1 to 65, described method is included in the antibody that produces anti-supramolecular constructs in the suitable hosts organism, but monoclonal antibody particularly, described supramolecular constructs comprises and beta-amyloid peptide, particularly beta-amyloid peptide A β _1-15, A β _1-16With A β _1-16 _{(Δ 14)}The corresponding antigenic peptides of aminoacid sequence, described antigenic peptides hydrophobic part, for example, such as Palmic acid, or hydrophilic segment, for example, such as Polyethylene Glycol (PEG), or both combinations modify, and wherein said hydrophobic and hydrophilic segment passes through aminoacid respectively, for example, such as lysine or any other can covalently be connected to each end as the suitable aminoacid of link molecule or amino acid analogue; And separation antibody.

87. according to each described monoclonal antibody of claim 1 to 65 and/or its funtion part and/or according to each described pharmaceutical composition of claim 76 to 79, according to Claim 80 to 85 each described mixture preparation treatment or palliate a disease and the medicine of the effect of disease in purposes, wherein said disease is caused by amyloid or amyloid sample albumen with disease or is relevant with it, comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, such as following disease, include but not limited to sacred disease such as Alzheimer (AD), and be the disease or the disease of feature with the forfeiture of cognitive memory ability, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.

Prepare according to each described pharmaceutical composition of claim 76 to 79 88. use according to each described monoclonal antibody of claim 1 to 65 and/or its funtion part, or the method for 0 to 85 each described mixture according to Claim 8, be used for the treatment of or alleviate by amyloid or the influence that cause or relative disease and disease of amyloid sample albumen, described disease and disease comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, such as following disease, include but not limited to sacred disease such as Alzheimer (AD), and be the disease or the disease of feature with the forfeiture of cognitive memory ability, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.

89. use according to each described monoclonal antibody of claim 1 to 65 and/or its funtion part or function equivalent antibody, according to each described pharmaceutical composition of claim 76 to 79, or 0 to 85 each described mixture preparation is used to prevent or treats or alleviate method by the medicine of amyloid or amyloid sample albumen effect that cause or relative disease and disease according to Claim 8, described disease and disease comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, such as following disease, include but not limited to sacred disease such as Alzheimer (AD), and be the disease or the disease of feature with the forfeiture of cognitive memory ability, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula.

90. use according to each described monoclonal antibody of claim 1 to 65 and/or its funtion part or function equivalent Antibody Preparation according to the method for each described pharmaceutical composition of claim 76 to 79, comprise with the described antibody of the acceptable form preparation of pharmacy.

91. according to the method for claim 90, wherein antibody is contained in the compositions with the treatment effective dose.

92. reduce to suffer from and cause the speckle load increases in the brain the disease or the animal of disease, mammal particularly, but the method for speckle load in the Ren Lei brain especially, comprise to the animal of this treatment of needs, particularly mammal, more especially human grant the treatment effective dose according to each described monoclonal antibody of claim 1 to 65, or according to each described compositions of claim 76 to 79, or 0 to 85 each described mixture according to Claim 8.

93. according to the method for claim 92, speckle load has reduced at least 20% in its midbrain, and especially at least 25%, more particularly at least 30%, even more particularly more than 30%.

94. reduce to suffer from and cause the speckle load increases in the brain the disease or the animal of disease, mammal particularly, but the method for plaque volumes in the Ren Lei brain especially, comprise to the animal of this treatment of needs, particularly mammal, more especially human grant the treatment effective dose according to each described monoclonal antibody of claim 1 to 65, or according to each described compositions of claim 76 to 79, or 0 to 85 each described mixture according to Claim 8.

95. according to the method for claim 94, the plaque volumes in its midbrain has reduced at least 10%, and especially at least 15%, more particularly at least 20%.

96. reduce to suffer from and cause soluble A β concentration increases in the brain the disease or the animal of disease, mammal particularly, but the method for soluble A β total amount in the Ren Lei brain especially, comprise to the animal of this treatment of needs, particularly mammal, more especially human grant the treatment effective dose according to each described monoclonal antibody of claim 1 to 65, or according to each described compositions of claim 76 to 79, or 0 to 85 each described mixture according to Claim 8.

97. by granting antibody, but monoclonal antibody or comprise according to the compositions of each described antibody of claim 1 to 39 or mixture and prevent for the animal or human's class that is subjected to sickness influence particularly, treatment or alleviate method by amyloid or amyloid sample albumen effect that cause or relative disease and disease, wherein said disease and disease comprise amyloidosis, this is one group of disease and the disease relevant with the formation of amyloid speckle, comprise secondary amyloidosis and age related amyloidosis, such as following disease, include but not limited to sacred disease such as Alzheimer (AD), comprise that with cognitive memory ability forfeiture be the disease or the disease of feature, for example, such as mild cognitive impairment (MCI), dementia with Lewy body, mongolism, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam hirano disease; And other is based on amyloid sample albumen or relative disease, such as progressive supranuclear plasy, multiple sclerosis, Creutzfeldt-Jakob disease, parkinson, the relevant dementia of HIV-, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), grow up morbidity type diabetes, senile cardiac amyloidosis, endocrine tumors, and other disease, comprise degeneration of macula; Described method comprise to the animal of this treatment of needs, particularly mammal, more especially human grant the treatment effective dose according to each described monoclonal antibody of claim 1 to 65, or according to each described compositions of claim 76 to 79, or 0 to 85 each described mixture according to Claim 8.

98. keep or increase the method for the mammiferous cognitive memory ability that shows amyloid related diseases or disease, comprise to the animal of this treatment of needs, particularly mammal, more especially human grant the treatment effective dose according to each described monoclonal antibody of claim 1 to 65, or according to each described compositions of claim 76 to 79, or 0 to 85 each described mixture according to Claim 8.

99. according to the method for claim 98, wherein monoclonal antibody or the compositions that comprises monoclonal antibody are granted with the treatment effective dose.

100. hybridoma cell line is characterised in that it produces according to each described monoclonal antibody of claim 1-65.

101. hybridoma cell line, be characterised in that its generation comprises the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has by December in 2005 features characteristic with the antibody of the hybridoma cell line FP 12H3 generation of DSM ACC2752 preservation on the 1st.

102. hybridoma cell line, be characterised in that its generation comprises the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has by December in 2005 features characteristic with the antibody of the hybridoma cell line FP 12H3-C2 generation of DSM ACC2750 preservation on the 1st.

103. hybridoma cell line, be characterised in that its generation comprises the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has by December in 2005 features characteristic with the antibody of the hybridoma cell line FP 12H3-G2 generation of DSM ACC2751 preservation on the 1st.

104. hybridoma cell line, be characterised in that its generation comprises the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has by December in 2005 features characteristic with the antibody of the hybridoma cell line ET 7E3 generation of DSM ACC2755 preservation on the 8th.

105. hybridoma cell line, be characterised in that its generation comprises the monoclonal antibody of any function equivalent antibody or its funtion part, described antibody has by December in 2005 features characteristic with the antibody of the hybridoma cell line EJ 7H3 generation of DSM ACC2756 preservation on the 8th.

106. hybridoma cell line, December in 2005 hybridoma cell line FP 12H3 with DSM ACC2752 preservation on the 1st.

107. hybridoma cell line, December in 2005 hybridoma cell line FP 12H3-C2 with DSM ACC2750 preservation on the 1st.

108. hybridoma cell line, December in 2005 hybridoma cell line FP 12H3-G2 with DSM ACC2751 preservation on the 1st.

109. hybridoma cell line, December in 2005 hybridoma cell line ET 7E3 with DSM ACC2755 preservation on the 8th.

110. hybridoma cell line, December in 2005 hybridoma cell line EJ 7H3 with DSM ACC2756 preservation on the 8th.

111. the method for diagnosis amyloid related diseases or disease in the patient comprises and detects monoclonal antibody or its active fragment in the sample or the immunologic opsonin combination of the amyloid epi-position of original position, said method comprising the steps of:

A) make and suspect and to contain antigenic sample of amyloid or given body part or body region and to contact described antibodies amyloid epi-position with the antibody of description hereinbefore according to the present invention;

B) allow antibodies to amyloid antigen to form immune complex;

C) formation of detection immune complex; And

D) with the existence of immune complex or do not exist with the antigenic existence of amyloid or do not exist in sample or given body part or the zone and be associated.

112. determine the degree methods of the speckle load of generation amyloid in the tissue, comprising:

(a) sample of the tissue of being studied is represented in acquisition;

(b) use according to the antigenic existence of amyloid in the described sample of each described antibody test of claim 1-65;

(c) determine to be attached to the amount of antigenic antibody; And

(d) load of the speckle in the computation organization.

113. according to the method for claim 112, wherein determine the formation of the immune complex in step c), be associated thereby make the existence of immune complex or do not exist with the antigenic existence of amyloid or do not exist.

114. diagnosis is to the method for the quality of amyloid related diseases or disease in the patient, comprises detecting monoclonal antibody or its active fragment in the sample or the immunologic opsonin combination of the amyloid epi-position of original position, it may further comprise the steps:

(a) make and suspect and to contain antigenic sample of amyloid or given body part or body region and to contact described antibodies amyloid epi-position with the antibody of description hereinbefore according to the present invention;

(b) allow antibodies to amyloid antigen to form immune complex;

(c) formation of detection immune complex; And

(d) with the existence of immune complex or do not exist with the antigenic existence of amyloid or do not exist in sample or given body part or the zone and be associated,

(e) amount of described immune complex is compared with the normal control value,

The increase that the amount of wherein said aggregation is compared with the normal control value shows that described patient suffers from or have the risk that develops into amyloid related diseases or disease.

115. the method for monitoring according to the small residual disease of the patient after the antibody of aforementioned each claim or the vaccine combination treatment, wherein said method comprises:

(b) allow antibodies to amyloid antigen to form immune complex;

(c) formation of detection immune complex; And

(d) with the existence of immune complex or do not exist with the antigenic existence of amyloid or do not exist in sample or given body part or the zone and be associated;

(e) amount and the normal control value with described immune complex compares,

The increase that the amount of wherein said aggregation is compared with the normal control value shows that described patient still suffers from small residual disease.

116. the method for prediction according to the reaction of the antibody of aforementioned each claim or vaccine combination treatment comprises:

(b) allow antibodies to amyloid antigen to form immune complex;

(c) formation of detection immune complex; And

(e) relatively before the treatment beginning and the amount of the described immune complex after the treatment beginning,

The minimizing of the amount of wherein said aggregation shows that described patient has treating aitiogenic high potentiality.

117. be used to detect and diagnose the test kit of amyloid related diseases and disease, comprise each described antibody according to claim 1-65.

118. test kit according to claim 114, comprise and hold one or more containers according to antibody of the present invention, and use antibody to be used in conjunction with amyloid antigen to form immune complex, and the formation that detects immune complex, there is or do not exist the description that is associated thereby make the existence of immune complex or do not exist with amyloid antigen.